The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

Karl R. Wotton§, Kathryn E.M. French, Sebastian M. Shimeld

Department of Zoology, University of Oxford, The Tinbergen Building, South Parks

Road, Oxford, OX1 3PS

§Corresponding author

Email addresses:

KRW: karl.wotton@balliol.oxford.ac.uk

Keywords: FoxL2; forkhead box; BPES; POF; dogfish; catshark; Scyliorhinus canicula; gill buds; Rathke's pouch; pituitary; mandibular arch; mandibular mesoderm; head mesoderm; eye; eyelid; development; evolution

* Manuscript

Abstract

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is

mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome

(BPES), which is characterized by eyelid malformations, and Premature Ovarian

Failure (POF). In the mouse expression is seen in the perioptic mesenchyme,

developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the

dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing

the characterisation of this gene’s sequence and expression from a lineage that

diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis

strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily.

We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular

head mesoderm and later in continuous populations of mandibular arch cells and

mandibular head mesenchyme cells around the developing pituitary. As development

proceeds expression decreases in the mesenchyme of the head but is seen in the

mesenchyme around the eye and later in the developing eyelids. Additionally

expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from

which gill buds will form. This expression is maintained in the developing and

elongating gill buds. Thus, S.canicula foxl2 is a marker for the mandibular mesoderm

and gill buds and its expression is conserved in the perioptic mesenchyme, developing

eyelids and pituitary.

1. Results and discussion

The Fox genes are a family of transcription factors characterised by a 110 amino acid

DNA binding domain. They are involved in a wide range of biological processes in

the adult and during development and have been implicated in a number of diseases

(Carlsson and Mahlapuu, 2002). FOXL2 is mutated in the disorder Blepharophimosis,

Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid

malformations, and Premature Ovarian Failure (POF) (Crisponi et al., 2001).

Consistent with this phenotype, mouse Foxl2 expression is seen in the perioptic

mesenchyme, developing eyelids and ovary, and also in the pituitary (Crisponi et al.,

2001; Treier et al., 1998). Foxl2 is one of the earliest markers of ovary development

and its expression in this tissue has been reported from the mouse, chicken, goat,

turtle, and the teleost fishes medaka (Oryzias latipes), rainbow trout (Oncorhynchus

mykiss) and the Nile tilapia (Oreochromis niloticus) (Baron et al., 2004; Crisponi et

al., 2001; Loffler et al., 2003; Nakamoto et al., 2006; Pailhoux et al., 2001; Wang et

al., 2004). Additionally in the Nile tilapia RT-PCR has revealed expression in the

brain, pituitary, and gills (Wang et al., 2004). Sequence analysis has shown high

conservation in mammals, including a polyalanine tract, the expansion of which is

associated with eyelid malformations without POF (type II BPES). Analysis of non-

mammalian foxl2 sequences has shown an absence of a polyalanine tract (Cocquet et

al., 2002; Wang et al., 2004). Amongst the invertebrates, foxl2 genes have been

identified from various genomes (Mazet et al., 2003) but have only been studied in

the sponge, Suberites domuncula (Adell and Muller, 2004)

1.1 Isolation of S. canicula foxl2 and sequence analysis

Foxl2 appears in a single copy gene in the genomes of mammals, Gallus gallus and

Xenopus tropicalis while a second diverged duplicate has been identified in the

genomes of some teleosts (Baron et al., 2004). The S. canicula foxl2 cDNA is 1726bp

long and encodes a 198 amino acid protein (Fig.1). It shows the highest protein

sequence identity to X. tropicalis foxl2, 77% over the whole length and 96% in the

forkhead domain. Like sequences from other non-mammalian species the polyalanine

tract is absent, however a polyalanine tract was found in foxl2 from the Opossum

(Monodelphis domestica) sugesting this feature arose in the mammals before the

seperation of the placental and marsupial lineages. Phylogenetic analysis places S.

canicula foxl2 with the vertebrate FoxL2 sequences with a high bootstrap value.

