Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development

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Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development Eugene Curtin a,b,1, Graham Hickey Eric C. Liao a,b,* a b

a,b,1

, George Kamel

a,b

, Alan J. Davidson b,

Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA

A R T I C L E I N F O

A B S T R A C T

Article history:

Wnt activity is critical in craniofacial morphogenesis. Dysregulation of Wnt/b-catenin sig-

Received 14 April 2010

naling results in significant alterations in the facial form, and has been implicated in cleft

Received in revised form

palate phenotypes in mouse and man. In zebrafish, we show that wnt9a is expressed in the

6 November 2010

pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and

Accepted 11 November 2010

ectodermal cells superficial to the lower jaw structures. Alcian blue staining of morpho-

Available online 18 November 2010

lino-mediated knockdown of wnt9a results in loss of the ethmoid plate, absence of lateral and posterior parachordals, and significant abrogation of the lower jaw structures. Analysis

Keywords: Wnt

of cranial neural crest cells in the sox10:eGFP transgenic demonstrates that the wnt9a is required early during pharyngeal development, and confirms that the absence of Alcian blue staining is due to absence of neural crest derived chondrocytes. Molecular analysis

wnt9a

of genes regulating cranial neural crest migration and chondrogenic differentiation suggest

wnt9b

that wnt9a is dispensable for early cranial neural crest migration, but is required for chon-

Cleft

drogenic development of major craniofacial structures. Taken together, these data corrob-

Lip

orate the central role for Wnt signaling in vertebrate craniofacial development, and reveal

Palate Craniofacial Neural crest

that wnt9a provides the signal from the pharyngeal epithelium to support craniofacial chondrogenic morphogenesis in zebrafish.

Vertebrate

 2010 Elsevier Ireland Ltd. All rights reserved.

Zebrafish

1.

Introduction

Neural crest cell migration and morphogenesis to form the facial skeleton involves intricate spatial and temporal choreography of molecular cues. When this process is perturbed during embryogenesis, congenital craniofacial phenotypes result. Craniofacial anomalies constitute a significant fraction of human birth defects, where clefts of the lip and palate (CLP) are among the most common congenital malforma-

tions, affecting about 1/700 births. Through complementary studies in human and mouse genetics, an increasing number of genetic mutations have been implicated in CLP malformation (Juriloff and Harris, 2008; Kimmel et al., 2001; Koillinen et al., 2005; Murray, 2002). The palate serves to separate the nasal and oral cavities of amniotes, such as mouse and humans. Genes that govern palatogenesis are also conserved across vertebrates, such as sonic hedgehog (Shh), fibroblast growth factor 8 (Fgf8),

* Corresponding author. Address: Center for Regenerative Medicine, Massachusetts General Hospital, CPZN 4256, 185 Cambridge Street, Boston, MA 02114, USA. Tel.: +1 617 643 5975; fax: +1 617 643 5963. E-mail address: [email protected] (E.C. Liao). 1 These authors contributed equally to this work. 0925-4773/$ - see front matter  2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2010.11.003

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transcription factor AP-2 (Tfap2), and plate-derived growth factor receptor alpha (Pdgfra) (Juriloff and Harris, 2008; Roessler et al., 1996; Shi et al., 2009; Soriano, 1997; Tallquist and Soriano, 2003). Although zebrafish does not form a palate that excludes the oral cavity from the nasal passage, detailed fate mapping of neural crest cells have demonstrated that the ethmoid plate is analogous to the amniote palate (Eberhart et al., 2006; Wada et al., 2005). In vertebrates, the most anterior neural crest cells migrate between the developing eyes to form the anterior neurocranium and the primary palate (Hilliard et al., 2005; Kontges and Lumsden, 1996). In zebrafish, neural crest cells anterior of the optic vesicle form the median ethmoid plate, whereas the neural crest cells posterior of the optic vesicle form the trabeculae that span either side of the median ethmoid (Wada et al., 2005). Molecular and cell fate mapping studies have identified the discrete cranial neural crest cell populations that contribute to the trabeculae versus the median ethmoid plate, and have shown that signals such as shh from the stomodeum epithelium is important for the development of the median ethmoid plate (Eberhart et al., 2006; Wada et al., 2005). Wnt activity is critical in facial morphogenesis, as demonstrated in the Lef1//Tcf4/ mouse embryos, where failure of Wnt/b-catenin signal resulted in dramatic transformation of the murine upper lip contour, where the typical infranasal depression become more broad, as seen in the human upper lip contour (Brugmann et al., 2007). In the mouse, Wnt9b is involved in fusion of the medial and lateral nasal palatal shelves (Juriloff et al., 2006; Lan et al., 2006). The Wnt9b gene is expressed along the leading edge of palatal shelf epithelium and is mutated in the clf1 mouse, resulting in cleft palate. Variation of Wnt genes is associated with nonsyndromic CLP in human and mouse studies (Chiquet et al., 2008; Juriloff et al., 2006). The gene expression of zebrafish wnt9b has been examined but limited signal was detected in the first and second pharyngeal arches (Jezewski et al., 2008). Study of vertebrate gene synteny identified conservation of wnt9a across human, mouse, Xenopus and zebrafish genomes (Garriock et al., 2007). Mouse Wnt9a is expressed in joint cartilage progenitor cells and appears to function specifically in synovial chondrogenesis, whereas palatogenesis appears unaffected in the wnt9a/ embryos (Koyama et al., 2008). Zebrafish wnt9a gene expression was recently described in the gut, pronephros and neural tissues such as the optic tract (Cox et al., 2010). However, the functional role of wnt9a during zebrafish emrbyogenesis has not been reported. There are several examples of species-specific segregation or redundancy of function among orthologous genes of related family members across vertebrates. For example, Tfap2 family genes have variations of function in frogs, mice and zebrafish. Tfap2a is required for specification of neural crest in frogs but appears to be dispensable in zebrafish and mice (Barrallo-Gimeno et al., 2004; Knight et al., 2003; Schorle et al., 1996; Zhang et al., 1996). In zebrafish, redundant activities of Tfap2a and Tfap2c are necessary for neural crest induction. A similar situation is observed for the Msx gene family. Targeted disruption of Msx1 leads to a cleft palate phenotype in the mouse, whereas simultaneous knockdowns of msxB, msxC and msxE are necessary to appreciably disrupt ethmoid

