The Role of the Transcriptional Regulatory Protein BCL11B ...

AN ABSTRACT OF THE DISSERTATION OF

Kateryna Kyrylkova for the degree of Doctor of Philosophy in Pharmacy

presented on January 16, 2014

Title: The Role of the Transcriptional Regulatory Protein BCL11B in Dental and

Craniofacial Development

Abstract approved:

Mark E. Leid

BCL11B is a transcriptional regulatory protein that plays essential roles during

mouse embryonic development. BCL11B is expressed and functions in the

immune and nervous systems as well as within ectodermal organs. Multiple

studies have characterized the roles of BCL11B in T cells, brain, and skin.

However, very little is known about the mechanistic role of BCL11B during tooth

development, and data are not available on the function of BCL11B in the

craniofacial skeleton.

BCL11B is expressed widely within the or al cavity during development, and mice

lacking BCL11B exhibit a spectrum of to oth developmental defects. The most

striking feature of the Bcl11b-/- dental ph enotype is a defect in development of

enamel-secreting cells, known as ameloblasts, in the mouse incisor. Ameloblasts

are localized exclusively on the labial aspect of the mouse incisor in wild-type

mice. In contrast, Bcl11b-/- mice exhibit defective ameloblasts on the labial and

develop ectopic, ameloblast-like cells on the lingual aspect of the tooth.

BCL11B regulates asymmetric ameloblast formation by regulating the

development of epithelial stem cell niches in the posterior part of the incisor.

Specifically, BCL11B induces proliferation and differentiation of epithelial stem

cells into ameloblasts in the labial cervical loop, whereas BCL11B suppresses

these processes within the lingual epithelium. Such bidirectional actions of

BCL11B are mediated by spatio-specific regulation of a large gene network

comprised of genes that encode members of fibroblast growth factor (FGF) and

transforming growth factor β (TGFβ) superfamilies, Sprouty proteins, and sonic

hedgehog (SHH). In addition, my data integrate BCL11B into FGF and SHH

signaling pathways revealing the molecular mechanisms that suppress

development of ectopic ameloblast-like cells in the lingual epithelium.

In the second half of this dissertation, I show that BCL11B is expressed in the

osteogenic mesenchyme of developing craniofacial skeleton, and loss of

BCL11B in these tissues has striking effects on craniofacial development.

-/-Bcl11b mice exhibit accelerated mineralization of the skull during embryonic

development and synostosis of facial and coronal sutures. My results

demonstrate that BCL11B normally functions to suppress proliferation and

premature differentiation of osteoblasts in the craniofacial complex. I suggest that

the principal mechanistic basis of these actions of BCL11B is the repression of

Fgfr2c expression within the osteogenic mesenchyme.

Taken together, my data demonstrate that BCL11B plays an important role in

proliferation and differentiation of ameloblast and osteoblast lineages. In addition,

my work implicates BCL11B in regulation of FGF and TGFβ signaling pathways.

Therefore, these studies contribute to a better understanding of the molecular

and cellular functions of BCL11B in vivo.

The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development

by Kateryna Kyrylkova

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the

degree of

Doctor of Philosophy

Presented January 16, 2014 Commencement June 2014

Doctor of Philosophy dissertation of Kateryna Kyrylkova presented on

January 16, 2014.

APPROVED:

Major Professor, representing Pharmacy

Dean of the College of Pharmacy

Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of

Oregon State University libraries. My signature below authorizes the release of

my dissertation to any reader upon request.

Kateryna Kyrylkova, Author

ACKNOWLEDGEMENTS

Dr. Mark Leid, who was not only my graduate advisor, but a real mentor and a

good friend

Dr. Chrissa Kioussi, who supervised my first steps in developmental biology, and

provided an example of a confident and successful woman

Dr. Michael Freitag, Dr. John Fowler, and Dr. Siva Kolluri, my committee

members, who challenged me throughout the entire PhD program

Dr. Mark Zabriskie, who always believed in me and shared the joy of my

achievements

Dr. Taifo Mahmud, who was always patient and supportive with all my

organizational endeavors and multiple applications

Dr. Urszula Iwaniec, our precious collaborator, without whom our craniofacial

project would not be possible

Dr. Ophir Klein and Dr. Brian Biehs (from UCSF), our collaborators for the tooth

project

Dr. Oleh Taratula, who inspired me to continuously aim for better and achieve

every goal I set

Dr. Andriy Morgun, who was a constant source of encouragement in my

professional development, as well as the postdoctoral position search

Dr. Aleksandra Sikora, who tremendously supported me when Mark was away,

and made me feel as a part of her lab-family

Dr. Jane Ishmael, Dr. Theresa Filtz, Dr. Olena Taratula, Dr. Natalia Shulzhenko,

Drs. Gitali and Arup Indra, Dr. Michael Gross, Dr. Adam Alani, who were always

there for me when I needed any help or support

All students, postdocs, and technicians (especially Dr. Walter Vogel, Dr. Kenneth

Philbrick, Sam Bradford, Jeff Serrill, Katya Distanova, Lina Thomas and

numerous others) for being supportive and creating a great environment at the

department

Former lab members, including Dr. Olga Golonzhka, Dr. Ling-Juan Zhang,

Dr. Anand Venkataraman, for setting an example of what I should aim for

College of Pharmacy and Graduate School for supporting me financially

My family, Volodymyr Kyrylkov, Oksana Kyrylkova, Sergiy Kyrylkov, Hanna

Vayvala, who always emphasized the importance of education as the first priority

of my early life

And finally to a person, who has been with me and supported me in every

possible way throughout my entire long PhD journey, my husband Sergiy

Kyryachenko

CONTRIBUTION OF AUTHORS

Chapter 1: Kateryna Kyrylkova wrote the chapter. Kateryna Kyrylkova and Mark

Leid edited the chapter.

Chapter 2: Kateryna Kyrylkova, Sergiy Kyryachenko, Brian Biehs, Ophir Klein,

Chrissa Kioussi, and Mark Leid conceived and designed experiments. Kateryna

Kyrylkova and Sergiy Kyryachenko performed the experiments and data

analysis. Brian Biehs, Ophir Klein, Chrissa Kioussi, and Mark Leid contributed

reagents and materials. Kateryna Kyrylkova wrote the chapter. Kateryna

Kyrylkova, Ophir Klein, Chrissa Kioussi, and Mark Leid edited the chapter.

Chapter 3: Kateryna Kyrylkova, Urszula Iwaniec, and Mark Leid conceived and

designed experiments. Kateryna Kyrylkova and Urszula Iwaniec performed the

experiments and data analysis. Urszula Iwaniec and Mark Leid contributed

reagents and materials. Kateryna Kyrylkova wrote the chapter. Kateryna

Kyrylkova and Mark Leid edited the chapter.

Chapter 4: Kateryna Kyrylkova wrote the chapter. Kateryna Kyrylkova and Mark

Leid edited the chapter.

Page

Chapter 1. Transcriptional Regulation and in vivo functions of BCL11B ........... 1

BCL11B as a Transcriptional Regulatory Protein ........................................ 3

BCL11B Function in vivo ............................................................................. 6

BCL11B in T Cell Development ............................................................. 6

BCL11B in Neural Development ............................................................ 8

BCL11B in Development of Ectodermal Organs .................................. 11

References ................................................................................................ 13

Chapter 2. BCL11B Regulates Epithelial Proliferation and Asymmetric

Development of the Mouse Mandibular Incisor............................................... 20

Abstract ..................................................................................................... 21

Introduction ............................................................................................... 21

Materials and Methods .............................................................................. 23

Mouse Lines ........................................................................................ 23

Histological Analysis, RNA in situ Hybridization, and

Immunohistochemistry ......................................................................... 24

Cell Proliferation Assay ........................................................................ 24

Apoptosis Assay .................................................................................. 24

TABLE OF CONTENTS

Page

Results ...................................................................................................... 24

BCL11B is Expressed at all Stages of Incisor Development ................ 24

Reduced Epithelial Proliferation between Initiation and Bud Stage in

Bcl11b−/− Incisors ................................................................................. 25

Altered Development of Bcl11b−/− Incisors at Cap Stage ..................... 26

Reduced Size and Disruption of Labial-lingual Asymmetry at Bell

Stage in Bcl11b−/− Incisors ................................................................... 27

Delay in Ameloblast Development and Ectopic Formation of Lingual Ameloblast-like Cells in Bcl11b−/− Incisors ........................................... 28

Alteration of the FGF Signaling at Bell Stage in Bcl11b−/− Incisors ...... 29

−/− Disruption of TGFβ Signaling at Bell Stage in Bcl11b Mice .............. 31

Cell Autonomous Effects of BCL11B in Lingual Epithelium ................. 32

FGF Signaling Negatively Regulates BCL11B Expression in the

Lingual IEE and SR ............................................................................. 34

Discussion ................................................................................................. 36

BCL11B Regulates Proliferation of the Dental Epithelium ................... 36

BCL11B Controls the Expression of FGF and TGFβ Family

Members ............................................................................................. 37

BCL11B Controls Asymmetric Development of the Ameloblasts ......... 39

TABLE OF CONTENTS (Continued)

Page

Integration of BCL11B into FGF and SHH Signaling Pathways ........... 41

Acknowledgements ................................................................................... 42

References ................................................................................................ 67

Chapter 3. BCL11B Regulates Craniofacial Suture Patency through

Repression o f Fgfr2c Expression .................................................................... 71

Abstract ..................................................................................................... 72

Introduction ............................................................................................... 72

Materials and Methods .............................................................................. 74

Mouse Lines ........................................................................................ 74

Micro-CT Analysis ................................................................................ 74

Histological Analysis, RNA in situ Hybridization, BrdU Labeling and

Immunohistochemistry ......................................................................... 74

Results ...................................................................................................... 75

Neural Crest-Specific Ablation of Bcl11b Leads to Growth

Impairment and Craniofacial Synostoses ............................................. 75

Newborn Bcl11b-/- Mice Exhibit Facial and Coronal Synostoses .......... 76

Embryonic Skulls of Bcl11b-/- Exhibit Increased Osteoblast

Proliferation and Maturation, Premature Mineralization, and

Craniofacial Synostosis ....................................................................... 76



Page

BCL11B Is Expressed in Osteogenic Mesenchyme ............................. 77

Runx2 Expression I s Up-Regulated in Osteogenic Mesenchyme of

Bcl11b-/- Skulls ..................................................................................... 78

Fgfr2c is ectopically expressed within facial and coronal sutures of

Bcl11b-/- mice ....................................................................................... 78

Discussion ................................................................................................. 79

References ................................................................................................ 96

Chapter 4. Conclusion .................................................................................. 103

Regenerative Dentistry ............................................................................ 104

Targeted Treatment of Craniofacial Synostosis ...................................... 107

References .............................................................................................. 112

Bibliography .................................................................................................. 117

LIST OF FIGURES

Figure Page

2.1. BCL11B expression during incisor development .................................... 43

2.2. Epithelial invagination defect in Bcl11b−/− developing incisors between

initiation and early bud stage .......................................................................... 44

2.3. Alterations in Bcl11b−/− incisor development at cap stage ....................... 45

2.4. Morphological defects in Bcl11b−/− incisor development at bell stage ...... 46

2.5. Altered ameloblast development in Bcl11b−/− incisors ............................. 48

2.6. Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor .............................................................................................. 49

2.7. Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor ............... 50

2.8. Ectopic lingual expression of ameloblast markers and signaling

molecules in Bcl11bep−/− incisor ...................................................................... 51

2.9. Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/−

mice at E16.5 .................................................................................................. 52

2.10. Model for a reciprocal, inhibitory circuit on the lingual side of the

mouse incisor ................................................................................................. 53

S2.1. Expression of Bcl11b at early bell stage ............................................... 54

S2.2. Expression patterns of selected genes in Bcl11b−/− incisor at early

bud stage ........................................................................................................ 55

LIST OF FIGURES (Continued)

Figure Page

S2.3. Expression patterns of selected genes in Bcl11b−/− incisor at cap

stage ............................................................................................................... 56

S2.4. Delay in the initiation of apoptosis in Bcl11b−/− enamel knot at cap

stage ............................................................................................................... 57

S2.5. Size difference between wild-type and Bcl11b−/− incisors of newborn

mice ................................................................................................................ 58

S2.6. Labial to lingual reversal of expression of Gli1 in Bcl11b−/− incisors ...... 59

S2.7. Expression pattern of Lfrn and Notch1 in Bcl11b−/− incisors .................. 60

S2.8. Expression of ameloblast markers in Bcl11bep−/− and Bcl11bmes/−

incisors at E18.5 ............................................................................................. 61

S2.9. Morphology and mineralization of Bcl11bep−/− incisors at P21 ............... 62

S2.10. Expression patterns of ameloblast markers and signaling molecules in Bcl11bmes−/− incisors at E16.5 ..................................................... 63

S2.11. Labial to lingual reversal of expression of Tbx1 in Bcl11b−/− incisors .. 64

S2.12. BCL11B expression in Fgf3−/−; Fgf10+/− incisors ................................. 65

S2.13. Summary of direct or indirect BCL11B target genes at E16.5 ............. 66

3.1. Impaired postnatal growth, misshapen heads, and craniofacial

synostoses in Bcl11bncc-/- mice ....................................................................... 84

3.2. Craniofacial defects of Bcl11bncc-/- and Bcl11b-/- newborn mice ............... 85

LIST OF FIGURES (Continued)

Figure Page

3.3. Increased osteoblast maturation, mineralization, and craniofacial

synostoses in Bcl11b-/- embryonic skulls ........................................................ 86

3.4. BCL11B expression in osteogenic and sutural mesenchyme .................. 88

3.5. Increased Runx2 expression in the Bcl11b-/- embryonic faces and

coronal sutures ............................................................................................... 89

3.6. Ectopic Fgfr2c expression in facial and coronal sutures of Bcl11b-/-

embryos .......................................................................................................... 90

S3.1. Some Bcl11bncc-/- mice exhibit increased bone porosity in the skull at

P21 ................................................................................................................. 91

S3.2. Craniofacial phenotype development in Bcl11bncc-/- mice at postnatal

stages ............................................................................................................. 92

S3.3. Synostosis of facial sutures in newborn Bcl11b-/- mice .......................... 93

S3.4. Delayed elevation and fusion of palatal shelves in Bcl11b-/- mice ......... 94

S3.5. Neural crest-specific ablation of BCL11B expression in the facial

mesenchyme .................................................................................................. 95

S3.6. Expression patterns of FGF and FGFR genes in the faces of

Bcl11b-/- mice at E14.5 ................................................................................... 96


Chapter 1

Transcriptional Regulation and in vivo Functions of BCL11B

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Cell fates, development of tissues and organs, as well as complex body plans

are governed by large transcriptional and signaling networks that define precise

patterns of gene expression during mammalian embryogenesis (Peter and

Davidson, 2011; Spitz and Furlong, 2012). Loss of a single, key player from such

a system may disrupt the normal developmental program and lead to

morphogenetic and functional defects, as well as early death. Understanding the

precise function of each player and its interaction with the rest of the network

may provide us with invaluable information regarding the genetic, developmental,

and physiological processes. Furthermore, the knowledge obtained can be used

to manipulate genetic and environmental factors and ultimately aid in the

development of targeted therapies and regenerative medicine.

Regulation of transcriptional activity during embryonic development plays an

essential role in cell type specification and organogenesis. Transcriptional

regulatory proteins serve as sequence-specific, DNA-binding factors or adaptor

molecules that determine spatial and temporal expression of ~20,000 protein-

coding genes in humans. Mutations in components of trans-acting transcriptional

machinery or cis-acting regulatory DNA elements, including promoters and distal

elements, are associated with numerous human diseases and developmental

defects (Maston et al., 2006).

In order to study the effects of different genetic mutations that may underlie

human disorders, model organisms are used extensively in many areas of

biological research. Similarities in anatomy, physiology, and genetics between

mice and humans place the mouse at the foremost position as a model to study

biological processes, development, and pathology. Discovery of how

homologous recombination can be utilized to introduce specific, genetic

modifications in mice using embryonic stem cells allowed studying the functions

of individual genes in vivo (Capecchi, 2005). Recently, a new approach of

“genome editing” has emerged that utilizes engineered nucleases (i.e. zinc-finger

nucleases, transcription activator-like effector nucleases, and clustered

3

regulatory interspaced short palindromic repeat/Cas-based RNA-guided DNA

endonucleases) to quickly and efficiently produce mice with mutations in multiple

genes (Gaj et al., 2013; Urnov et al., 2010). This technology will dramatically

accelerate the development of animal models for diseases with simple Mendelian

or even polygenic inheritance. The hope is that linking phenotypic and genotypic

information will allow us to create a functional map of mammalian genome

(Nguyen and Xu, 2008).