1.2 Developmental expression of S.canicula foxl2

S. canicula embryos were staged according to Ballard et al. (1993) and the

developmental expression of foxl2 investigated between stages 17 and 31. S. canicula

foxl2 expression is detected at stage 17 (Ballard et al., 1993) in the mandibular head

mesoderm (Fig.2A). Sectioning shows expression is restricted to the medial wall of

the head cavity (Fig.2B). As development proceeds through stages 18 and 19

expression is seen in the mandibular mesenchyme of the first pharyngeal arch and

under the mesencephalon (Fig.2C). Serial sections through the head show expression

in the mesenchyme around Rathke's pouch (Fig.2F), the mandibular head cavities

(Fig.2F) and in the aortic blood vessels of the mandibular arches (Fig.2G). At stage

21 expression is still seen in the mesenchyme under the mesencephalon and can be

detected here up to stage 24 (Fig.2H, I, J, L). Also at stage 21 a new site of expression

is seen in the ectoderm of the hyoid and 3rd pharyngeal arches at sites from which the

first gill buds will form (Fig.2H). Sections of a stage 22 embryo shows expression in

the ectoderm of the hyoid and 3rd pharyngeal arch, and also in the mesodermal cores

of each arch (Fig.2J, K). At this stage weak expression is also seen in the hinge of the

mandibular arch and this maintained up to stage 27 (Fig.2J, L, M). Stages 21 onwards

sees the expression of foxl2 in the prospective gill bud tissue of each arch, including

those in the spiracular clefts (Fig.2M) and the mature gill filaments (Fig.2N, O, P). At

stage 29 expression is seen in the mesenchyme around the eyes (Fig.2O, Q) and at

stages 30 and 31 this expression restricts to the underlying mesenchyme at the outer

edges of the developing eyelids (Fig.2R, S, T).

In summary expression of S.canicula foxl2 marks the mandibular mesoderm, the

mesodermal cores of the pharyngeal arches, the gill buds, the perioptic mesenchyme

and the developing eyelids. Expression in the gill buds and mesodermal cores of the

pharyngeal arches may be specific to the chondrichthian lineage however the gills

have been identified as a site of expression by RT-PCR in the teleosts (Wang et al.,

2004). The expression of foxl2 in the perioptic mesenchyme, eyelids and around the

developing pituitary in mouse and S. canicula suggest these are conserved sites of

gene expression in the gnathostomes (Crisponi et al., 2001; Treier et al., 1998).

Despite this we note the apparent absence of eyelids from most fish. Finally foxl2 has

been reported as an early marker of ovary development in a range of tetrapods and

teleosts, however we did not detect expression in the stages examined (Baron et al.,

2004; Loffler et al., 2003; Nakamoto et al., 2006; Pailhoux et al., 2001; Wang et al.,

2004).

2. Experimental procedures

2.1 Obtaining S. canicula embryos

Eggs were collected from seaweed gardens at low tide from beaches in the Menai

Strait in Wales, UK. The eggs were kept in saltwater tanks and allowed to develop. At

selected developmental stages, see (Ballard et al., 1993), they were fixed in 4%

MOPS buffered paraformaldehyde at 4oC overnight then dehydrated into 100%

methanol and stored at -20oC.

2.2 cDNA library screening

An S. canicula cDNA library in the vector lambdaZap Express was screened with an

S. canicula foxc1 gene fragment using Roche’s DIG nucleic acid detection kit. One of

the recovered cDNAs contained a forkhead domain with high similarity to the FoxL2

subfamily and was fully sequenced.

2.3 Phylogenetic analysis

Protein sequences were collected from the NCBI or the JGI websites (see Fig.1 for

accession numbers). They were aligned and trimmed using BioEdit and ClustalX

(Hall, 1999; Thompson et al., 1997). Phylogenetic analyses were carried out using

maximum likelihood implemented by ClustalX, and by PHYML (Guindon and

Gascuel, 2003; Guindon et al., 2005; Thompson et al., 1997). Substitution models

were predicted with ProtTest (Abascal et al., 2005).

