Vertebrate Limb Development - University of Minnesota Duluth
Transcript of Vertebrate Limb Development - University of Minnesota Duluth
Tetrapod Limb Development
Biology 4361 – Developmental Biology
July 29, 2009
Tetrapod Limbs
© Vicki Lockard and Paul Barry
© Father Alejandro Sanchez
© Merlin D. Tuttle
© Anne Fischer
Limb Development - Overview
Patterning
Early development
Specification
Establishing limb axes
Morphogenic models
Cross-talk / regulation
Francesca V. Mariani and Gail R. Martin 2003 Nature 423:319-325 doi:10.1038/nature01655
Limb Patterning
Humerus
Radius
Ulna
Carpals Metacarpals
Phalanges
Human
arm
Chicken wing
Chicken
leg
Shoulder Finger
Pinkie
Thumb
Knuckle
Palm
Proximal Distal
Posterior
AnteriorDorsal
Ventral
Limb Field
Limb Bud
Formation
Vertebrate Limb Buds
Limb bud
So
mite
s
Ectoderm
Mesodermal
mesenchymeLateral
plate
mesoderm
Hox expression
determines limb
bud locationHox5
Hox6
Hoxc6 Specification of Limb Buds
Forelimb initiation:
- anterior-most point of
Hoxc6 expression
Tbx Genes Specify Limb Type
Tbx expression initiated by Wnts, FGFs
NOTE – Tbx genes are not the first step in forelimb/hindlimb specification
(initial step(s) unknown)
Fgf/Wnt - Limb Bud Initiation
Limb Bud Axes
Distal
Proximal
Anterior
Posterior
Dorsal
Ventral
Apical Ectodermal Ridge
Apical Ectodermal
Ridge (AER)
Fgf10 initiates AER via Wnt3a, β-catenin
AER expresses Fgf8, Fgf4; maintains Fgf10 expression
Proximal-Distal Axis
Apical Ectodermal Ridge (AER) forms at boundary
between dorsal and ventral ectoderm
Fgf8
lateral
plate
mesoderm
Fgf10Fgf10
Lateral plate mesoderm expresses Fgf10
Apical Ectodermal Ridge Manipulation
Extent of development depends
on time of AER removal
Distal structures
are duplicated;
- note structure
Degree of “legness” of wing
depends on placement of
leg mesenchyme
Progress Zone
Progress Zone – mesodermal mesenchyme; receives AER signals:
- promotes proliferation (mitosis)
- prevents differentiation into cartilage
- maintains expression of A/P and D/V-related signals
Progress Zone
(PZ)
Apical Ectodermal
Ridge (AER)
Fgf8
Fgf4
PZFgf8
Fgf10
PZ mesenchyme specifies proximal-distal axis
- transplantation experiments demonstrated that positional
information was carried by PZ cells
- PZs conveyed age-appropriate specification instructions
AER establishes
Progress Zone
~200 μm
Proximal-Distal Specification ModelsProgress zone model: Identity established by residence time in PZ
Pro
xim
al
Dis
tal
Early allocation and progenitor expansion: Elements specified early
Specifying mechanism - ??
Anterior-Posterior Specification
Zone of Polarizing
Activity (ZPA)
Apical Ectodermal
Ridge (AER)
Progress Zone (PZ)
Morphogen
Shh
Shh necessary and sufficient for establishing ZPA
(NOTE – Shh not necessary for polarity of styolpod)
Shh induced by dHAND and Hoxb8
ZPA maintained by feedback loop with AER
ZPA/AER Feedback Loop Model
1. dHAND - bHLH transcription factor
and Fgf8 from AER stimulate Shh
- Fgf8 (and Fgf4) maintains Shh
expression
2. Shh up-regulates Gremlin1 in
posterior mesenchyme
- Grem1 antagonizes BMP ligands
(BMPs repress Fgf expression in AER)
3. Wnt7a maintains Shh
Wnt7a determines the size of AER
Loss-of-function mutants (both Shh and Grem1) = syndactyly, loss of digits
ZPA Morphogen Gradient
ZPA
Shh gradient
12
3
4
5
[Morphogen]
High
Low
Posterior Anterior
5
4
32
ZPA Transplantation
Mirror-image duplication effects can be replicated by transplanting Shh bead
Posterior tissue transplant to anterior = duplicated autopod
Retinoic acid operates upstream of Shh
- implant RA-soaked bead =mirror-image duplication
- possible Hox gene involvement
C. Tickle, Nature Molecular Biology 7(2006)45-53
2
34
43
2
“new” posterior
Drosophila Hedgehog PathwayShh
(vertebrates)
/ Gli1, 2, 3
(vertebrates)
/ Gli1, 2, 3
(vertebrates)
Shh/Gli Interactions
Gli3 – proteolytic fragment – acts as a transcriptional repressor (Gli3R)
- represses e.g. dHAND, Gremlin, Fgf4, Hoxd13
Without Shh:
With Shh:
Gli’s retained in long form – acts as a transcriptional activator (Gli3A)
- e.g. Gli1 activates Shh
ZPA
Shh gradient
Gli3A
Gli3R
Shh/Gli Interactions
C. Tickle, Nature Molecular Biology 7(2006)45-53
Shh main function may be to relieve Gli3R repression in posterior region
Anterior – high Gli3R
Posterior – low Gli3R/ high Gli3A
Shh-/- = 1 digit;
- Gli3R prevails
Gli3-/- = polydactyly
- Shh prevails
~ 8 digits
- unpatterned
Shh Specifies Digit Identity
digit 4 progenitor cells
digit 5 precursors
(paracrine)
(autocrine & paracrine)
(autocrine)
BMPs Regulate Digital IdentityShh initiates BMP2 and BMP7 gradients
- BMPs in interdigital mesoderm specifies identity of digits anteriorly
Noggin – BMP
antagonist
Insert BMP antagonist
into interdigital webbing
NOTE – Fgfs from AER control phalange development;
Shh bead inserted between digits can add phalange; Shh
sustains Fgf signal; Fgf inhibitor = lack of phalange
Remove interdigital
mesoderm
- BMP targets unknown
NOTE – BMP effects probably only on tissue “primed” by Shh
Hox Genes in Early Limb Bud
5’ Hox Genes Pattern Limb ElementsForelimb
Hindlimb
Dorsal-Ventral Specification
Zone of
Polarizing
Activity
(ZPA)
Apical Ectodermal
Ridge (AER)
Progress Zone (PZ)
Ectoderm
Wnt7a – necessary and sufficient to dorsalize limb bud
- induces Lmx1 in dorsal mesenchyme
- Lmx1 knockouts = ventralized phenotype
- Wnt7a knockouts = ventral footpads on both surfaces
Dorsal
Ventral
Apoptosis in Limb Primordia
BMP signals