BCL11B as a Transcriptional Regulatory Protein

BCL11B (B-cell leukemia/lymphoma 11B) is a Krüppel-like Cys2His2 (C2H2) zinc

finger transcriptional regulatory protein. It is also known as CTIP2 (chicken

ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein

2), as it, along with its homolog CTIP1, was initially cloned in Dr. Leid’s laboratory

based on its ability to interact with all members of COUP-TF family of orphan

nuclear receptors in yeast two-hybrid screening (Avram et al., 2000). Later,

Bcl11a/Ctip1 was demonstrated to serve as a site of retroviral integration that

was associated with murine myeloid leukemia (Nakamura et al., 2000).

Moreover, human BCL11A was shown to be involved in B-cell lymphoid

malignancies through chromosomal translocation (Satterwhite et al., 2001).

Subsequently, Ctip2 was re-named to Bcl11b based on its sequence homology

to Bcl11a, although it has never been implicated in B-cell leukemia and is not

expressed in B cells (Avram et al., 2000; Satterwhite et al., 2001).

The Bcl11b gene is located on mouse chromosome 12 (52.0 cM) and human

chromosome 14 (q32.1). Both mouse and human genes contain four exons, and

three splice variants, composed of exons 1-2-4, 1-2-3-4 (long isoform), and 1-4

(short isoform), have been described (Wakabayashi et al., 2003).

BCL11B harbors seven C2H2 zinc finger motifs that represent the most common

DNA-binding domains found in eukaryotic transcription factors (Avram et al.,

2000). A single C2H2 zinc finger, composed of a ȕ-hairpin and an α-helix held

4

together by a tetrahedrally coordinated zinc ion, spans a DNA sequence of three

or four consecutive base pairs in the major groove. DNA recognition usually

requires two to four tandemly arranged zinc fingers. When only one or two finger

motifs are present, additional secondary structure elements are generally used to

augment DNA recognition (Wolfe et al., 2000). Extensive studies of zinc finger

binding to DNA made possible to generate “designer” zinc finger proteins that

can target specific genomic sites. Moreover, zinc finger motifs are now known to

have additional activities, such as recognition of RNA and proteins (Brown, 2005;

Gamsjaeger et al., 2007; Hall, 2005).

Initially, BCL11B was shown to act as a transcriptional repressor when recruited

to the template either by interaction with COUP-TF family members or by direct

sequence-specific DNA-binding to a GC-rich response element (Avram et al.,

2000; Avram et al., 2002). BCL11B-mediated transcriptional repression is

dependent on the trichostatin A (TSA)-insensitive class III histone deacetylase

(HDAC) sirtuin 1 as well as TSA-sensitive class I and/or II HDACs (Cismasiu et

al., 2005; Senawong et al., 2003; Topark-Ngarm et al., 2006). HDACs promote

the repressed transcriptional state by facilitating formation of a compact form of

chromatin, thereby making genes less accessible to the general transcriptional

machinery and/or activators (Dai and Faller, 2008). In addition, BCL11B was

shown to interact with the histone methyltransferase SUV39H1, which, along with

HDAC1 and HDAC2, promotes HIV-1 transcriptional silencing in the microglia

cells (Marban et al., 2007). Genes encoding the cyclin-dependent kinase

inhibitors p21 and p57, transcription factor FOXP3, anti-inflammatory cytokine IL

10, as well as ubiquitin ligase for p53 degradation HDM2 are transcriptionally

repressed by BCL11B (Cherrier et al., 2009; Obata et al., 2012; Topark-Ngarm et

al., 2006; Vanvalkenburgh et al., 2011). More recent reports suggest that

BCL11B can act not only as a transcriptional repressor, but also as an activator

of the target genes in a cell type and promoter context-dependent manner. For

example, BCL11B associates with the p300 co-activator and augments

expression from the IL2 promoter in T cells (Cismasiu et al., 2006).

5

BCL11B was found to associate with the nucleosome remodeling and

deacetylation (NuRD) co-repressor complex in T lymphocytes and

neuroblastoma cells (Cismasiu et al., 2005; Topark-Ngarm et al., 2006).

Multisubunit complexes, such as the NuRD complex, function in spatially and

temporarily coordinated manner by acting as “writers”, “erasers”, or “readers” of

the histone code. The “writer” and “eraser” functions reside in subunits containing

catalytic activity that add or remove the mark of histones, whereas the “reader”

function requires signature domains that specifically recognize and bind to these

marks (Ruthenburg et al., 2007; Taverna et al., 2007). For example, the NuRD

complex contains HDAC1 and HDAC2 that act as “erasers” of histone acetylation

marks, leading to transcriptional repression (Zhang et al., 1999). Non-enzymatic

subunits that perform “reader” function in the NuRD complex are methyl-CpG

binding domain 2 protein (MBD2) and MBD3 that convert the information

represented by DNA methylation patterns into the appropriate functional state

(Fatemi and Wade, 2006; Zhang et al., 1999). In addition, the NuRD complex

contains chromodomain-helicase-DNA-binding protein CHD3 or CHD4 that

participate in chromatin remodeling and facilitate transcriptional activation (Zhang

et al., 1998). Consistent with this, recent studies demonstrate that the NuRD

complex, traditionally known to regulate transcriptional repression, is also

required for transcriptional activation in certain contexts (Hung et al., 2012;

Miccio and Blobel, 2010; Miccio et al., 2010). This provides one possible

explanation for how BCL11B, in association with the NuRD complex, may act as

a transcriptional repressor or activator in a promoter context-dependent manner.

Bimodal function of BCL11B as a transcriptional repressor or activator depends

also on its post-translational modifications. ERK1/2-mediated phosphorylation of

BCL11B stimulates its repressive activity on the Id2 promoter, whereas

sumoylation of BCL11B results in recruitment of p300 to the promoter with

subsequent induction of transcription. These post-translational modifications are

mutually exclusive, which suggests the existence of a phospho-deSUMO switch

within the protein (Zhang et al., 2012b).

6

BCL11B Function in vivo

BCL11B is highly conserved at the amino acid level across a wide range of

vertebrate species, suggesting that it plays an important role in these organisms

(Satterwhite et al., 2001). It is expressed during mouse development and

adulthood, and the most notable expression is observed in the central nervous

system, thymus, and ectodermal structures (Enomoto et al., 2011; Golonzhka et

al., 2007; Golonzhka et al., 2009b; Leid et al., 2004). Mice without functional

Bcl11b (Bcl11b-/-) were generated by “floxing” and deletion of exon 4, which

encodes ~75% of the open reading frame and six zinc finger motifs. The mutants

exhibit perinatal death and severe phenotypes in the tissues that express Bcl11b

(Golonzhka et al., 2009a).

BCL11B in T Cell Development

The common lymphoid progenitor gives rise to the lymphocytes: T cells, B cells,

and natural killer cells. Progenitors of T cells are produced in the bone marrow,

but migrate to the thymus, in which they undergo maturation prior to release into

general circulation. The two major subtypes of T cells differ in their T-cell

receptor (TCR) structure and functional properties in the immune system: αȕ and

Ȗβ cells. T cells expressing Ȗβ TCR represent only a small fraction of thymocytes

(no more than 5%), largely mediate mucosal immunity, and are localized

predominantly in the epithelium of intestine, skin, lungs, and reproductive organs.

In contrast, αȕ T cells are abundant in lymphoid organs, play a major role in cell-

mediated, adaptive immunity, and require antigen presentation by major

histocompatibility complex (Mertsching et al., 2002).

Development of αȕ T cells proceeds through multiple, sequential stages that are

distinguished by the expression of cell surface markers and increasingly

restricted differentiation potential (Liu et al., 2010; Rothenberg and Taghon,

2005). Early, committed T cells lack expression of TCR and are termed double-

negative (DN; cluster of differentiation CD4 - CD8-) thymocytes. As they progress

7

through their development they become double-positive T cells (DP; CD4+ CD8+)

and finally mature to single-positive (CD4+ CD8- or CD4 - CD8+) thymocytes that

are then released from thymus to peripheral tissues (Germain, 2002). The DN

population can be further subdivided by the expression of CD44 and CD25:

CD44+ CD25− (DN1) cells differentiate into CD44+ CD25+ (DN2) cells, which give

rise to CD44− CD25+ (DN3) cells, which finally become the most mature

CD44− CD25− (DN4) DN population (Ceredig and Rolink, 2002).

Bcl11b is expressed in the mouse developing thymus as early as embryonic day

(E) 14.5, when most thymocytes are DN T-cell precursors (Leid et al., 2004).

Specifically, steep onset of Bcl11b expression is observed in the early DN2

stage, just preceding commitment of hematopoietic precursors to the T

lymphocyte lineage (Li et al., 2010a). The Bcl11b expression persists in thymus

throughout all stages of mouse development and in mature T lymphocytes (Leid

et al., 2004; Wakabayashi et al., 2003). Germline deletion of Bcl11b results in a

blockade of thymocyte development at the DN stage, enhanced susceptibility to

apoptosis, and a complete absence of αȕ T cells without impairment of the B

and Ȗβ T-cell lineages (Wakabayashi et al., 2003). The role of BCL11B in later

stages of T-cell development was examined by conditionally deleting Bcl11b in

DP thymocytes using CD4-cre transgenic mice. This approach revealed that

BCL11B also plays a critical role in a positive selection of both CD4+ and CD8+

lineages (Albu et al., 2007). Therefore, BCL11B serves as a key regulator of both

survival and differentiation during thymocyte development. Moreover, BCL11B is

necessary for T-cell lineage commitment in mice, as it is specifically required to

repress natural killer and stem cell-associated genes and induce T-cell-specific

gene expression (Li et al., 2010a; Li et al., 2010b).

Conditional deletion of Bcl11b at the DP stage of T-cell development or in

regulatory T cells causes inflammatory bowel disease (Vanvalkenburgh et al.,

2011). This condition is a chronic disorder that includes Crohn’s disease and

ulcerative colitis, both of which are characterized by infiltration of highly reactive

8

CD4+ T cells into the gut. Bcl11b-deficient regulatory T cells exhibit reduced

protein levels of a lineage specification factor FOXP3 and an anti-inflammatory

cytokine IL-10. At the same time, genes encoding pro-inflammatory cytokines are

up-regulated in the mutant CD4+ T cells that infiltrate the colon (Vanvalkenburgh

et al., 2011).

BCL11B was shown to function as a tumor suppressor gene in

lymphomagenesis. The Bcl11b locus was once named Rit1 (radiation-induced

tumor suppressor gene 1), because it was characterized by homozygous

deletions and point mutations in Ȗ-ray-induced mouse thymic lymphomas

(Wakabayashi et al., 2003). The paradigm for the role of tumor suppressors in

cancer is that they are trans-acting and recessive in nature (Cavenee et al.,

1983). Although Bcl11b+/- heterozygous mice rarely develop thymic lymphomas

spontaneously, Bcl11b+/-; p53+/- double heterozygotes are characterized by

significantly increased lymphomagenesis. This suggests cooperativity between

BCL11B and p53 in cancer development (Kamimura et al., 2007). BCL11B has

also been implicated in the pathogenesis of 9-16% of human T-cell acute

lymphoblastic leukemia (T-ALL) (De Keersmaecker et al., 2010; Gutierrez et al.,

2011). BCL11B is involved in recurrent cryptic t(5;14)(q35;q32) translocations

with the TLX3 locus, in which BCL11B gene regulatory elements drive aberrant

over-expression of the TLX3 oncogene (Bernard et al., 2001; Van Vlierberghe et

al., 2008). Moreover, BCL11B acts as a haploinsufficient tumor suppressor that

collaborates with all major T-ALL oncogenic lesions in human thymocyte

transformation (De Keersmaecker et al., 2010; Gutierrez et al., 2011).

BCL11B in Neural Development

High levels of Bcl11b expression are detected in the central nervous system

during the course of fetal development, predominantly through the hippocampal

subregions, olfactory bulb, limbic system, basal ganglia, frontal cortex of the

9

developing brain, and in dorsal cells of the spinal cord. The brain expression

domains of Bcl11b are also maintained into adulthood (Leid et al., 2004).

The mammalian cerebral cortex is organized into six layers. BCL11B is highly

expressed in corticospinal motor neurons that are located primarily in cortical

layer V and extend extremely long axons to precise locations within the spinal

cord. Developmental analysis of Bcl11b-/- mice showed defects in the

organization and fasciculation of subcerebral fiber tracts. Particularly, loss of

Bcl11b results in failure of the corticospinal motor neurons to extend projections

to the spinal cord, with striking pathfinding errors along the corticospinal tract

(Arlotta et al., 2005). Moreover, ectopic expression of Bcl11b in layer II/III cells

causes their axons to project subcortically. Therefore, BCL11B is both required

and sufficient to form subcortical projections (Chen et al., 2008).

The striatum is the largest and major receptive component of the basal ganglia

that control motor and cognitive functions. Within the striatum BCL11B is

uniquely expressed by medium-sized spiny neurons, specifically labeling this

neuronal population from early post-mitotic stages (Arlotta et al., 2008).

GABAergic, medium-spiny neurons account for the vast majority (~90-95%) of

striatal neurons and are critically involved in motor control (Gerfen, 1992).

Degeneration of this neuronal population is a critical component of Huntington’s

and Parkinson’s diseases (Albin et al., 1989). Loss of BCL11B results in a failure

of differentiation of medium-spiny neurons and distinct changes in the expression

of multiple genes. As a result, Bcl11b-/- medium-spiny neurons fail to aggregate

into patches, which results in invasion of heterotopic cellular aggregates (Arlotta

et al., 2008). Abundant striatal expression of BCL11B is also observed in

adulthood, and BCL11B is responsible for transcriptional control of important

genes in the adult striatum (Desplats et al., 2006). This has important

implications in Huntington's disease as sequestration of BCL11B by huntingtin

and down-regulation of Bcl11b expression results in dysregulation of striatal gene

expression in this disease (Desplats et al., 2008). BCL11B regulates the

10

expression of genes in the brain-derived neurotrophic factor signaling pathway,

alterations in which are implicated in several neurodegenerative diseases,

including Huntington’s, Alzheimer’s, and Parkinson’s disease (Tang et al., 2011).

The hippocampus plays an important role in memory and learning as well as in

emotional behavior. The dentate gyrus, the primary gateway for input information

into the hippocampus, is one of only two brain regions with continuous

neurogenesis in adult mammals. BCL11B is expressed in post-mitotic granule

neurons and is involved in the regulation of both progenitor proliferation as well

as granule cell differentiation. BCL11B directly suppresses the expression of

Desmoplakin, a gene that encodes an obligate component of desmosomes; and

re-expression of Desmoplakin in Bcl11b-/- mice rescues impaired neurogenesis

(Simon et al., 2012).

The vomeronasal organ is an auxiliary olfactory sense organ that functions by

detecting pheromones to mediate social and reproductive behavior and is found

in most terrestrial vertebrates (Keverne, 1999). Vomeronasal sensory neurons

are classified into two major types: vomeronasal 1 receptor/Gαi2- and

vomeronasal 2 receptor/Gαo-positive cells (Jia and Halpern, 1996). Bcl11b is

highly expressed in the developing vomeronasal system in mice and is required

for its proper development. Bcl11b-/- mice display various olfactory phenotypes,

such as disorganization of layer formation of the accessory olfactory bulb;

impaired differentiation and axonal projections of vomeronasal sensory neurons;

and impaired balance of the two neuronal types (Enomoto et al., 2011).

The hair cells are sensory neurons acting as receptors for auditory sensation in

the cochlea of the inner ear. Degeneration of these cells is a major cause of

deafness and age-related hearing loss. Outer hair cells are the mechanical

amplifiers for sound sensitivity and frequency selectivity, whereas inner hair cells

serve as passive detectors of the amplified vibratory signal (Fettiplace and

Hackney, 2006). BCL11B is expressed in the outer hair cells, and heterozygous

11

deletion of Bcl11b leads to degeneration of these cells and progressive age-

related hearing loss, which is evident at 3 months of age. Therefore, BCL11B

activity is required for maintenance of the outer hair cells and normal hearing

(Okumura et al., 2011).