2.4 In situ hybridisation

In situ hybridisation of S. canicula embryos is based on a chick in situ protocol (Nieto

et al., 1996) with modifications described in (Freitas and Cohn, 2004). The protocol

was carried out using a riboprobe derived from the whole cDNA and made using the

Roche DIG RNA labelling kit.

2.5 Histology

Tissue was embedded in gelatin/albumin (0.45% gelatin, 25% albumin, 20% sucrose

in PBS) and fixed with 2.5% glutaraldehyde. The embedded embryos were then

sectioned at 50µm using a vibratome.

3. Acknowledgements

We thank Dr. J. Begbie for allowing us to use her sectioning facilities and Dr. M.

Cohn for the S. canicula cDNA library. This work was supported by the BBSRC.

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Figure Legends

Figure 1. Sequence and phylogeny of FoxL2 proteins. (A) Sequence alignment of

FoxL2 proteins. The forkhead domain and polyalanine tracts are shown and

percentage identity values to the S. canicula sequence are indicated. (B) Phylogenetic

tree of FoxL2 subfamily proteins constructed using 106 amino acids of the forkhead

domain. We chose foxL1 and FoxI sequences as outgroups as they often associate

with the FoxL2 branch in analyses including all Fox classes (Kaestner et al., 2000;

Mazet et al., 2003). S. canicula foxl2 groups within the vertebrate FoxL2 sequences

with high bootstrap support. Accession numbers are shown and can be retrieved from

the following genome websites: NCBI http://www.ncbi.nlm.nih.gov/, JGI

http://www.jgi.doe.gov/ or Ensembl http://www.ensembl.org/index.html (indicated by

the bracketed abbreviation En).

Figure 2. Developmental expression of S. canicula foxl2. Stage numbers (Ballard et

al., 1993) are shown in the bottom right corner, arrows indicate planes of section. (A)

Stage 17 expression in the mandibular head mesoderm (mm). (B) Section of embryo

in (A) showing expression in the mandibular mesoderm (mm) and the mandibular

head cavity (mhc). (C) Stage 19 expression in the mandibular arch (ma) and

mandibular head mesenchyme (mm). (D, E, F, G) Serial sections of embryo in (C),

(D, E) show expression in the mandibular head mesoderm (mm) (F) shows expression

in the mesenchyme around the head cavities (mhc) and Rathke's pouch (rp) and (G)

shows expression in the mandibular arch blood vessels (aa). (H) Stage 21 expression

in the future gill bud forming regions of the hyoid arch (ha) and arch 3 (a3). (I)

Sagittal section of embryo in (H) showing expression in the head mesenchyme

relative to the developing brain. (J) Stage 22 expression in the gill bud forming

regions of the hyoid arch and arch 3. (K) Section of embryo in (J) showing expression

can be seen in the mesodermal cores of the pharyngeal arches (mc) and the ectoderm

(ec) of the gill bud forming regions. (L) Stage 24 embryo showing expression in arch

2 and 3 gill buds (gb) and arch 4 and 5 gill buds forming regions. Expression is also

seen in mandibular arch hinge region (h). (M) Stage 27 embryo showing expression in

the mandibular hinge region (h) and gill buds (gb) on arches 2-5 and the early forming

gill buds of the mandibular arch in the spiracular cleft (sgb). Expression is also seen in

the gill buds forming region of arch 6. (N) Stage 28 expression in the lengthening gill

buds and gill filaments (gf). (O) Stage 29 expression in the gill filaments and in the

mesenchyme around the eye (pom). (P, Q) Sections of embryo in (N) showing

expression in the gill filaments and the perioptic mesenchyme. (R, S, T) Expression in

the developing eyelid (el) at stages 30 and 31. (S) Section through the eye of the

embryo in (R), note that the lens has been removed. Labels: (aa) aortic arch, (bg)

buccal groove, (c1) hyomandibular or spiracular cleft, (c2) pharyngeal cleft 2, (di)

diencephalon, (ec) ectoderm, (el) eyelid, (en) endoderm, (gb) gill buds, (gf) gill

filaments, (h) hinge of mandibular arch, (ha) hyoid arch, (in) infundibulum, (lp) lens

placode, (ma) mandibular arch, (mc) mesodermal core of pharyngeal arch, (me)

mesencephalon, (mhc) mandibular head cavity, (mm) mandibular mesoderm, (mt)

metencephalon, (my) myelencephalon, (olf) olfactory placode, (ot) otic placode, (ov)

optic vesicle, (p1-p5) pharyngeal pouches, (ph) pharynx, (rp) Rathke's pouch (sgb)

spiracular cleft gill bud, (te) telencephalon.