BCL11B in Development of Ectodermal Organs

The ectoderm is the outermost of the three primitive germ layers of an animal

embryo. In vertebrates, the ectoderm gives rise to the epidermis, hair, teeth,

olfactory epithelium, and many exocrine glands. Bcl11b is expressed in most of

these tissues including the developing skin, hair follicles, teeth, mammary glands,

and olfactory epithelium (Golonzhka et al., 2007; Golonzhka et al., 2009b; Leid et

al., 2004).

BCL11B expression in the ectoderm is first detected at E10.5, and becomes

increasingly restricted to the basal cells of the developing epidermis at later

stages of fetal development and in adult skin. BCL11B expression is also

detected is suprabasal layer of developing epidermis, dermal cells, as well as

developing and adult hair follicles (Golonzhka et al., 2007). BCL11B plays critical

roles in epidermal proliferation and terminal differentiation, as well as barrier

formation during mouse embryonic development in both cell autonomous and

non-cell autonomous manners (Golonzhka et al., 2009a). BCL11B functions

through interaction with the promoter regions of several genes involved in lipid

metabolism and epidermal development, such as epidermal growth factor

receptor 1 and Notch1 (Wang et al., 2013; Zhang et al., 2012a). Moreover,

BCL11B modulates cell migration, proliferation and differentiation, as well as

maintains expression of hair follicle stem cell markers during cutaneous wound

healing (Liang et al., 2012). Selective deletion of Bcl11b in epidermal

keratinocytes (Bcl11bep-/- mice) reveal cell-autonomous role of BCL11B in barrier

maintenance and epidermal homeostasis in adult mouse skin. In addition,

12

BCL11B suppresses inflammatory responses in skin by repressing expression of

T-helper 2-type cytokines and thymic stromal lymphopoietin (Wang et al., 2012).

BCL11B is highly expressed in the ectodermal components of the developing

tooth, including inner and outer enamel epithelia, stellate reticulum, stratum

intermedium, and the ameloblasts. Bcl11b-/- molars and incisor are poorly

developed and exhibit hypoplastic stellate reticulum. Ameloblasts do not

differentiate properly on the labial side, and ectopic, ameloblast-like cells form on

the lingual side of the Bcl11b-null incisor. Therefore BCL11B is required for

proper tooth development and differentiation of ameloblast lineage (Golonzhka et

al., 2009b).

In the studies described below, I further elucidate the role of BCL11B in mouse

incisor development and describe molecular mechanisms that underlie the dental

phenotype of Bcl11b-/- mice. In addition, I describe a novel role of BCL11B in the

craniofacial development, and show that germline and neural crest-specific

ablation of Bcl11b leads to craniofacial synostoses in mice.

13

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Chapter 2

BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor

Kateryna Kyrylkova, Sergiy Kyryachenko, Brian Biehs, Ophir Klein, Chrissa Kioussi, Mark Leid

PLoS One Published May 22, 2012 PLoS One 7(5):e37670 doi: 10.1371/journal.pone.0037670

http://www.ncbi.nlm.nih.gov/pubmed/22629441

21

Abstract

Mouse incisors grow continuously throughout life with enamel deposition

uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition

is due to the presence of enamel-secreting ameloblasts exclusively within the

labial epithelium of the incisor. We have previously shown that mice lacking the

transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted

ameloblast formation in the developing incisor. We now report that BCL11B is a

key factor controlling epithelial proliferation and overall developmental

asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the

labial epithelium and development of the epithelial stem cell niche, which gives

rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and

development of stem cells and ameloblasts on the inner, or lingual, side of the

incisor. This bidirectional action of BCL11B in the incisor epithelia appears

responsible for the asymmetry of ameloblast localization in developing incisor.

Underlying these spatio-specific functions of BCL11B in incisor development is

the regulation of a large gene network comprised of genes encoding several

members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic

hedgehog. Our data integrate BCL11B into these pathways during incisor

development and reveal the molecular mechanisms that underlie phenotypes of

both Bcl11b−/− and Sprouty mutant mice.

Introduction

Tooth initiation in the mouse is characterized by a thickening of the oral

epithelium at embryonic day (E) 11.5. The proliferating epithelium invaginates

into the underlying neural crest-derived mesenchyme and forms a bud at E12.5–

E13.5 (bud stage). The epithelium expands and folds around the condensed

mesenchyme to form a cap-like structure at E14.5 (cap stage). The cap stage is

characterized by formation of the enamel knot, a critical signaling center, and

lateral protrusions of the epithelium, known as cervical loops (CLs). CLs extend

22

during bell stage (E16.5–E18.5), at which point cytodifferentiation begins (Kerley,

1975; Lumsden, 1988; Peters and Balling, 1999; Tucker and Sharpe, 2004).

Continuous growth of the rodent incisor requires the presence of epithelial and

mesenchymal stem cells that provide a continuous supply of enamel-producing

ameloblasts and dentin-producing odontoblasts, respectively. Epithelial stem

cells (EpSCs) are slow-cycling cells located in the CLs (Harada et al., 1999; Klein

et al., 2008; Seidel et al., 2010). The labial CL consists of a core stellate

reticulum (SR) and stratum intermedium cells surrounded by basal epithelial

cells, known as the inner and outer enamel epithelium (IEE and OEE,

respectively) (Tummers and Thesleff, 2009). EpSCs reside in the labial CL and

give rise to transit amplifying cells that migrate anteriorly along the IEE while

sequentially differentiating to mitotic pre-ameloblasts, post-mitotic secretory

ameloblasts, and mature ameloblasts (Ryan et al., 1999). The lingual CL

contains a smaller EpSC niche, which does not give rise to ameloblasts, resulting

in a complete lack of enamel deposition on the lingual aspect of the rodent

incisor. Thus, enamel, the hardest substance in the body, is secreted uniquely on

the labial aspect of the incisor. This leads, to preferential abrasion of the lingual

incisor surface during feeding, counteracting the continuous growth of the mouse

incisor to produce an incisor of fixed length (Tummers and Thesleff, 2003).

Tooth development is regulated by sequential and reciprocal signaling between

the epithelium and mesenchyme and is accompanied by patterning and

differentiation of specialized cell types at distinct anatomical locations. A complex

network of fibroblast (FGFs) and transforming (TGFβ) growth factors regulates

proliferation and differentiation of EpSCs during development. The antagonists of

these pathways, Sprouty (Spry) proteins and Follistatin (FST), respectively, also

regulate EpSC niche development, and growth and asymmetry of the mouse

incisor (Klein et al., 2008; Tummers and Thesleff, 2009).

23

CTIP2/BCL11B (BCL11B hereafter) is a transcription factor that plays essential

roles in the development of the immune (Li et al., 2010; Wakabayashi et al.,

2003), central nervous (Arlotta et al., 2005; Arlotta et al., 2008), and cutaneous

(Golonzhka et al., 2009a) systems and is required for perinatal survival

(Wakabayashi et al., 2003). Bcl11b−/− incisors and molars are poorly developed,

and exhibit a hypoplastic SR. Ameloblasts do not differentiate properly on the

labial side, and ectopic ameloblast-like cells form on the lingual side of the

Bcl11b-null incisor (Golonzhka et al., 2009b).

Our analyses of Bcl11b−/− mice revealed that BCL11B plays important roles

throughout incisor development. Mice lacking Bcl11b exhibit epithelial

proliferation defects early in development, which ultimately impact incisor size

and shape. BCL11B also controls formation of both labial and lingual epithelial

stem cell niches and differentiation of ameloblasts. However, BCL11B does so in

a bidirectional manner: promoting development and differentiation of the

epithelium on the labial side while suppressing that on the lingual side, which

strongly enforces asymmetric ameloblast development in the mouse mandibular

incisor.

Materials and Methods

Mouse Lines

/− cl11b− B and Bcl11bL2/L2 mice have been described (Golonzhka et al., 2009a).

Lines carrying mutant alleles of Spry2 (Shim et al., 2005), Spry4 (Klein et al.,

2006), Fgf3 (Alvarez et al., 2003), and Fgf10 (Sekine et al., 1999), as well as

K14-cre (Dassule et al., 2000) and Wnt1-cre (Danielian et al., 1998) transgenes,

were maintained as reported. Animal experiments were approved by the Oregon

State University Institutional Animal Care and Use Committee, protocol 4279.

24

Histological Analysis, RNA in situ Hybridization, and

Immunohistochemistry

Embryonic heads were fixed in 4% paraformaldehyde, cryopreserved in 30%

sucrose, and frozen in O.C.T. Hematoxylin and eosin (H&E) staining and RNA in

situ hybridization (ISH) with digoxigenin-labeled probes were performed

according to standard protocols on 16 µm-thick sagittal sections.

Immunohistochemistry using anti-BCL11B (Abcam, 1:300) was performed as

described (Golonzhka et al., 2007).

Cell Proliferation Assay

Pregnant mice (E11.5–E16.5) were injected intraperitoneally with 100 µl of 5

mg/ml BrdU solution per 100 g of body weight and sacrificed after 2 h.

Cryopreserved heads were serially sectioned (10 µm), and an anti-BrdU antibody

(Accurate Chemical, 1:100) was used to detect BrdU incorporation. The BrdU

index was calculated as the mean relative amount of BrdU-positive cells as a

fraction of total, DAPI-positive cells. An unpaired, two-tailed Student’s t-test was

used to determine statistical significance. At least six sections from a minimum of

three animals per genotype and age were analyzed.

Apoptosis Assay

Apoptosis in sagittal sections (16 µm) was determined with the DeadEnd

Colorimetric TUNEL System (Promega) using Cy3-conjugated streptavidin (SA

Cy3; Sigma 1:250).

Results

BCL11B is Expressed at all Stages of Incisor Development

BCL11B is expressed in the ectoderm of the first branchial arch at E9.5 and

E10.5 and in the molar at all stages of development (Golonzhka et al., 2009b).

25

To determine the function of BCL11B in the developing incisor, we analyzed

BCL11B expression on sagittal sections of the mandibular incisor from E11.5 to

birth. At initiation (E11.5) and early bud (E12.5) stages, BCL11B was expressed

in the thickened epithelium; lower levels of BCL11B were detected in the

underlying mesenchyme (Figs. 2.1A and B). High levels of BCL11B persisted in

the dental epithelium at cap stage (E14.5), whereas mesenchymal cells

surrounding CLs and the follicle continued to express lower levels (Fig. 2.1C). At

early (E16.5) and late (E18.5) bell stages, BCL11B was detected in the lingual

epithelium and in the labial OEE, and at lower levels in the papillary

mesenchyme surrounding both CLs, dental follicle, SR, and ameloblasts at all

stages of differentiation (Figs. 2.1D, E and S2.1). BCL11B was expressed in the

tissue surrounding the tip of the incisor and the vestibular lamina, an invagination

of the oral epithelium that gives rise to the oral vestibule (Figs. 2.1C and D).

Reduced Epithelial Proliferation between Initiation and Bud Stage in

Bcl11b−/− Incisors

The first morphological sign of tooth development is the thickening of the oral

epithelium at E11.5. At this stage, wild-type and Bcl11b−/− incisors were

morphologically indistinguishable (Figs. 2.2A, B, E, F, and I; wild-type BrdU index

= 33.2±5.4%; Bcl11b−/− BrdU index = 33.2±4.0%). By E12.5 the mutant

epithelium appeared approximately one-half the thickness of the wild-type, and

cells at the leading edge of the mutant epithelium were less elongated and poorly

polarized (Fig. 2.2C, D). A 2.7-fold decrease in epithelial proliferation of the

Bcl11b−/− incisor was detected at E12.5 (Figs. 2.2G, H, and J; wild-type BrdU

index = 61.9±3.8%; Bcl11b−/− BrdU index = 23.2±1.4%).

Several signaling molecules and transcription factors orchestrate invagination of

the epithelium between initiation and early bud stages. For example, BMP4, a

critical signaling molecule that regulates tooth initiation and morphogenesis

(Zhang et al., 2005), is expressed in the dental epithelium and underlying

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037670#pone-0037670-g001







26

mesenchyme during the initiation of tooth development at E11.5 (Fig. 2.2K).

Bmp4expression largely shifts to the dental mesenchyme by early bud stage in

wild-type mice (Fig. 2.2M) (Peters and Balling, 1999; Zhang et al., 2000).

Alterations in Bmp4 expression were not detected in Bcl11b−/− incisors at E11.5

(Fig. 2.2L). However, the Bcl11b−/− epithelium failed to down-regulate expression

of Bmp4 at E12.5 (Fig. 2.2N). The expression patterns of other critical signaling

molecules and transcription factors, including Activin, Shh, Pax9, and Msx1, were

not altered in Bcl11b−/− incisors at the bud stage (Fig. S2.2).

− Altered Development of Bcl11b /− Incisors at Cap Stage

The wild-type, mandibular incisor is characterized by a cap-like shape of the

dental epithelium at E14.5, with an enamel knot in the center and protruding CLs

(Fig. 2.3A). The enamel knot is a transitory signaling center that is characterized

by minimal proliferation and clearly defined apoptosis (Vaahtokari et al., 1996a;

Vaahtokari et al., 1996b). In contrast, the CLs are highly proliferative with a low

−/− apoptotic index (Wang et al., 2007). The Bcl11b incisor exhibited a delay in

epithelial invagination and protrusion of both CLs at E14.5 (Fig. 2.3B). BrdU

labeling studies revealed that the incisor epithelium of Bcl11b−/− mice was

hypoproliferative compared to that of wild-type mice (27.6±4.1% and 51.8±2.7%

BrdU-positive cells, respectively), whereas mesenchymal proliferation appeared

unchanged from controls (Fig. 2.3D and E).

Proliferation of the dental epithelium at cap stage is controlled in part by FGF10,

which is derived from mesenchymal cells of the dental papilla (Kettunen et al.,

2000). The Bcl11b−/− papillary mesenchyme was essentially devoid of Fgf10

transcripts at early E14 and ectopic Fgf10 expression was noted between the

dental epithelium and vestibular lamina, the latter of which exhibited impaired

invagination (Fig. 2.3F, G). The delay of initiation of mesenchymal Fgf10

expression may contribute to decreased dental epithelial proliferation, delayed





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27

invagination of the dental epithelium, and subsequently decreased size of the

mutant incisor.

The expression patterns of Shh, Gli1, Fgf3, Fgf9, Spry2, Spry4, Bmp4, activin,

Fst, and Tbx1, were unaltered in Bcl11b−/− incisors at cap stage (Fig. S2.3).

Apoptotic cells were predominantly localized in the enamel knot of wild-type

incisors at E14.5 (Fig. S2.4A). However, very few apoptotic cells were detected

in the Bcl11b−/− enamel knot (Fig. S2.4B), consistent with delayed incisor

development in Bcl11b−/− mice.

Reduced Size and Disruption of Labial-lingual Asymmetry at Bell Stage in

Bcl11b−/− Incisors

Wild-type incisors at E16.5 are characterized by a large labial CL, which contains

stem cells that give rise to ameloblasts (Figs. 2.4A and B). In contrast, the lingual

CL of wild-type incisors is relatively smaller, consistent with reduced

developmental potential on the lingual side of the incisor (Fig. 2.4C). Bcl11b−/−

incisors were reduced in size by approximately half at this stage (compare Figs.

2.4A and E) and were characterized by a hypocellular labial CL (compare Figs.

2.4B and F), an enlarged lingual CL (compare Figs. 2.4C and G), and elongated

cells resembling ameloblasts along the length of the lingual epithelium (compare

Figs. 2.4D and H).

The posterior basal epithelium of the labial CL of Bcl11b−/− mice was

hypoproliferative relative to that of wild-type mice at E16.5 (23.2±4.9% and

42.0±5.0% BrdU-positive cells, respectively; Figs. 2.4I-K). However, neither the

apoptotic index (Figs. S2.4C and D) nor proliferation in the lingual CL (Fig. 2.4K)

of mutant incisors was significantly different from wild-type.

By E18.5, wild-type incisors developed a large labial (region I, Figs. 2.4L and P)

and a small lingual (Figs. 2.4L and M) CL. Ameloblast differentiation occurs

















Bcl11 −/− b incisors were reduced in size at E18.5 (compare Figs. 2.4L and N).

However, the mutant lingual CL was enlarged (compare Figs. 2.4M and O), and

the labial CL was markedly hypoplastic (compare Figs. 2.4P and U) to the point

of resembling the lingual CL of wild-type mice in both size and morphology

(compare Figs. 2.4M and U). Mutant ameloblasts were smaller and disorganized

at all stages of differentiation along the labial epithelium (compare Figs. 2.4Q-S

and V-X), and an abnormal layer of polarized cells resembling ameloblasts was

28

sequentially in the labial IEE with mitotic pre-ameloblasts (Figs. 2.4L and Q),

post-mitotic secretory ameloblasts (Figs. 2.4L and R), and mature ameloblasts

(Figs. 2.4L and S) in regions II, III, and IV, respectively (Ryan et al., 1999).

Ameloblasts are not present on the lingual side of the wild-type incisor, but rather

a thin layer of non-polarized epithelial cells is found on this aspect of the

developing tooth (Figs. 2.4L and T).

observed in the anterior region of the lingual epithelium (compare Figs. 2.4T and

Y). Finally, Bcl11b−/− incisors were approximately one-half the length of wild-type

incisors at birth and correspondingly narrower across the entire tooth (Fig. S2.5).

These results demonstrate that BCL11B plays an important role in development

of the labial CL and differentiation of ameloblasts, while simultaneously

suppressing these processes on the lingual side of the incisor.

Delay in Ameloblast Development and Ectopic Formation of Lingual

Ameloblast-like Cells in Bcl11b−/− Incisors

To determine if labial ameloblasts and lingual ameloblast-like cells underwent

differentiation in Bcl11b−/− incisors, we examined expression of sonic hedgehog

(Shh) and amelogenin (Amelx), markers of pre-ameloblasts (Bitgood and

McMahon, 1995; Klein et al., 2008) and mature ameloblasts (Zeichner-David et

al., 1995), respectively. Shh expression was observed in a gradient along the

length of the labial IEE of wild-type incisors at E16.5 and E18.5, with the most

intense staining in the posterior region (Fig. 2.5A, C). Shh expression was greatly















29

reduced in the labial epithelium of Bcl11b−/− mice, and ectopic Shh transcripts

were detected in the lingual epithelium at E16.5 and E18.5 (Figs. 2.5B and D).

The expression pattern of Gli1, a mediator of SHH signaling (Ruiz i Altaba,

1999), reflected changes in Shh expression in the mutant incisor (Fig. S2.6).

Amelx expression was greatly reduced in the labial epithelium of Bcl11b−/− mice

at E16.5 (compare Figs. 2.5E and F) but recovered to a level similar to that of

wild-type mice by E18.5 (Fig. 2.5G). Ectopic Amelx expression was observed in

the anterior lingual IEE of Bcl11b−/− mice at E18.5 (compare Figs. 2.5G and H),

consistent with the presence of terminally differentiated ameloblasts in the lingual

epithelium.

These results demonstrate that BCL11B plays a key role in the establishment

and/or enforcement of developmental incisor asymmetry and cellular

differentiation within the ameloblast lineage.

− Alteration of the FGF Signaling at Bell Stage in Bcl11b /− Incisors

Asymmetric development of the CLs is controlled by several signaling pathways.

FGFs and their intracellular antagonists, the Sprouty proteins, are crucial for

proper development of the labial and lingual CLs (Klein et al., 2008). FGF3 and

FGF10 are key mesenchymal instructive signals that cooperatively stimulate

proliferation of the incisor epithelium at bell stage (Harada et al., 1999; Harada et

al., 2002; Yokohama-Tamaki et al., 2006). Fgf3 is expressed exclusively within

the posterior labial mesenchyme in wild-type incisors (Figs. 2.6A and C),

whereas Fgf10 transcripts are more widely distributed around the labial CL and

to a lesser extent in the lingual mesenchyme (Figs. 2.6E and G).

The Fgf3 expression domain, which is located in the mesenchyme just anterior to

the labial CL in wild-type mice, was absent in Bcl11b−/− mutants at E16.5 and

E18.5. However, Fgf3 was ectopically expressed in the mesenchyme adjacent to

the lingual CL in Bcl11b−/− mice (Figs. 2.6B and D). The expression pattern of









30

Fgf10 was altered in Bcl11b−/− incisors in a manner that was qualitatively similar

to that of Fgf3 (Figs. 2.6F and H).

Epithelial FGF9 forms a positive-feedback signaling loop with mesenchymal

FGF3 and FGF10 on the labial side of the wild-type incisor (Klein et al., 2008).

Fgf9 RNA was detected anterior to the labial CL of the wild-type incisor (Figs.

2.6I and K). This Fgf9-positive domain was reduced in Bcl11b−/−incisors, and

ectopic expression of Fgf9 was detected in the lingual epithelium at E16.5 and

E18.5 (Figs. 2.6J and L).

Sprouty proteins are responsible, in part, for inhibition of ameloblast

differentiation in the lingual epithelium (Klein et al., 2006). Spry4 RNA was

detected in the mesenchyme adjacent to the labial CL, and at lower levels in the

posterior lingual and labial epithelium in wild-type mice at E16.5 and E18.5 (Figs.

2.6M and O). Spry2 expression was detected predominantly in the posterior

lingual and labial epithelium of the wild-type incisor (Figs. 2.6Q and S).

Spry4 expression was up-regulated in the lingual basal epithelium and underlying

mesenchyme of Bcl11b−/− incisors at E16.5 and E18.5, and down-regulated on

the labial side of the developing Bcl11b−/− incisor at both developmental stages

(Figs. 2.6N and P). Spry2 expression was up-regulated in the lingual CL and

slightly down-regulated in the labial epithelium of Bcl11b−/−mice at E16.5 and

E18.5 (Figs. 2.6R and T).

Mesenchymal FGF10 stimulates expression of Lunatic Fringe (Lfrn), which

encodes a secretory molecule that modulates the Notch pathway (Harada et al.,

1999). To determine if the Notch pathway was altered inBcl11b−/− incisors at bell

stage, we examined expression patterns of Lfrn and Notch1. Lfrn RNA was

detected predominantly along the length of IEE and in the posterior OEE of wild-

type incisors at E16.5 and E18.5 (Figs. S2.7A and C). Lfrn expression was down-

regulated at the posterior end of the labial CL of Bcl11b−/− incisors at both E16.5

and E18.5 (Figs. S2.7B and D). Ectopic Lfrn expression was detected in the












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posterior part of the mutant lingual epithelium at E18.5 (Fig. S2.7D). Loss of

BCL11B did not affect the level of expression or localization of Notch1

transcripts. However, Notch1 expression reflected the morphological expansion

and contraction of lingual and labial SR, respectively, in Bcl11b−/− incisors (Figs.

S2.7E-H).

These findings highlight dysregulation of the FGF signaling pathways as being

central to the incisor phenotype of Bcl11b−/− mice. As asymmetric expression of

Fgf3 and Fgf10 contribute to asymmetric development of labial and lingual EpSC

niches (Wang et al., 2007). Thus, the complete reversal of asymmetric Fgf3 and

Fgf10 expression, together with that of Fgf9, likely underlies the enhanced and

repressed development of the lingual and labial CLs, respectively, in Bcl11b−/−

mice.

Dis ruption of TGFβ Signaling at Bell Stage in Bcl11b−/− Mice

The TGFβ family members, BMP4 and activin βA, and the antagonist FST play

key roles in the generation and maintenance of asymmetric ameloblast

localization during incisor development. For example, FST inhibits ameloblast

differentiation on the lingual side of the incisor, whereas BMP4 promotes it on the

labial side. In contrast, activin enhances development of the labial CL, whereas

BMP4 limits CL growth (Wang et al., 2007; Wang et al., 2004; Zhang et al.,

2005).

Bmp4 expression was detected predominantly in the labial mesenchyme, anterior

to the labial CL, in wild-type mice at E16.5. Lower levels of Bmp4 transcripts

were present in the mesenchyme underlying the lingual epithelium and in an

anterior region of the ameloblast layer (Fig. 2.7A). The boundaries of

mesenchymal Bmp4 expression were disrupted in Bcl11b−/−incisors at E16.5,

with ectopic expression noted in the mesenchyme posterior to the lingual CL.

Expression of Bmp4 in the ameloblast layer appeared reduced in mutants at this

stage (Fig. 2.7B). At E18.5, Bmp4 transcripts were detected predominantly in the






32

labial epithelium, in a wide region of labial mesenchyme, and at lower levels on

the lingual side of the wild-type incisor (Fig. 2.7C). Bmp4 expression increased

uniformly in all of these domains in Bcl11b−/− mutants at E18.5 (Fig. 2.7D).

Activin expression was restricted to the labial mesenchyme directly underlying

the posterior epithelium, within the tip of the labial CL, and in the posterior part of

the dental follicle in wild-type mice at E16.5 and E18.5 (Figs. 2.7E and G). Activin

expression was lost within the labial mesenchyme and epithelium in Bcl11b−/−

mice at both developmental stages. However, ectopic mesenchymal expression

of activin was observed around the lingual CL and follicular expression of activin

appeared to be delocalized in the Bcl11b−/− incisors at E16.5 and E18.5

(asterisks in Figs. 2.7F and H).

Fst transcripts were observed in the OEE on the labial and lingual sides at E16.5

(Fig. 2.7I; see also (Wang et al., 2004)). Fst expression in the OEE persisted at

E18.5, and Fst transcripts were also detected in highly-defined domains at the

anterior epithelial tip of the incisor on both labial and lingual sides (Fig. 2.7K; data

not shown). In contrast, Fst transcripts were diffusely distributed throughout the

labial and lingual epithelium of Bcl11b−/− incisors, particularly at the anterior

(incisal) tip of the epithelium, and in the papillary mesenchyme at E16.5 (Fig.

2.7J). Fst expression within the posterior region of the wild-type incisor at E18.5

was indistinguishable from that of Bcl11b−/−mice (data not shown). However, we

noted a dramatic expansion of the Fst expression domain within the anterior

labial epithelium at E18.5. Additionally, Bcl11b−/− incisors failed to extinguish Fst

expression along the length of the labial OEE at E18.5 (Fig. 2.7L).

Cell Autonomous Effects of BCL11B in Lingual Epithelium

BCL11B is expressed in both ectodermal-derived epithelium and neural crest-

derived mesenchyme (Fig. 2.1). We created lines conditionally null for Bcl11b

expression in both germinal layers to determine the expression domain

responsible for BCL11B-mediated suppression of ameloblast differentiation in the











33

lingual epithelium. Mice harboring an epithelial-specific deletion of Bcl11b

(Bcl11bep−/−), which were created by crossing floxed Bcl11bL2/L2 mice with the

K14-cre deleter strain (Dassule et al., 2000), clearly lacked BCL11B in the entire

dental epithelium (Figs. 2.8A and B). However, specific BCL11B expression

persisted in the dental mesenchyme and other non-epithelium-derived tissues.

Bcl11bep−/− mice expressed the pre-ameloblast marker Shh with an ectopic

gradient along the length of the lingual epithelium at E16.5 (Figs. 2.8C and D),

and this persisted at a lower level at E18.5 (Fig. S2.8B) However, Bcl11bep−/−

mice did not express Amelxin the lingual epithelium at either E16.5 (Fig. 2.8F) or

E18.5 (Fig. S2.8E). Considered together, these results suggest that Bcl11bep−/−

mice initiate but do not complete ameloblast differentiation within the lingual

dental epithelium.

Next, we examined the expression of several genes encoding signaling

molecules to determine the effect of epithelium-specific inactivation of Bcl11b on

generation of labial-lingual asymmetry at E16.5. A low level of ectopic expression

of Fgf3, Fgf9, and activin was detected on the lingual side of the Bcl11bep−/−

incisor, and this was qualitatively similar to Bcl11b−/− incisors (compare Figs.

2.8G-L, 2.6B and F, and 2.7F). However, we did not observe alterations in the

expression patterns of these signaling molecules on the labial side of the

Bcl11bep−/− incisor (Figs. 2.8G-L), as described previously for Bcl11b−/− mice (see

Figs. 2.6 and 2.7).

The size and shape of the Bcl11bep−/− incisors were similar to control incisors

(see Fig. 2.8), and the slight variations in lingual gene expression in Bcl11bep−/−

incisors did not result in altered amelogenesis as determined by X-ray micro-CT

radiography performed on P21 mandibles (Fig. S2.9).

Excision of the Bcl11b locus in neural crest-derived mesenchyme using the

Wnt1-cre deleter line (Bcl11bmes−/−; see Figs. S2.10A and B) did not result in















34

altered morphology or disrupted gene expression patterns (Shh, Amelx, Fgf3,

and activin; Figs. S2.10C-J; see also S2.8C and F).

These data indicate that epithelial, but not mesenchymal Bcl11b expression is

required for suppression of ectopic pre-ameloblast formation in the lingual

epithelium. However, loss of Bcl11b in the epithelium is not sufficient for the

lingual pre-ameloblasts to persist or to undergo further differentiation into mature,

Amelx-positive ameloblasts.

FGF Signaling Negatively Regulates BCL11B Expression in the Lingual IEE

and SR

−/− The ectopic development of lingual pre-ameloblasts expressing Shh in Bcl11b

mice is similar to that reported in Spry4−/−; Spry2+/− mice. Loss of Sprouty gene

expression results in abnormal FGF gene expression and establishment of a

FGF positive-feedback signaling loop on the lingual side of the incisor. In

addition, Spry4−/−; Spry2+/− mice were characterized by up-regulated expression

of Etv4 and Etv5 (previously known as Pea3 and Erm), which are considered to

be transcriptional targets of FGF signaling, and indicative of activation of the FGF

signaling pathway(s) in mutant incisors (Klein et al., 2008; O'Hagan and Hassell,

1998; Roehl and Nusslein-Volhard, 2001). We assessed expression of BCL11B

in incisors from Spry4−/−; Spry2+/− embryos in order to determine if the FGF

signaling pathway(s) regulates BCL11B expression.

BCL11B was highly expressed in the entirety of the wild-type lingual epithelium at

E16.5, including the CL, anterior OEE, and IEE (Fig. 2.9A; see also 2.1D). In

contrast, BCL11B protein levels were dramatically decreased in the Spry4−/−;

Spry2+/− incisor, particularly within the lingual IEE and SR, while BCL11B levels

in the lingual OEE and labial epithelium were largely unaffected (Fig. 2.9B).

Expression of Tbx1, which is also important for incisor developmental

asymmetry, was increased in the lingual epithelium of Spry4−/−; Spry2+/− incisors





35

(Caton et al., 2009). These findings prompted us to examine Tbx1 expression in

Bcl11b−/− mice. Tbx1 was predominantly expressed in the posterior basal

epithelium on the labial side of wild-type incisors at both E16.5 and E18.5, and

diffusely at a much lower level in the lingual epithelium (Figs. S2.11A and C). We

observed striking up-regulation of Tbx1 expression in the lingual IEE of Bcl11b−/−

mice at E16.5 and E18.5 (Figs. S2.11B and D), suggesting that BCL11B directly

or indirectly represses the Tbx1 expression in the lingual epithelium, and that up-

regulation of Tbx1 expression in Spry4−/−; Spry2+/− mice (Caton et al., 2009) may

occur through down-regulation of BCL11B protein levels. These findings place

BCL11B downstream of FGF signaling and upstream of Tbx1 expression in the

lingual epithelium of the developing incisor. Tbx1 expression was severely

decreased in the labial epithelium of Bcl11b−/− mice at E16.5 (Fig. S2.11B).

Labial expression of Tbx1 in the Bcl11b−/− incisor recovered by E18.5 (Fig.

S2.11D), suggesting that another factor(s) may compensate for loss of BCL11B

expression in the control of expression of Tbx1 in the labial epithelium.

These above findings suggest that the FGF signaling pathways regulate BCL11B

expression in the lingual epithelium, and we hypothesized that inactivation of

FGF signaling may lead to up-regulation of BCL11B expression within the labial

IEE. In order to test this hypothesis, we assessed BCL11B expression in Fgf3−/−;

Fgf10+/− incisors; however, BCL11B immunostaining was indistinguishable from

wild-type incisors (Fig. S2.12). It is conceivable that another FGF family

member(s) may compensate for loss of Fgf3 expression and partial loss of Fgf10

expression by enforcing the repression of BCL11B expression within the labial

IEE (Porntaveetus et al., 2011). Indeed, Fgf3−/−; Fgf10+/− and wild-type incisors

are nearly identical in size (Fig. S2.12), suggesting that loss or partial loss of

these two signaling molecules did not compromise proliferation during incisor

development. Finally, it is possible that regulation of BCL11B expression within

the labial epithelium may not involve the FGF signaling pathways, as was clearly

evident on the lingual side (Fig. 2.9B).









36

Discussion

The studies reported here demonstrate that the transcription factor BCL11B

participates in several essential aspects of mouse incisor development. First,

BCL11B controls epithelial proliferation, which ultimately impacts the size and

shape of the incisor. Second, BCL11B plays a key role in the establishment and

maintenance of labial-lingual asymmetry by regulating the expression of several

key signaling molecules and transcription factors. Third, BCL11B is essential for

the proper formation, differentiation, and localization of ameloblasts.