A

B

Gallus gallus L2 (NCBI) NP_001012630.1

Xenopus tropicalis L2 (JGI) 319742

Monodelphis domestica L2 (En) ENSMODP00000023199

Homo sapiens L2 (NCBI) AAY21823

Danio rerio L2 (NCBI) AAI16586.1

Scyliorhinus canicula L2 (NCBI) ABP63571

Branchiostoma floridae L2 (JGI) 68667

Suberites domuncula L2 (NCBI) ABB89483.1

Mus musculus I1 (NCBI) Q922I5

Mus musculus L1 (NCBI) NP_032050.2

76

66

69

82

98

78

0.05

Forkhead domain

Poly-Ala tract

Sequence Identities: 56%60%74%77%73%100%

10 20 30 40 50 60 70. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Homo sapiens M M A S Y P E P E D A A GA L L A P E - - T G R T V K - - E P E G P - - - - - P P S P G K GG G GG G GT A P E K P D P A Q K P P Y S Y V AM onodelphis domesti ca M M A S Y P E P E N D S GA L L A P E S A T G R S G K D S E P R G P R D C K E E L S P E K S G G GG G - - V P E K P D P S Q K P P Y S Y V AGall us gal lus M M S GY A D G E E D A V A M L A H D G GG S K E P E R GK E E L S - - - - - - - - A E K G - - - - - - - - P E K P D P S Q K P P Y S Y V AX enopus t ropica lis - M A S F Q S P E QG T V A L M T H N S N G N K E A E R S K E D L L - - - - - - - - P E K G - - - - - - - - Q E K P D P S Q K P P Y S Y V ADanio r eri o M M A T Y P GH E D N GM I L M - D T T S S S A E K D R T K D E A P - - - - - - - - P E K G - - - - - - - - P D K S D P T Q K P P Y S Y V AScyl ior hinus ca nicula - M A T Y Q H P E D D T L V L M T D N I S T P S A K D R V K S E S S - - - - - - - - P D K T - - - - - - - - A E K P D P T Q K P P Y S Y V A

80 90 100 110 120 130 140. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Homo sapiens L I A M A I R E S A E K R L T L S G I Y QY I I A K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L DM onodelphis domesti ca L I A M A I R E S A E K R L T L S G I Y QY I I G K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L DGall us gal lus L I A M A I R E S A E K R L T L S G I Y QY I I S K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L DX enopus t ropica lis L I A M A I R E S A E K R L T L S A I Y QY I I S K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L DDanio r eri o L I A M A I R E S S E K R L T L S G I Y QY I I S K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L DScyl ior hinus ca nicula L I A M A I R E S P E K R L T L S G I Y QY I I T K F P F Y E K N K K GW QN S I R H N L S L N E C F I K V P R E G G G E R K G N Y W T L D

150 160 170 180 190 200 210. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Homo sapiens P A C E D M F E K GN Y R R R R R M K R P F R P P P A H F Q P GK G - L F GA G GA A G G C G V A G A GA D G Y GY L A P P K Y L QS G F LM onodelphis domesti ca P A C E D M F E K GN Y R R R R R M K R P F R P P P A H F Q P GK G - L F GP G GA GA G G - - - A G GS D G Y GY L A P P K Y L QS G F LGall us gal lus P A C E D M F E K GN Y R R R R R M K R P F R P P P T H F Q P GK S - L F GP - - - - - - - - - - - - - - D G Y GY L S P P K Y L QS T FMX enopus t ropica lis P A C E D M F E K GN Y R R R R R M K R P F R P P P T H F Q A GK S - L F S S - - - - - - - - - - - - - - D T Y GY L S P P K Y L QS T FMDanio r eri o P A C E D M F E K GN Y R R R R R M K R P F R P P P T H F Q P GK S - L F GG - - - - - - - - - - - - - - E G Y GY L S P P K Y L QS G F IScyl ior hinus ca nicula P A C E D M F E K GN Y R R R R R M K R P F R P P P T H F Q H S K A A L F A S - - - - - - - - - - - - - - D S Y GY I GP P K Y L QS T FM