To our knowledge, this is the first report of a transcription factor that integrates

developmental control of both labial and lingual EpSC niches and ameloblasts.

Such regulation appears to be bidirectional: BCL11B stimulates the development

of the labial CL by enhancing the expression of key signaling molecules on the

labial side while limiting development of the lingual CL by repressing the

expression of the same signaling molecules on the lingual aspect. Subsequently,

BCL11B promotes differentiation of the labial, EpSC-derived IEE cells into

mature ameloblasts and blocks ectopic formation and differentiation of the lingual

IEE into cells of the ameloblast lineage.

BCL11B Regulates Proliferation of the Dental Epithelium

BCL11B is initially required for proper transition from initiation to early bud stage

of tooth development. Specifically, BCL11B is necessary for the proper timing of

epithelial proliferation, invagination, and down-regulation of epithelial Bmp4

expression. During tooth initiation, BMP4 is secreted by the epithelium and

induces the mesenchymal expression of genes (Msx1, Msx2, and Bmp4) that

further direct incisor formation (Tucker and Sharpe, 2004). While the significance

of down-regulation ofBmp4 expression in the dental epithelium at bud stage is

unknown, overexpression of Bmp4 in the distal respiratory epithelium results in

decreased epithelial proliferation (Bellusci et al., 1996). Thus, a delay in down

37

regulation of epithelial Bmp4 expression may contribute to reduced proliferation

of the dental epithelium between initiation and bud stages.

The size and shape of the incisor is tightly regulated by the opposing forces of

cellular proliferation and apoptosis, both of which are controlled by signaling

pathways. FGF10 induces a mitogenic response in dental epithelial cells

(Kettunen et al., 2000), and a delay in induction of Fgf10 in Bcl11b−/−incisors may

further contribute to the proliferation defect in Bcl11b−/− dental epithelium.

Therefore, a combination of at least two molecular dysregulations, a delay in

down-regulation of epithelial Bmp4 expression at E12.5 and in the induction of

mesenchymal Fgf10 expression at E14, may contribute to decreased proliferation

of Bcl11b−/− dental epithelium. We further propose that altered epithelial

proliferation in the absence of BCL11B may account for the slowed invagination

of this tissue, resulting in an incisor that is approximately one-half of the size of a

wild-type incisor at birth.

Apoptosis of epithelial cells comprising the enamel knot at cap stage also

regulates the overall shape of the incisor (Vaahtokari et al., 1996a). Thus,

delayed initiation of apoptosis in Bcl11b−/− incisors may also contribute to altered

morphology in the mutant.

BCL11B Controls the Expression of FGF and TGFβ Family Members

Transition from the cap to bell stage of incisor development is accompanied by

establishment of an asymmetric shape. The size and asymmetry of the mouse

incisor is dictated by transcription factor networks, which control the expression

of genes encoding components of various signaling pathways that play

deterministic roles in cellular specification and organogenesis. The FGF and

TGFβ signaling pathways, and their respective antagonists–the Sprouty proteins

and FST–are particularly important in incisor development.

38

FGF3 and FGF10, both of which are expressed predominantly in the labial

mesenchyme, maintain proliferation of EpSCs and, thus, directly contribute to the

asymmetric shape of the incisor (Harada et al., 1999; Harada et al., 2002; Wang

et al., 2007). Furthermore, both FGF3 and FGF10, together with epithelial FGF9,

form a positive-feedback loop on the labial aspect of the tooth. This FGF

feedback loop is inhibited by Sprouty proteins on the lingual side, resulting in the

limited development of the lingual EpSC niche (Klein et al., 2008). In turn, the

expression of the Sprouty genes can be induced by FGF signaling (Hacohen et

al., 1998).

In the absence of BCL11B, a remarkable inversion of the expression patterns of

FGF genes relative to the labial-lingual axis occurred, such that these genes

were expressed predominantly on the lingual side, with no or little expression

was observed on the labial side. Therefore, BCL11B may function as a spatial

switch governing expression of FGF signaling pathway members (Fig. S2.13).

Expression of Sprouty genes was altered in a similar manner in Bcl11b−/−incisors,

possibly in a compensatory or feedback manner. Consistent with this, expression

of Tbx1, which is positively regulated by FGF signaling in the developing incisor

(Caton et al., 2009), was similarly altered. Our findings strongly suggest that

complete loss of expression of FGF family members on the labial side coupled

with ectopic expression of these signaling proteins on the lingual side of the

Bcl11b−/− incisor underlies the abnormal morphology of the Bcl11b−/− tooth.

The FGF and TGFβ signaling pathways are closely interweaved during incisor

development. For example, BMP4 represses Fgf3 expression in the

mesenchyme; however, activin abrogates this repression on the labial side of the

incisor, which allows FGF3 expression within this domain. Low activin expression

in the lingual mesenchyme allows BMP4 to inhibit Fgf3 expression on the lingual

side in an unopposed fashion (Kettunen et al., 2000). In addition, BMP4

promotes ameloblast differentiation within the labial epithelium, possibly by

inducing expression of p21 and ameloblastin. Ameloblast-inducing activity of


39

BMP4 is inhibited on the lingual side by FST, but the relative lack of

Fstexpression in the labial epithelium facilitates terminal differentiation of

ameloblasts (Wang et al., 2004). Alteration of Bmp4 expression in Bcl11b−/−

incisor generally paralleled those observed with FGF family members. Thus,

BCL11B appears to be required for Bmp4 expression on the labial side of the

developing incisor and for suppression of Bmp4 expression in the lingual

mesenchyme.

Expression of activin also exhibited complete reversal in the mutants at the bell

stage. Activin expression was completely lost on the labial side of the mutant

incisor, perhaps allowing BMP4 to inhibit Fgf3 expression within this domain. In

contrast, activin transcripts were detected in the lingual mesenchyme of Bcl11b−/−

incisors, suggesting that this ectopic expression domain allows activin to block

the repressive action of BMP4 on Fgf3 expression in this tissue. Because FGF3

functions in a positive-feedback loop with FGF10 and FGF9 (Klein et al., 2008),

the expression patterns of the latter were also altered. FGF3, and possibly FGF

family members, could then induce the development of the lingual EpSC niche in

Bcl11b−/− mice. Thus, we propose that activin contributes to the establishment of

new borders of expression of FGF family members in the Bcl11b−/− incisor.

BCL11B Controls Asymmetric Development of the Ameloblasts

The asymmetric pattern of expression of FGF and TGFβ signaling molecules is

thought to lead to the asymmetric development of ameloblasts in wild-type

incisors. Therefore, dysregulated expression of these signaling pathways in

Bcl11b−/− incisors likely contributes to the ectopic development of lingual,

ameloblast-like cells and delayed development of the labial ameloblasts. The

expanded lingual EpSC niche in Bcl11b−/− incisors gave rise to ectopic Shh-

expressing pre-ameloblasts, which further differentiated into mature, Amelx

positive ameloblasts. The mutant labial EpSC niche also gave rise to some pre

ameloblasts, which were characterized by down-regulated Shh expression.

40

These pre-ameloblasts failed to differentiate into mature ameloblasts at the early

bell stage. Although the pool of Shh-positive pre-ameloblasts was greatly

reduced in Bcl11b−/− incisors at late bell stage, it was remarkable that

differentiation to Amelx-positive ameloblasts occurred in the labial epithelium by

E18.5. This observation suggests that another transcription factor(s) may

compensate for loss of Bcl11b expression in the ameloblast lineage, allowing

ameloblast development to occur, albeit in a delayed manner.

Ectopic Shh-positive pre-ameloblasts were abundant on the lingual aspect of the

Bcl11bep−/−incisor at early bell stage. Low levels of ectopic lingual expression of

Fgf3, Fgf9, and activin might contribute to such differentiation of IEE. However,

the ectopic, lingual Shh-positive domain was dramatically reduced in size and

was present only in the posterior epithelium by late bell stage, and mature

ameloblast-like cells were not observed on the lingual aspect of the Bcl11bep−/−

incisor, suggesting that other factors and/or mesenchymal BCL11B may be

sufficient to suppress terminal differentiation in the ameloblast lineage within the

lingual epithelium. Deletion of Bcl11b in either the epithelium or mesenchyme did

not affect labial expression of ameloblast markers and key signaling molecules,

the morphology of the labial CL, ameloblast development or amelogenesis,

suggesting that both epithelial and mesenchymal BCL11B may contribute to

developmental processes on the labial side of the incisor. BCL11B regulates

expression of signaling molecules and transcription factors that are essential for

establishment and maintenance of asymmetric incisor development. The majority

of these changes in expression of key genes in Bcl11b−/− mice were qualitatively

similar and characterized by down-regulation on the labial side and up-regulation

on the lingual aspect of the developing incisor. The single exception to this

observation was Fst, the expression domain of which appeared to be maintained

by BCL11B. Collectively, these data suggest that BCL11B regulates the

expression of downstream signaling molecules and transcription factors

bidirectionally, activating expression on the labial side while repressing

expression of the same genes on the lingual side (Fig. S2.13).


41

Integration of BCL11B into FGF and SHH Signaling Pathways

Combined deletion of Spry4 and one allele of Spry2 (Klein et al., 2008) also

results in ectopic development of Shh-positive pre-ameloblasts along the lingual

epithelium of the developing incisor, suggesting a possible convergence in the

FGF signaling pathways and BCL11B-dependent transcriptional regulation. This

interpretation was supported by the strongly decreased expression of BCL11B

within the lingual IEE of Spry4−/−; Spry2+/− incisors, indicating that unrestrained

activity of the FGF signaling pathways results in pronounced down-regulation of

BCL11B expression in the lingual IEE and subsequent de-repression of Shh

expression. Based on these findings, we propose a model (Fig. 2.10) to explain

the role of BCL11B in the FGF signaling pathways within the lingual epithelium at

E16.5 (Fig. 2.10A) and in the Spry4−/−; Spry2+/− incisor (Fig. 2.10B). This model

posits that:

1. SPRY4 and SPRY2 inhibit establishment of a FGF positive-feedback

signaling loop (Klein et al., 2008) and the repressive effect of FGFs on

BCL11B expression in the wild-type lingual IEE.

2. As a result, BCL11B is highly expressed in the entire lingual epithelium in

wild-type mice and directly or indirectly inhibits Fgf9 and Shh expression in

the lingual epithelium, as was demonstrated in both Bcl11b−/− and Bcl11bep−/−

incisors. FGF9 acts in the mesenchyme to induce expression of Fgf3 and

Fgf10 (Klein et al., 2008; Wang et al., 2007). Therefore, the repression of Fgf9

expression by BCL11B in the lingual epithelium prevents activation of Fgf3

and Fgf10 expression in the dental mesenchyme. (Fig. 2.10A).

3. In contrast, Sprouty gene inactivation leads to up-regulation of FGF gene

expression on the lingual side (Klein et al., 2008) and subsequent repression

of BCL11B expression in the lingual IEE. This down-regulation of BCL11B

expression leads to up-regulation of FGF family members, as well as that of

Shh, which induces development of ectopic pre-ameloblasts in the lingual

epithelium (Seidel et al., 2010; Takahashi et al., 2007) (Fig. 2.10B).






42

These data, which suggest that FGFs and BCL11B form a reciprocal, inhibitory

circuit that is upstream of SHH, integrate FGFs, BCL11B, and SHH in a single

pathway and provide insight into the molecular mechanisms underlying both the

Sprouty and Bcl11b−/− phenotypes.

Acknowledgments

We thank I. Thesleff, A. P. McMahon, M.-J. Tsai, S. M. K. Lee, P.-T. Chuang,

R. G. Kelly, S. Bellusci, and J. Smith for activin, Shh, Notch1, Bmp4, Gli1, Tbx1,

Fgf10, and Fst probes, respectively. We are also grateful to O. Golonzhka for

valuable contributions and support.

43

Figure 2.1. BCL11B expression during incisor development. BCL11B immunostaining in sections of wild-type mice at indicated developmental stages. The epithelium is outlined by white dots. Scale bars: (A-B) 100 µm; (C) 200 µm; (D-E) 500 µm. a, ameloblasts; an, anterior; cl, cervical loop; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; iee, inner enamel epithelium; lab, labial; lin, lingual; oee, outer enamel epithelium; pm, papillary mesenchyme; po, posterior; sr, stellate reticulum; vl, vestibular lamina.

44

Figure 2.2. Epithelial invagination defect in Bcl11b−/− developing incisors between initiation and early bud stage.

(A-D) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined in black. (E-H) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (I-J) BrdU index of wild-type and Bcl11b−/− dental epithelium; *** denotes statistical significance at p ≤ 0.001, n = 3. (K-N) RNA ISH using a Bmp4 probe in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows denote epithelial staining. Scale bar, 100 µm.

Figure 2.3. Alterations in Bcl11b−/− incisor development at cap stage.

(A-B) H&E staining in sections of wild-type and Bcl11b−/− mice at E14.5. The

epithelium is outlined in black. (C-D) BrdU immunostaining (green) in sections of wild-type andBcl11b−/− mice at E14.5. All sections were counterstained with DAPI

(blue). The epithelium is outlined in white. (E) BrdU index of wild-type and Bcl11b−/− CLs; *** denotes statistical significance at p ≤ 0.001, n = 3. (F-G)

RNA ISH using an Fgf10 probe in sections of wild-type and Bcl11b−/− mice at E14. Black arrows indicate mesenchymal staining; black asterisks denote the staining between dental epithelium and vestibular lamina. Scale bar, 200 µm. cl, cervical loop; ek, enamel knot; vl, vestibular lamina.

45

47

Figure 2.4. Morphological defects in Bcl11b−/− incisor development at bell stage.

(A-H, L-Y) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined in black. Brackets indicate the posterior end of the incisors in A, E, L, N. Panels D, H, T, Y are higher magnification images of boxes in A, E, L, N, respectively. White arrows indicate lingual epithelium. I denotes the labial CL; II-IV denote stages of progression of ameloblast differentiation in L, N, P-S, U-X. The Bcl11b−/− labial CL was smaller than that of wild-type mice by 60% at E16.5 and by 69% at E18.5 (p ≤ 0.001, n = 11), whereas the mutant lingual CL was enlarged by 35% at E16.5 and by 64% at E18.5 relative to wild-type tissue (p ≤ 0.001, n = 11). (I-J) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice at E16.5. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (K) BrdU index of wild-type and Bcl11b−/− basal epithelium of labial and lingual CLs; ns, not significant; *** denotes statistical significance at p ≤ 0.001, n = 3. Scale bars: (AS, U-X) 200 µm; other panels, 100 µm. a, ameloblasts; cl, cervical loop; lab, labial; lin, lingual.

48

Figure 2.5. Altered ameloblast development in Bcl11b−/− incisors.

RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.

49

Figure 2.6. Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor.

RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/−

incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Scale bars: (A-B, E-F, I-J, M-N, Q-R) 500 µm; other panels, 200 µm.

Figure 2.7. Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor.

RNA ISH using the indicated probes in sections of wild-type

and Bcl11b−/− incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Black and red asterisks denote staining in the posterior part of the dental follicle and epithelial tip of the incisor, respectively. Scale bars: (A-B, E-F, I-J) 500 µm; other panels, 200 µm.

50

Figure 2.8. Ectopic lingual expression of ameloblast markers and signaling molecules in Bcl11bep−/− incisor.

(A-B) BCL11B immunostaining in sections of Bcl11bL2/L2 and Bcl11bep−/− mice at E16.5. The epithelium is outlined by white dots. (C-L) RNA ISH using the

indicated probes in sections of Bcl11bL2/L2 and Bcl11bep−/− incisors at E16.5. The epithelium is outlined by red dots. Black and red arrowheads denote lingual mesenchymal and epithelial staining, respectively. Scale bar, 500 µm.

51

52

Figure 2.9. Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/− mice at E16.5.

BCL11B immunostaining in sections of wild-type and Spry4−/−; Spry2+/− mice at E16.5. The epithelium is outlined by white dots. White arrowheads denote lingual epithelial staining. Scale bar, 500 µm.

53

Figure 2.10. Model for a reciprocal, inhibitory circuit on the lingual side of the mouse incisor. (A) Proposed model for integration of BCL11B into FGF signaling pathways in the lingual epithelium of wild-type incisors. This model suggests that BCL11B expression in the lingual epithelium is facilitated by SPRY2- and SPRY4mediated inhibition of the FGF signaling pathways. The resulting, high levels of BCL11B directly or indirectly suppress expression of Shh and subsequently lead to disruption of pre-ameloblast differentiation and ameloblast formation. It is also proposed that BCL11B inhibits expression of Fgf9 in the lingual epithelium, and this serves to enforce disruption of the FGF signaling loop that is mediated by the Sprouty proteins. The repressive effect of BCL11B on Fgf3 and Fgf10expression is most likely a secondary effect and is depicted by a dashed line. Therefore, BCL11B participates in the suppression of both FGF-mediated stimulation of lingual EpSC proliferation and differentiation of EpSC into Shh-positive preameloblasts within the lingual IEE. (B) In the absence of Spry4 and one allele of Spry2, FGF signaling is enhanced, which extinguishes expression of BCL11B in the lingual IEE. Loss of BCL11B results in derepression of Fgf9 and Shh expression in the epithelium. FGF9 drives Fgf3 and Fgf10 expression in the lingual mesenchyme, whereas Shh expression is required for development of ectopic pre-ameloblasts within the lingual IEE.

54

Figure S2.1. Expression of Bcl11b at early bell stage. (A) RNA ISH using Bcl11b probe in sections of wild-type mice at E16.5. (B-D) Sections of wild-type mice stained with DAPI and immunostained for BCL11B. Scale bar, 500 µm.

55

Figure S2.2. Expression patterns of selected genes in Bcl11b−/− incisor at early bud stage.

RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at E12.5. The epithelium is outlined by red dots. Scale bar, 100 µm.

56

Figure S2.3. Expression patterns of selected genes in Bcl11b−/− incisor at cap stage.

RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at E14.5. The epithelium is outlined by red dots. Scale bar, 200 µm.

57

Figure S2.4. Delay in the initiation of apoptosis in Bcl11b−/− enamel knot at cap stage.

TUNEL immunostaining in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by white dots. White arrows denote apoptosis in the enamel knot. Scale bars: (A-B) 200 µm; other panels, 500 µm.

58

Figure S2.5. Size difference between wild-type and Bcl11b−/− incisors of newborn mice.

Alizarin red staining of wild-type and Bcl11b−/− mandibular incisors at P0. Blue brackets indicate the posterior end of the incisor. Scale bar, 500 µm.

59

Figure S2.6. Labial to lingual reversal of expression of Gli1 in Bcl11b−/−

incisors.

RNA ISH using aGli1 probe in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and red arrowheads indicate lingual epithelial staining. Scale bars: (A-B) 500 µm; other panels, 200 µm.

60

Figure S2.7. Expression pattern of Lfrn and Notch1 in Bcl11b−/− incisors.

RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.

61

Figure S2.8. Expression of ameloblast markers in Bcl11bep−/− and Bcl11bmes/− incisors at E18.5.

RNA ISH using the indicated probes in sections of Bcl11bL2/L2 , Bcl11bep−/−,

and Bcl11bmes−/−mice at E18.5. The epithelium is outlined by red dots. Red arrowheads denote lingual epithelial staining. Scale bar, 200 µm.

62

Figure S2.9. Morphology and mineralization of Bcl11b incisors at P21. Micro-CT analysis of Bcl11bL2/L2 and Bcl11bep−/− jaws at P21.

ep−/−

63

Figure S2.10. Expression patterns of ameloblast markers and signaling molecules in Bcl11bmes−/− incisors at E16.5.

(A-B) BCL11B immunostaining (red) in sections of Bcl11bL2/L2 and Bcl11bmes−/−

mice at E16.5. The epithelium is outlined by white dots. White asterisks denote BCL11B staining in the posterior mesenchyme. (C-J) RNA ISH using the indicated probes in sections of Bcl11bL2/L2 and Bcl11bmes−/− mice at E16.5. The epithelium is outlined by red dots. Scale bar, 500 µm.

64

Figure S2.11. Labial to lingual reversal of expression of Tbx1 in Bcl11b−/−

incisors.

RNA ISH using aTbx1 probe in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B) 500 µm; other panels, 200 µm.

65

Figure S2.12. BCL11B expression in Fgf3−/−; Fgf10+/− incisors.

BCL11B immunostaining in sections of wild-type and Fgf3−/−; Fgf10+/− mice at E16.5. The epithelium is outlined by white dots. Scale bar, 500 µm.

66

Figure S2.13. Summary of direct or indirect BCL11B target genes at E16.5.

This model is based on RNA ISH studies presented in Figs. 5, 6, 7 and Suppl. Figs. S5, S6, and S10. The red staining of the incisor is a pseudo-color representation of BCL11B immunohistochemical staining experiment. The epithelium is outlined by black dots. Green and red arrows indicate induction and inhibition of gene expression, respectively; blue dots denote the enforcement of gene expression domains by BCL11B.

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Chapter 3

BCL11B Regulates Craniofacial Suture Patency through Repression of Fgfr2c Expression

Kateryna Kyrylkova, Urszula Iwaniec, Mark Leid

To be submitted to: Development

72

Abstract

BCL11B is a transcription factor that plays essential roles during development of

the immune, nervous and cutaneous systems. Here we show that BCL11B is

expressed in the osteogenic and sutural mesenchyme of the developing

craniofacial complex. Bcl11b-/- mice exhibit increased proliferation of

osteoprogenitors, premature osteoblast differentiation, and skull mineralization.

These processes lead to synostosis of facial and coronal sutures in the Bcl11b-/-

skulls. Expression of Fgfr2c, a gene implicated in craniosynostosis in mice and

humans, was up-regulated and Fgfr2c transcripts were detected ectopically

within these sutures in Bcl11b-/- mice. These data suggest that overexpression of

Fgfr2c in the sutural mesenchyme, without concomitant changes in the

expression of FGF ligands, leads to craniosynostosis in Bcl11b-/- mice.

Introduction

Flat bones of the skull and face develop by intramembranous ossification of

mesenchyme derived from the mesoderm and neural crest (Jiang et al., 2002). In

this process, mesenchymal cells condense, differentiate, and form ossification

centers (Opperman, 2000; Rice, 2008). Craniofacial sutures are formed when the

margins of the developing bones approach each other. While human sutures

remain patent, they harbor proliferating progenitors that give rise to osteoblasts

at the osteogenic fronts of the skull bones. Therefore, craniofacial sutures serve

as important sites of facial and calvarial bone growth during fetal development

and into young adulthood, when sutures undergo ossification (Rice, 2008).

Suture development is precisely controlled and synchronized with brain growth.

Premature osteoblast differentiation and suture ossification underlie the condition

known as craniosynostosis, which occurs in 1 out of 2,200 live births.

Craniosynostosis is associated with restricted skull expansion, increased

intracranial pressure, and craniofacial dysmorphologies, all of which may

negatively impact respiration, vision, hearing, and cognition. Midfacial hypoplasia

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(MFH) often accompanies craniosynostosis and until recently was thought to

result from premature ossification of sutures at the cranial base (Nie, 2005;

Rosenberg et al., 1997). However, it was shown that MFH, in the context of

syndromic craniosynostosis, likely arises from premature fusion of facial sutures

(Purushothaman et al., 2011).

Mutations in fibroblast growth factor receptors (FGFRs) are the most common

cause of syndromic cranyosynostosis, of which the most prevalent forms are

Crouzon and Apert syndromes (Purushothaman et al., 2011). Both conditions are

the result of activating mutations within FGFR2 isoform IIIc (FGFR2c). The

FGFR2c-C342Y mutation, which affects immunoglobulin-like (Ig) domain III,

results in constitutive activation of the receptor and leads to development of

Crouzon syndrome. In contrast, Apert syndrome is caused by FGFR2c-S252W

mutation in Ig II-III linker region and is characterized by increased ligand affinity

and altered specificity of FGFR2 (Cunningham et al., 2007). FGFRs act through

the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)

pathways to stimulate transcription factor RUNX2, a master-regulator of

osteogenesis (Kim et al., 2003a; Park et al., 2010). As a result, constitutive

activation of FGFR2c leads to enhanced osteoblast differentiation and premature

ossification of the sutures. However, little is known about molecular control of

FGFR2c expression and function during craniofacial development.

We showed previously that the transcription factor BCL11B controls mouse

incisor development by regulating the expression of some of the individual

components of the FGF signaling network in the labial and lingual cervical loops

(Kyrylkova et al., 2012a). Our analyses of neural crest-specific and germline

Bcl11b knockouts (Bcl11bncc-/- and Bcl11b-/-, respectively) reveal that BCL11B

plays an essential role in craniofacial development and maintenance of suture

patency. Mice lacking Bcl11b exhibit craniosynostosis of facial and coronal

sutures, severe midfacial hypoplasia, and malocclusion. We show that BCL11B

regulates timely proliferation and differentiation of osteoprogenitors by repressing

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Fgfr2c expression directly or indirectly in the craniofacial sutures.

Materials and Methods

Mouse Lines

− cl11 − B b / , Bcl11bL2/L2 , and Bcl11bncc-/- (same as Bcl11bmes-/-) mice have been

described (Golonzhka et al., 2009; Kyrylkova et al., 2012a). Lines carrying Wnt1

cre transgene were maintained as reported (Danielian et al., 1998). Animal

experiments were approved by the Oregon State University Institutional Animal

Care and Use Committee, protocol 4279.

Micro-CT Analysis

Mouse skulls were scanned using a Scanco μCT40 scanner (Scanco Medical

AG, Basserdorf, Switzerland) at a voxel size of 12 to 20 μm. The threshold for

analysis was determined empirically and set at 245 (scale 0 – 1000). At least

three mouse skulls were scanned for each genotype and age group.

Histological Analysis, RNA in situ Hybridization, BrdU labeling, and

Immunohistochemistry

Embryonic heads were fixed in 4% paraformaldehyde, cryopreserved in 30%

sucrose, and frozen in O.C.T. Von Kossa as well as hematoxylin and eosin

(H&E) stainings were performed according to standard protocols. RNA in situ

hybridization using digoxigenin-labeled probes and immunohistochemistry using

anti-BCL11B (Abcam, 1:300) were performed on 16 µm-thick sagittal (for coronal

suture) and horizontal (for facial suture) sections as described previously

(Kyryachenko et al., 2012; Kyrylkova et al., 2012b). At least three mice were

used for each genotype and experiment. At least ten serial sections across the

facial and coronal sutures were analyzed for each experiment.

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Results

Neural Crest-Specific Ablation of Bcl11b Leads to Growth Impairment and

Craniofacial Synostoses

In our previous study on the role of BCL11B in tooth development, we generated

a mouse line conditionally null for Bcl11b expression in neural crest-derived

dental mesenchyme (Bcl11bmes-/-; referred to as Bcl11bncc-/- in a current

manuscript) by crossing floxed Bcl11bL2/L2 mice with the Wnt1-cre deleter strain

(Danielian et al., 1998; Kyrylkova et al., 2012a). Wnt1-expressing neural crest

cells contribute not only to the tooth, but also to a plethora of different tissues and

organs, including components of enteric and peripheral nervous system,

endocrine and cardiac derivatives, melanocytes, as well as cephalic

Bcl11bncc-/-mesenchyme (Pietri et al., 2003). mice did not exhibit dental

abnormalities, as the incisor morphology and gene expression patterns were

indistinguishable from those of control mice (Kyrylkova et al., 2012a). However,

-/- ncc-/-unlike Bcl11b mice, Bcl11b mice survived after birth for around three weeks

(P21). At this age, Bcl11bncc-/- mice were characterized by abnormally small and

misshapen heads, severe MFH, and malocclusion (compare Figs. 3.1A, C with B,

D). We observed fusion of multiple craniofacial sutures, including the interfrontal,

internasal, temporal, premaxillary-maxillary, and premaxillary-nasal sutures. In

addition, the Bcl11bncc-/- nasofrontal suture exhibited abnormal morphology and

lacked its canonical, fractal interdigitation (compare Figs. 3.1E, G with F, H).

Bcl11bncc-/-Some skulls were characterized by a high degree of porosity, which

was accompanied by up-regulated expression of pro-inflammatory cytokines in

frontal and parietal bones (Fig. S3.1 and data not shown).

We traced back the developmental dynamics of the conditional knockout

Bcl11bncc-/-phenotype by conducting micro-CT analyses of multiple skulls at

different postnatal stages. At P5, synostosis of temporal and frontomaxillary

Bcl11bncc-/-sutures was observed in 50% of mice. In addition, the skulls of

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Bcl11bncc-/- mice were characterized by expansion of sagittal, lambdoid, and

coronal sutures, and these expansions appeared to be of a compensatory

nature. The onset of bone porosity was visible at P5 in the anterior region of

frontal bones and at the base of the Bcl11bncc-/- skulls. A similar but more severe

craniofacial phenotype was observed in the conditional mutants at P10. The

progression of synostosis was further evident in the anterior part of interfrontal

and posterior part of internasal sutures in Bcl11bncc-/- mice at P14 (Fig. S3.2).

Newborn Bcl11b-/- Mice Exhibit Facial and Coronal Synostosis

Premaxillary-maxillary and premaxillary-nasal sutures in Bcl11bncc-/- mice were

already fused at birth, at which time mild MFH and a reduced gap between

frontal and maxillary bones were also evident (compare Figs. 3.2A, D with B, E).

Bcl11bncc-/-The early onset of the craniofacial phenotype of mice led us to

examine Bcl11b-/- mice, which harbor a germline deletion of Bcl11b and die

perinatally (Golonzhka et al., 2009). Bcl11b-/- skulls exhibited facial synostoses at

ncc-/-P0 that were indistinguishable from those of Bcl11b mice (compare Figs.

3.2B, E with C, F). However, premature fusion of the temporal and coronal

sutures was also evident in Bcl11b-/- mice at P0, a phenotype that we have never

Bcl11bncc-/-observed in mice (compare Figs. 3.2A, D with C, F). Moreover,

enhanced ossification within the anterior fontanelle was noted in Bcl11b-/- mice

Bcl11bncc-/-relative to either control or mice (compare Figs. 3.2A, B with C).

Enhanced mineralization of the facial and cranial skeleton in Bcl11b-/- mice was

further confirmed by von Kossa staining at P0 (Fig. S3.3). Collectively, the above

findings indicate that facial and cranial synostoses in mice lacking Bcl11b initiate

during embryonic development.

Embryonic Skulls of Bcl11b-/- Exhibit Increased Osteoblast Proliferation and

Maturation, Premature Mineralization, and Craniofacial Synostosis

Mineralization within control and Bcl11b-/- heads was observed at E14.5 as

detected by von Kossa staining. However, Bcl11b-/- mice exhibited increased

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mineralization within the facial skeleton (compare Figs. 3.3A, C with B, D).

Moreover, expression of bone sialoprotein (Bsp), a marker of osteoblast

maturation, was dramatically up-regulated within Bcl11b-/- faces (compare Figs.

3.3E, G with F, H). Osteoblasts of Bcl11b-/- facial mesenchyme exhibited ~30%

increase in the proliferation rate compared to the control cells (data not shown).

Micro-CT analyses of Bcl11b-/- skulls at E16.5 and E18.5 also revealed increased

mineralization within the facial skeleton and calvaria of the mutant skulls (Figs.

3.3I-P). The premaxillary-maxillary suture was fused in the mutants at this

developmental stage (compare Figs. 3.3I, K with J, L). However, initiation of

coronal and temporal synostoses was not observed until E18.5 in the Bcl11b-/-

skulls (compare Figs. 3.3M, O with N, P).

Palatogenesis is a key component of midface development (Weinzweig et al.,

2006). Bcl11bncc-/- embryos did not exhibit defects in palatal development (data

not shown). However, we detected a delay in palatal shelf elevation and fusion in

-/-Bcl11b mice at E14.5 but this defect was completely resolved by E15.5.

BCL11B expression was localized predominantly in the palatal epithelium prior to

and after shelf elevation and fusion (Fig. S3.4).

BCL11B Is Expressed in Osteogenic and Sutural Mesenchyme

BCL11B expression within osteogenic progenitors of the face was first detected

at E13.5 (Fig. S3.5C). High levels of BCL11B expression persisted at E14.5 and

E16.5 within facial and calvarial osteogenic and sutural mesenchyme, including

osteogenic fronts of frontal and parietal bonas as well as the coronal suture

(Figs. 3.4A-F). However, by E18.5 BCL11B expression was down-regulated and

barely detectable within either facial or cranial bones and sutures (Figs. 3.4G-I),

suggesting that BCL11B functions predominantly during embryonic stages of

skull development.