220 230 240 250 260 270 280. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Homo sapiens N N S WP L P Q P P S P M P Y A S C QM A A A A A A A A A A A A A A A A A A A A A A A A A GP G S P G A A A V V K G L A G P A A S Y G P Y TM onodelphis domesti ca N N S WP L T Q P P S P M P Y A S C QM A A A A A A A A A A A A - - - - - - - - - A A A A GP G S P G G - - - V K G I P G A P A S Y S P Y SGall us gal lus N N S WP L P Q P P A P V P Y A S C QM S G - - - - - - - - - - - - - - - - - - - - - - - GS V S P V N - - - V K G L S - G P A S Y G P Y SX enopus t ropica lis N N S WP L A Q P P A P M S Y T S C QM A G - - - - - - - - - - - - - - - - - - - - - - - GN V S P V N - - - V K G L S - A S S S Y S P Y SDanio r eri o N N S WS - - - - P A P M S Y T S C Q V S S - - - - - - - - - - - - - - - - - - - - - - - GS V S P V N - - - M K G L S - A P S S Y N P Y SScyl ior hinus ca nicula N N S WP L GQ P P A P M A Y A S C QM A A - - - - - - - - - - - - - - - - - - - - - - - GN V GS V N - - - V K G L S - G H A S Y N P Y T

290 300 310 320 330 340 350. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Homo sapiens R V Q S M A L P P GV V N S Y N G L G G P P A A P P P P P H P H P H P H A H H L H A A A A P P P A P P H H GA A A P P P G Q L S P A S P A TM onodelphis domesti ca R V Q S M A L P - GV V N S Y N GM A G H H P A P H P H P H A H - H P H A H H L H P A A A A A H H P P H H P A A A P P - A Q L S P A S P T AGall us gal lus R V Q S V A L P - GM V N S Y N G V A - - - - - - - - - - - - - - - - H P H H P H - - - - - - - - - - - - - - - A H H P Q Q L G P A S - - PX enopus t ropica lis R V Q S M S L P - S M V N S Y N GM S - - - - - - - - - - - - - - - - H H H H P H - - - - - - - - - - - - - - - A H H A Q Q L S P A S - - PDanio r eri o R V Q S I G L P - S M V N S Y N G I S - - - - - - - - - - - - - - - - H H H H H H H T H P H A - - - - - - - - - L P H A Q Q L S P A T - - AScyl ior hinus ca nicula R V Q S M A L P - S M V N S Y N GM S - - - - - - - - - - - - - - - - H H H H - - - - - - - - - - - - - - - - - - P H P Q Q L S P - - - - -

360 370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

Homo sapiens A A P P A P A P T S A - - - - - - P G L Q F A C A R QP - E L AM M H C S Y W D H D S K T GA L H S R L D LM onodelphis domesti ca A A P P A P A P A QA N A T G P P P G L Q F A C A R QP - D L S M M H C S Y W D H D S K H S A L H S R I D IGall us gal lus A P P A A P A A N GA - - - - - - - G L Q F A C A R QP A E L S V M H C S Y W E H D S K H GA L H S R I D IX enopus t ropica lis A P A P P A P P N - - - - - - - - - G V Q F T C A R QP S D L S M M H C S Y W D H D A K H S A L H P R I D LDanio r eri o A A P P V T T G N GT - - - - - - - G L Q F A C S R QP A E L S M M H C S Y W D H E S K H S A L H A R I D IScyl ior hinus ca nicula - A P P P S S S N GA - - - - - - - G L Q F P C A R QP A D L S M M H C S Y W D H D S K H S A L H T R I D I

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