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BCL11B expression was ablated specifically in neural crest-derived facial

mesenchyme, including dermal and osteogenic mesenchyme, in Bcl11bncc-/- mice

at E12.5. The expression of BCL11B within the osteogenic mesenchyme of the

face was also absent in Bcl11bncc-/- at E13.5. At the same time, levels of BCL11B

protein were unaltered in all epithelial structures in Bcl11bncc-/- mice (Fig. S3.5).

Runx2 Expression Is Up-Regulated in Osteogenic Mesenchyme of Bcl11b-/-

Skulls

RUNX2 is the transcripition factor required for determination of the osteoblast

lineage and is a master regulator of osteogenesis (Komori, 2010; Komori et al.,

1997; Otto et al., 1997). Runx2 expression was strongly detected in

differentiating osteoblasts of the facial and calvarial osteogenic mesenchyme at

E14.5 and E16.5, respectively, in control mice. At the same time, low levels of

Runx2 transcripts were observed in the premaxillary-maxillary and coronal

sutures of control skulls (Figs. 3.5A, C, E). An expansion of Runx2-positive cells

-/-was observed in the osteogenic mesenchyme of the Bcl11b face, along with

ectopic Runx2 transcripts within coronal suture of these mutants at E16.5 (Figs.

3.5B, D, F). This observation suggests that BCL11B limits osteoblast expansion

and restricts Runx2 expression within sutural mesenchyme.

Fgfr2c Is Ectopically Expressed Within Facial and Coronal Sutures of

Bcl11b-/- Mice

The FGF signaling pathways play important roles in regulation of craniofacial

suture patency, and activating mutations within FGFR receptors are associated

with syndromic and non-syndromic craniosynostosis in mice and humans (Nie et

al., 2006). We showed previously that BCL11B regulates FGF signaling during

tooth development (Kyrylkova et al., 2012a). Therefore, we analyzed Bcl11b-/-

mice for expression of multiple FGF ligands and receptors that play roles during

craniofacial development. Surprisingly, we did not detect alterations in

expression of any genes encoding FGF or FGFR family members in Bcl11b-/-

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mice (Fig. S3.6), with the exception of Fgfr2c (Fig. 3.6) Fgfr2c expression, was

detected widely within differentiating osteoblasts of the face and head of control

mice but was generally excluded from sutural mesenchyme (Fig. 3.6). Fgfr2c

transcripts were ectopically expressed within facial and coronal sutures of

Bcl11b-/- mice at E14.5 and E16.5, respectively (compare Figs. 3.6A, C, E with B,

D, F). However, Fgfr2c transcripts were excluded from the more differentiated

mesenchyme within central osteoids of Bcl11b-/- facial bones. These results

suggest that BCL11B normally represses Fgfr2c expression within craniofacial

sutures, and its absence is associated with ectopic expression of this key Fgfr2

splice variant.

Discussion

Data presented in this paper demonstrate that BCL11B plays an important role in

maintaining sutural patency from the earliest stages of craniofacial development.

BCL11B is expressed in osteogenic and sutural mesenchyme beginning at E13.5

and is down-regulated by E18.5, suggesting that it functions primarily during a

finite window of embryogenesis. BCL11B controls timely proliferation and

differentiation of osteoprogenitors, limits premature bone mineralization, and

suppresses ossification of facial and coronal sutures. We speculate that the

principal mechanistic basis of these actions of BCL11B is the repression of

Fgfr2c expression within osteogenic and sutural mesenchyme.

The craniofacial skeleton is derived from two distinct embryonic sources: neural

crest cells and the paraxial mesoderm. Neural crest cells give rise to the facial

skeleton, frontal and squamosal bones, as well as central part of the interparietal

bone, as determined by lineage tracing analyses (Jiang et al., 2002). In contrast,

the parietal bone and most of the interparietal bone are mesodermal in origin

(Yoshida et al., 2008). Therefore, the coronal suture, a major growth center of the

skull vault, is formed at the interface of the neural crest-derived frontal bone and

mesoderm-derived parietal bone. However, the mesenchyme of the coronal

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suture is derived from the mesoderm (Yoshida et al., 2008), and this likely

explains our observation of coronal synostosis in Bcl11b-/-, but not in Bcl11bncc-/-,

mice.

Bcl11bncc-/- mice survive after birth and exhibit severe MFH, a condition that often

accompanies syndromic craniosynostosis in humans. Until recently, the most

common theory on etiology of MFH was premature ossification of one or more

sutures in the cranial base (Rosenberg et al., 1997; Stewart et al., 1977).

However, analyses of mice harboring constitutively active mutations in Fgfr1 and

Fgfr2 genes, which underlie Apert and Pfeiffer syndromes, revealed that MFH is

a consequence of synostoses of facial sutures (Purushothaman et al., 2011).

Consistent with this, Bcl11bncc-/- mice did not exhibit premature ossification of the

cranial base. Instead, Bcl11bncc-/- mice were characterized by synostoses of facial

sutures, including premaxillary-maxillary and premaxillary-nasal, further

supporting the role of facial skeletal pathology in etiology of MFH. In comparison

to the biology of cranial sutures, development of facial sutures is understudied

and poorly understood. While growth at the osteogenic fronts of the facial bones

is similar to that of the cranial vault bones, the facial bones differ in that they

overlie the cartilaginous elements of the facial complex rather than the dura

mater. Moreover, development of some facial sutures, such as frontonasal, is

characterized by complex interdigitation patterns that are not observed within any

sutures of the cranial vault (Nelson and Williams, 2004). Therefore, Bcl11bncc-/-

mice may serve as a valuable model to study pathogenesis of facial synostoses

and development of MFH.

The FGF signaling serves as a positive regulator of osteogenesis and plays a

major role in craniofacial development. The FGF family is comprised of 22 genes

encoding structurally related proteins (Ornitz and Itoh, 2001). Fgf2, Fgf9, Fgf10

and Fgf18 are expressed in developing bones and/or sutural mesenchyme and

have been directly or indirectly associated with enhanced ossification and

associated skeletal defects including craniosynostosis (Behr et al., 2010;

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Hajihosseini et al., 2009; Harada et al., 2009; Kim et al., 1998; Liu et al., 2002;

Montero et al., 2000; Ohbayashi et al., 2002; Rice et al., 2000). In addition,

although Fgf4 is not expressed in the developing facial or cranial skeleton, direct

application of FGF4 in the vicinity of sutures promotes osteoblast proliferation

and/or differentiation and subsequent suture closure (Greenwald et al., 2001;

Kim et al., 2003b; Kim et al., 1998; Nagayama et al., 2013). It is noteworthy that

neither the expression level nor localization of Fgf transcripts that are relevant for

craniofacial development was altered in the osteogenic mesenchyme of Bcl11b-/-

mice.

FGFs bind four high-affinity, receptor tyrosine kinases (FGFR1 to FGFR4). The

ligand-binding specificity of FGFRs is mediated by specific extracellular regions

of these receptors known as immune globulin-like (Ig) domains. Alternative

splicing of transcripts derived from Fgfr1, Fgfr2 and Fgfr3 results in two different

versions of Ig-domain III within each receptor (referred to as domain IIIb and IIIc

or b and c isoforms, respectively). These alternative splicing events have

functional consequences as the b and c isoforms of FGFRs are characterized by

unique ligand-binding properties (Ornitz et al., 1996). Expression of Fgfr2c, as

well as low levels of Fgfr1c, Fgfr2b, and Fgfr3c, has been reported in developing

calvarial bones and particularly at the osteogenic fronts of these bones (Rice et

al., 2003). Mutations in genes encoding FGFR1-3, but not FGFR4, are

associated with craniosynostosis in mice and humans (Chim et al., 2011). In

general, all of these mutations result in receptors with enhanced and/or

constitutive activity (Cunningham et al., 2007). With the exception of Fgfr2c, we

did not observe alterations of expression of Fgfr family members in Bcl11b-/-

mice.

Fgfr2c is highly expressed in proliferating osteoprogenitors at the osteogenic

fronts (Iseki et al., 1997; Rice et al., 2003) and is of particular relevance in the

etiology of craniosynostosis. Expression of Fgfr2 is mutually exclusive with that of

Secreted phosphoprotein 1 (Spp1), a marker of differentiating osteoblasts (Iseki

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et al., 1997), suggesting that FGFR2 isoforms are expressed early in the

osteoblast lineage and are down-regulated as osteoprogenitors mature toward

terminal differentiation. Loss-of-function mutation in the IIIc exon of Fgfr2 gene

results in defective ossification and reduced expression of Runx2 and Spp1,

indicating altered osteoblast differentiation (Eswarakumar et al., 2002). In

contrast, mice harboring gain-of-function mutation of Fgfr2c (Fgfr2cc342y/+) exhibit

craniosynostosis, cleft lip and palate, MFH, and other skeletal defects. The mice

are characterized by increased proliferation of osteoprogenitor cells and up-

regulation of Runx2 and Spp1 expression (Eswarakumar et al., 2004). RUNX2 is

an indispensible transcription factor for osteogenesis and skeletal development

(Kim et al., 2003a; Park et al., 2010). FGF signaling induces Runx2 expression

as well as enhances RUNX2 stability, DNA-binding, and transcriptional activity

through PKC and extracellular signal-regulated kinase MAPK pathways (Kim et

al., 2003a; Park et al., 2010; Twigg et al., 2013; Xiao et al., 2002). Some effects

of FGF activity on RUNX2 activity are mediated by a complicated cascade of

downstream factors, including the transcription factors Twist1, TCF12, and ERF

(Connerney et al., 2006; Fitzpatrick, 2013; Sharma et al., 2013; Twigg et al.,

2013).

Fgfr2c expression was up-regulated and Fgfr2c transcripts were ectopically

localized in the facial and coronal sutures of Bcl11b-/- mice. However, the central

osteoid of both facial and cranial bones was largely devoid of Fgfr2c transcripts,

consistent with enhanced osteoblast differentiation in these areas of the Bcl11b-/-

craniofacial skeleton. In addition, we observed enhanced osteoprogenitor

proliferation and increased Runx2 expression, both of which phenocopy defects

Fgfr2cc342y/+ of mice. However, we assume that Bcl11b-/- mice over- and

ectopically-express a wild-type form of Fgfr2c, yet exhibit a craniosynostosis

phenotype that is much more severe than that of mice heterozygously expressing

Fgfr2cc342y/+ a constitutive active form of the receptor, e.g., . This may be

explained by the fact that Fgfr2 over-expression results in ligand-independent

activation of the receptor in other cell systems (Turner et al., 2010). In addition,

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overexpression of Fgfr1, driven by transient transfection, results in FGF2

mediated proliferation in neonatal cardiac myocyte cultures (Sheikh et al., 1999).

In the latter case, the over-expressed FGFR1 would appear to be stimulated by

an FGF ligand(s) that is in the vicinity. Therefore, we suggest that overexpression

of Fgfr2c in the sutural mesenchyme, without concomitant changes in the

expression of FGF ligands, leads to craniosynostosis in Bcl11b-/- mice.

Craniosynostosis affects 1 in 2,200 individuals and may be isolated

(nonsyndromic) or associated with other clinical signs as part of a syndrome.

Plausible causative mutations were identified only in ~30% of the cohort that

comprised over 300 cases of craniosynostosis. Genes that are predominantly

affected in craniosynostosis are FGFR2, FGFR3, TWIST1, and EFNB1

(Fitzpatrick, 2013; Twigg et al., 2004; Wilkie et al., 2010). Recently, heterozygous

loss-of-function mutations in TCF12 and ERF, both of which encode transcription

factors, were also found in the cohort (Sharma et al., 2013; Twigg et al., 2013).

However, ~70% of craniosynostosis cases still have unknown genetic etiology

(Fitzpatrick, 2013). We do not presently know if mutations at the BCL11B locus

contributes to craniosynostosis in humans as less than optimal exome coverage

of this locus hinders identification of possible mutations in humans. Given the

severity of the craniosynostosis phenotype of the Bcl11b-/- mice, it seems

plausible to hypothesize that dysregulated expression of the transcription factor

BCL11B may be implicated in craniosynostosis in humans.

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Figure 3.1. Impaired postnatal growth, misshapen heads, and craniofacial synostoses in Bcl11bncc-/- mice. (A-D) Ventral (A, B) and lateral (C, D) views of mouse heads show abnormal shape, short snouts, and severe malocclusion in Bcl11bncc-/- mice at P21. (E-H) Dorsal (E, F) and lateral (G, H) views of the micro-CT images of control and Bcl11bncc-/-mouse skulls at P21. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrow points to the lack of fractal interdigitation in Bcl11bncc-/- fronto-premaxillary suture. Red lines indicate dental occlusion. Scale bar: (E-H) 1 mm.

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Figure 3.2. Craniofacial defects of Bcl11bncc-/- and Bcl11b-/- newborn mice. Dorsal (A-C) and lateral (D-F) views of the micro-CT images of control, Bcl11bncc-/-, and Bcl11b-/- mouse skulls at birth. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- and Bcl11b-/- skulls and corresponding patent sutures in the control mice. Arrowhead points to increased mineralization and reduced anterior fontanelle within Bcl11b-/- calvaria. Red lines indicate dental occlusion. Scale bars: 1 mm.

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Figure 3.3. Increased osteoblast maturation, mineralization, and craniofacial synostoses in Bcl11b-/- embryonic skulls.

(A-D) Von Kossa staining for mineralization with Nuclear Fast Red counterstain in horizontal sections of control and Bcl11b-/- embryos at E14.5. Arrowhead points to increased mineralization in Bcl11b-/- facial mesenchyme. (E-H) RNA in situ hybridization using a Bsp probe in sections of control and Bcl11bncc-/- heads at E14.5. Arrowheads point to enhanced osteoblast maturation in Bcl11b-/- facial mesenchyme. (I-P) Dorsal (third row) and lateral (fourth row) views of the micro-CT images of control and Bcl11b-/- mouse skulls at E16.5 (A-D) and E18.5 (E-H). Arrowheads point to increased mineralization in cranial bones in Bcl11b-/- skulls. Asterisks denote sutures affected by synostosis in Bcl11b-/- skulls and corresponding patent sutures in the control mice. Scale bars: (A-H) 500 um; (IL), (M-P) 1 mm.

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Figure 3.4. BCL11B expression in osteogenic and sutural mesenchyme. BCL11B immunostaining in sections of embryonic faces (two upper rows represent sections from two different planes) and coronal sutures (lower row) at indicated stages. Osteogenic mesenchyme is denoted by asterisk in the facial sections and outlined by dashed line in the section of coronal suture. Scale bars: (two upper rows) 500 um; (C), (F, I) 200 um.

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Figure 3.5. Increased Runx2 expression in the Bcl11b-/- embryonic faces and coronal sutures. RNA in situ hybridization using a Runx2 probe in sections of embryonic faces at E14.5 (two upper rows represent sections from two different planes) and coronal sutures at E16.5 (lower row). Arrowheads denote up-regulation and expansion of Runx2 expression in Bcl11b-/- face. Asterisks indicate ectopic expression of Runx2 in Bcl11b-/- coronal suture. Scale bars: (A-D) 500 um; (E-F) 200 um.

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Figure 3.6. Ectopic Fgfr2c expression in facial and coronal sutures of Bcl11b-/- embryos. RNA in situ hybridization using an Fgfr2c probe in sections of embryonic faces at E14.5 (two upper rows represent sections from two different planes) and coronal sutures at E16.5 (lower row). Arrowheads denote up-regulation and expansion of Fgfr2c expression in Bcl11b-/- face. Asterisks indicate ectopic expression of Fgfr2c in Bcl11b-/- facial and coronal sutures. Scale bars: (A-D) 500 um; (E-F) 200 um.

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Figure S3.1. Some Bcl11bncc-/- mice exhibit increased bone porosity in the skull at P21. (A-F) Dorsal (A-B), lateral (C-D), and ventral (E-F) views of the micro-CT images of control and Bcl11bncc-/-mouse skulls at P21. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrows point to increased porosity in multiple Bcl11bncc-/- skull bones. Red lines indicate dental occlusion. Scale bar: (A-F) 1 mm.

Figure S3.2. Craniofacial phenotype development in Bcl11bncc-/- mice at postnatal stages. Dorsal (upper row), lateral (middle row), and ventral (bottom row) views of the micro-CT images of control and Bcl11bncc-/- at indicated stages. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrows point to increased porosity in multiple Bcl11bncc-/- skull bones. Red lines indicate dental occlusion. Red brackets indicate compensatory expansion in the sagittal suture of Bcl11bncc-/- heads. Scale bars: (A-F), (G-L), (M-R) 1 mm.

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Figure S3.3. Synostosis of facial sutures in newborn Bcl11b-/- mice.

Von Kossa staining for mineralization with Nuclear Fast Red counterstain in horizontal sections of control and Bcl11b-/- newborn mice. Arrows and arrowheads point to premaxillary-nasal and premaxillary-maxillary sutures, respectively, in control mice and synostosis of these sutures in Bcl11b-/- skulls. Scale bar: 500 um.

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Figure S3.4. Delayed elevation and fusion of palatal shelves in Bcl11b-/-

mice.

(A-D) Hematoxylin and eosin staining in sections of wild-type and Bcl11b-/- mice at indicated stages. Arrows point to the palatal shelves that are fused in control embryos at E14.5, and exhibit delay in elevation and fusion in Bcl11b-/- mice. (EF) Immunostaining for BCL11B (red) in the sections of palatal shelf at E13.5 and in the palate at E15.5. The sections were counterstained with DAPI (blue). BCL11B is expressed predominantly in the palatal epithelium. Scale bars: (A-D) 500 um; (E), (F) 100 um.

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Figure S3.5. Neural crest-specific ablation of BCL11B expression in the facial mesenchyme.

Bcl11bncc-/-BCL11B immunostaining in horizontal sections of control and embryonic faces at indicated stages. Asterisk denotes BCL11B expression in osteogenic mesenchyme at E13.5. Notice the lack of BCL11B staining in neural

Bcl11bncc-/-crest-derived facial mesenchyme in ; whereas BCL11B expression persists in all epithelial structures. Scale bar: 500 um.

97

Figure S3.6. Expression patterns of FGF and FGFR genes in the faces of Bcl11b-/- mice at E14.5.

RNA in situ hybridization using indicated probes in sections of control and Bcl11b-/- embryonic faces at E14.5. Scale bar: 500 um.

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Chapter 4

Conclusion

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A deeper understanding of developmental biology can foster greater progress in

regenerative medicine and treatment of congenital disorders. I elucidated the role

of the transcriptional regulatory protein BCL11B during tooth development and

showed that BCL11B functions in craniofacial development. Lack of Bcl11b

results in abnormal incisor morphology and craniofacial synostoses in mice.

BCL11B represses cell proliferation and premature differentiation of epithelial

cells in the lingual cervical loop of the incisor and osteogenic mesenchymal cells

of the craniofacial complex. BCL11B is simultaneously necessary to promote

these processes in the cells of the labial cervical loop. This observation

demonstrates context-dependent function of BCL11B. In all instances, ablation of

BCL11B function results in dysregulation of FGF signaling, and in the case of

tooth development TGF-β signaling is also affected. Moreover, ectopic FGF

signaling results in loss of BCL11B localization in the lingual. This is the first

report that integrates BCL11B in the FGF signaling network and illuminates the

molecular mechanisms that underlie dental and craniofacial phenotypes of

Bcl11b-/- mice. The studies described here are of significance because they will

contribute to a broader understanding of how the activities of transcription factors

and growth factors are regulated and how these pathways can be modulated as

an approach to therapy for developmental disorders that are not well treated by

current drugs.

Regenerative Dentistry

Substantial effort has been devoted to the understanding of the mechanisms of

tooth development, pathology, and therapy over the last several decades. The

immense interest in this subject is quite justified, as congenital disturbances in

tooth formation, acquired dental diseases, and odontogenic tumors affect millions

of people. Tooth agenesis is one of the most commonly inherited disorders,

which affect up to 20% of the population, whereas oral pathology is ranked as the

second most frequent clinical problem. In addition, tooth loss affects almost 10%

of the population and adversely influences mastication, articulation, facial

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esthetics, and psychological health (Kapadia et al., 2007; Koussoulakou et al.,

2009; Tucker and Sharpe, 2004).

Historically, surgeons have used several procedures to replace lost and repair

damaged teeth, including tooth allotransplantation, autotransplantation, and

artificial dentures (Ferreira et al., 2007; Koussoulakou et al., 2009). The current

state-of-the-art approach to replace a missing tooth is by means of a dental

implant. During the last half of the 20th century, dental implantation procedures

have improved significantly due to innovations and discoveries from basic and

translational research, material sciences, and clinical techniques (Chai and

Slavkin, 2003). Traditionally, the implants have been made from titanium alloys,

and the implant surface has undergone numerous modifications over the years

aiming to promote successful osseointegration within the host bone. However, in

comparison to the natural dentition, the metal implant is more prone to

mechanical and biological failure because it lacks periodontal ligament and

crevicular sulcus, which provide mechanical stress absorption and local anti-

bacterial defense, respectively. In addition, dental implants require a minimum

level of bone, and in cases of severe bone loss the use of the implants is very

limited and must be preceded by bone grafts (Ferreira et al., 2007; Volponi et al.,

2010). Technological advances in dental implantology have slowed substantially

since inception of this procedure, and relatively more effort is currently devoted to

development of biological methods of tooth replacement that could overcome

difficulties associated with traditional dental implants.

The future practice of dentistry is likely to be revolutionized by regenerative

therapies, which will rely on the bioengineering of dental tissues and even a

whole tooth. Bioengineered teeth will serve as a genuine replica of the damaged

tooth. In addition, because teeth are nonessential for life, bioengineered teeth will

be an excellent model to optimize new, cell-based treatments that can also be

used for replacement of major, internal organs (Sartaj and Sharpe, 2006; Volponi

et al., 2010).

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The development of new approaches in regenerative dentistry relies significantly

on our understanding of embryonic development, stem cell biology, and tissue

engineering technology. The mouse has proven to be an invaluable research

model for study of the molecular mechanisms that control tooth organogenesis.

Tooth development in mice is similar to that of humans, with the same set of

genes being expressed during tooth organogenesis; and mutations in the

counterpart genes often cause similar defective phenotypes (Fleischmannova et

al., 2008; Koussoulakou et al., 2009). However, unlike human teeth, mouse

incisors grow continuously throughout the life of the animal and are characterized

by the presence of epithelial and mesenchymal stem cell niches in the posterior

part of the tooth (Harada et al., 1999). This makes the mouse incisor an excellent

model to study several aspects of the dental stem cells.

Several methods have been proposed to achieve biological tooth replacement in

mice and other model organisms. These include stimulation of the formation of a

third dentition, construction of a tooth by bioengineering different component

parts separately, seeding of tooth-shaped biodegradable scaffolds with stem

cells, and producing embryonic-like tooth primordia from cultured cell populations

(Duailibi et al., 2004; Ferreira et al., 2007; Ohazama et al., 2004; Young et al.,

2005a; Young et al., 2005b). The latter approach has been used successfully by

several groups, which demonstrated that embryonic tooth primordia can form

teeth following dissociation into single-cell populations, subsequent

recombination, and transplantation into the adult mouse jaw (Angelova Volponi et

al., 2013; Oshima et al., 2011). However, embryonic cell sources have little rel-

evance for the development of a clinical therapy. Therefore, it is important to

identify the adult sources of human epithelial and mesenchymal cells that can be

obtained in sufficient numbers to render bioengineered teeth a viable alternative

to dental implants. To date, the only two non-dental sources of adult cells that

have been successfully used in tooth bioengineering are mesenchymal cells

derived from bone marrow stroma and human gingival epithelial cells that were

recombined with embryonic dental epithelium or mesenchyme, respectively

107

(Angelova Volponi et al., 2013; Ohazama et al., 2004). In each case, embryonic

tissue produced instructive signals to the non-embryonic counterpart. Therefore,

it is important to understand the complex networks of signaling molecules and

transcription factors that regulate embryonic tooth morphogenesis in order to

induce odontogenic potential in adult non-dental tissue in vitro.

Due to the rapid progress of research in molecular and developmental biology,

we are starting to understand how the development of dental tissues is regulated

at the level of proteins and signaling molecules. The transcriptional mechanisms

underlying tooth development are only beginning to be understood. We

demonstrate that BCL11B is a key component of the complex genetic network,

which regulates the development of the dental epithelial stem cells and their

differentiation into enamel-secreting ameloblasts in the mouse incisor. Therefore,

our findings on the role of BCL11B in tooth development is equally applicable to

research in emerging areas of regenerative dental medicine as it is in

developmental biology.

Targeted Treatment of Craniofacial Synostosis

With recent strides in medical genetics and developmental biology, it is evident

that advances in the treatment of craniosynostosis are dependent upon

elucidating the molecular events leading to this condition (Wan et al., 2008).

Craniosynostosis, defined as the premature fusion of one or more sutures in the

skull, is a common congenital craniofacial abnormality, with an overall incidence

of 1 in 2,200 live births (Fitzpatrick, 2013).

Craniofacial sutures serve as the major sites of bone growth along the leading

margins of the bones in the skull (Opperman, 2000). During childbirth, the

sutures allow the calvarial bones to overlap and mold, allowing for the passage of

the head through the birth canal (Warren and Longaker, 2001). Normal growth of

the skull within the sutures matches the spatial requirements of the developing

brain during fetal development and after birth. In the first year of life, head

108

circumference grows at an average of 2.4 mm per week and then drops down to

0.5 and 0.24 mm per week, respectively, in the second and third years of life

(Fitzpatrick, 2013). Therefore, one of the greatest concerns of the

craniosynostosis is elevated intracranial pressure that can result in deafness,

blindness, seizures, and cognitive deficits (Warren and Longaker, 2001).

Midfacial hypoplasia often accompanies craniosynostosis and can result from

premature ossification of facial sutures (Purushothaman et al., 2011). The most

common and debilitating concerns associated with midfacial hypoplasia are

malocclusion, respiratory difficulty related to nasopharyngeal stenosis, and ocular

conditions, such as ocular keratitis, corneal ulcers, globe herniation, and

blindness (Panchal and Uttchin, 2003; Raulo and Tessier, 1981).

Current treatment of craniofacial synostosis consists almost exclusively of

surgical modalities. Surgical approaches involve excision of the prematurely

fused suture(s) and/or parts of cranial bones, as well as advancement

procedures to remodel the skull. In addition, external and internal devices can be

used to separate the components of the skull after surgery (Cohen et al., 2008).

Ultimately, these invasive procedures are designed to increase intracranial

volume and to prevent possible complications associated with elevated

intracranial pressure. As a result, surgical correction of craniosynostosis should

be performed in infancy, within the first six months of life to maximize therapeutic

outcomes at an early stage of development (Panchal and Uttchin, 2003).

However, surgical treatment of craniofacial synostosis remains an invasive

procedure with commensurate risks, including infection, optic nerve ischemia,

seizures, and bleeding (Whitaker et al., 1979). Some 80-100% of patients who

undergo surgical correction of craniosynostosis need blood transfusions (Meara

et al., 2005). In addition, more than 6% of infants require a second operation to

separate the bones again, and 25% of those require third surgery (Panchal and

Uttchin, 2003). For these reasons, the development of minimally invasive,

biologically-based and targeted therapies offer an attractive option to be pursued

for the treatment of craniofacial synostoses (Wan et al., 2008).

109

The study of human sutural material obtained after surgeries is not sufficient for

experimentation to understand the etiology of craniofacial synostoses, but use of

mouse models of craniofacial synostoses obviates this concern. In mice, all

sutures, with the exception of the posterior frontal suture, remain patent

throughout life (Holmes, 2012). Mouse and human genetics revealed that

interactions between growth factors, their receptors, and transcription factors all

play integral roles in craniofacial suture biology. At least 150 syndromes have

been identified with specific mutations resulting in craniosynostosis, and most of

the common ones exhibit dominant inheritance (Cohen, 2005; Wan et al., 2008;

Wilkie, 1997). Many of the syndromes, such as Apert, Pfeiffer, and Crouzon

syndromes, can be traced to gain-of-function mutations in FGFRs (Wilkie, 1997).

TGFβ signaling pathway also has a well-established role in bone biology and

regulation of suture patency (Rawlins and Opperman, 2008). However, unlike the

FGF signaling, TGFβ family members have not been closely associated with

craniosynostosis in humans (Senarath-Yapa et al., 2012). Other less frequent

mutations linked to this condition are found in TWIST, MSX2, EFNB1, RAB23,

GLI3, TCF12, ERF and other genes (el Ghouzzi et al., 1997; Howard et al., 1997;

Jabs et al., 1993; Jenkins et al., 2007; Sharma et al., 2013; Twigg et al., 2004;

Twigg et al., 2013; Vortkamp et al., 1992; Wieland et al., 2005).

The goal of the biologically-based targeted treatment of craniofacial synostosis

would be to suppress the expression of genes promoting sutural fusion or to

increase the expression of those which support sutural patency. Gene therapy

holds great promise for development of such treatment for craniofacial synostosis

(Wan et al., 2008). Several attempts have been made to utilize gene therapy in

mice. Gene transfer of bone morphogenetic proteins-2 and -9 using adenoviral

vectors demonstrated promising effect in the promotion of osteogenesis,

specifically for craniofacial bone repair in rats (Alden et al., 2000; Lindsey, 2001).

Local adenoviral delivery of noggin, a TGF antagonist, to the posterior frontal

suture in mice prevented normal sutural fusion and resulted in broad snouts and

widely-spaced eyes (Warren et al., 2003). Similar experiments were performed

110

with adenoviral delivery of the dominant-negative forms of the TGF-β receptor II

and Fgfr1 to prevent fusion of the posterior frontal suture in organ culture

(Greenwald et al., 2001; Mehrara et al., 2002; Song et al., 2004). Conversely,

adenoviral infection of the normally patent coronal suture with an Fgf2 expression

vector resulted in pathologic suture fusion (Greenwald et al., 2001). It is

important to note that viral vectors suffer from several drawbacks, such as

toxicity, imunnogenicity, and the lack of cell-specific targeting. Therefore, delivery

of gene products using non-viral techniques would be desirable in humans. Such

approaches may involve direct injection of DNA, liposomes, electroporation,

particle bombardment, and nanoparticles (Schmidt-Wolf and Schmidt-Wolf,

2003).

Prevention of craniosynostosis in mouse models was also demonstrated using

pharmacologic strategies. Mouse calvaria obtained from the Crouzon-like

Fgfr2C342Y/+ mouse model cultured in the presence of FGFR tyrosine kinase

inhibitors (PD173074 or PLX052) exhibited coronal suture patency, as opposed

to the premature suture fusion seen in untreated mutants (Eswarakumar et al.,

2006; Perlyn et al., 2006). An in vitro calvarial culture model of the Apert mouse

harboring the Fgfr2 P253R mutation showed that treatment with MEK1 inhibitor

PD98059 partially alleviated fusion of the coronal suture (Yin et al., 2008).

Furthermore, treatment of the Apert Fgfr2S252W/+ mice with U0126, an inhibitor of

MEK1/2, during pregnancy and early postnatal development significantly

inhibited development of craniosynostosis in vivo (Shukla et al., 2007). These

studies suggest that small-molecule inhibitors can be used for treatment of

specific gain-of-function mutations observed in syndromic craniofacial synostosis

(Melville et al., 2010).

Several other strategies were used to modulate protein levels involved in

aberrant FGFR or TGFβ signaling. Infection of the posterior frontal suture of fetal

rats by introduction of the dominant-negative Fgfr1 construct in utero abrogated

overactive FGFR signaling and prevented suture closure (Greenwald et al.,

111

2001). shRNA targeting a mutant form of Fgfr2 was shown to prevent an Apert-

like syndrome in mice (Shukla et al., 2007). Delivery of neutralizing antibodies to

TGFβ3 resulted in synostosis of coronal suture, whereas antibodies targeted to

TGFβ2 prevented sutural closure (Opperman et al., 1999). Overall, these

strategies further demonstrate that targeted treatment may be promising for

prevention of the craniofacial synostosis.

As our understanding of the molecular and genetic basis of craniofacial

synostosis improves, the prospect of genetic and pharmacological therapy for the

treatment and prevention of premature suture fusion becomes more tangible.

However, such targeted treatment has yet to overcome significant obstacles,

such as development of targeted methods of delivery (including in utero), as well

as establishment of proper dosage and timing. In addition, more investigations

are still required to determine which protein or group of proteins would serve as

the optimal target in each case. Studies presented here demonstrate that

BCL11B plays an important role in maintaining of sutural patency in mice.

Abrogation of BCL11B function leads to accelerated mineralization of the skull

during development and subsequent facial and coronal synostoses that are

already evident at birth. Although BCL11B has not been previously associated

with the craniosynostosis in humans, the data presented here will be useful in

defining alternative therapeutic approaches to maintain sutural patency in patiens

with this condition.

112

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