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plate development in zebrafish (Satokata and Maas, 1994; Phillips et al., 2006). Analysis of wnt9a and wnt9b transcripts during zebrafish fin development suggested that segmentation function of wnt9a has been co-opted by wnt9b during teleost evolution (Crotwell and Mabee, 2007). Therefore, we evaluated both wnt9a and wnt9b to determine whether one or both of these related genes participate in zebrafish palatal development. Here we present expression and functional analysis of wnt9a in zebrafish. Consistent with earlier observation, wnt9a expression was found in the developing pronephros and optic tract (Cox et al., 2010). However, we also show that wnt9a is expressed in the pharyngeal arches, oral epithelium, and epithelium associated with craniofacial skeletal structures. Knockdown of wnt9a, but not wnt9b, disrupts craniofacial development. Cranial neural crest cells are formed in the wnt9a morphant but are segregated laterally, and the ethmoid plate and lower jaw structures fail to form. These results corroborate a central role for wnt9a in zebrafish craniofacial morphogenesis, and illustrate conservation of Wnt signaling in vertebrate craniofacial development.

2.

Results

2.1. Zebrafish wnt9a gene and peptide phylogenetic comparison The isolation of wnt9a and wnt9b genes has been previously described (Crotwell and Mabee, 2007; Jezewski et al., 2008). We cloned a full-length wnt9a cDNA from whole embryo cDNA by RT-PCR. Synteny of the wnt9a locus is conserved between vertebrates, located near wnt3a on chromosome 2 (based on the Zv7 genome sequence map). The predicted Wnt9a amino acid sequence from Danio rerio demonstrates a high degree of conservation, with percentage identity of 76 to human, 77 to mouse, 81 to chick, and 82 to Xenopus (Fig. 1). Areas of peptide identify conserved across vertebrates are in areas of key cysteine residues, where all 23 are preserved (Jezewski et al., 2008; Qian et al., 2003). Phylogenetic relationships of these wnt9 genes are summarized (Fig. 1b).

2.2.

Expression analysis of wnt9a

Whole mount RNA in situ hybridization was carried out to delineate the spatiotemporal expression of wnt9a during early embryogenesis. Prior to 36 h, wnt9a expression is diffuse and difficult to discern from background staining, whereas RT-PCR detects transcripts from the early somite stages onwards (Fig. S1). The first discrete expression of wnt9a occurs at approximately 36 hpf, concentrated in paired groups of cells in the pharyngeal arches (Fig. 2a and b). At 48 hpf, the pharyngeal arch cells expressing wnt9a continues anteriorly corresponding the arch 1, and divides into a dorsal and ventral populations (Fig. 2b, arrowheads). There is also strong staining along the region that correspond to the more posterior pharyngeal arches (Fig. 2b, bracket). By 3 dpf, wnt9a expression is detected in the epithelial tissue adjacent to the ethmoid plate and lower jaw skeletal structures. This staining

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Fig. 1 – Conservation of wnt9a across vertebrates. (a) Wnt9a peptide alignment from Homo sapien (Hs), Mus musculus (Mm), Gallus gallus (Gg) Xenopus tropicalis (Xt) and Danio rerio (Dr) performed using Clustal W algorithm. Wnt9a shares the 23 cysteine residues conserved across Wnt proteins (underlined in red). The region of potential signal peptidase cleavage site as predicted by SignalIP v2.0 is underlined in black. (b) Phylogenetic relationship of Wnt9a across vertebrate species.

is specific but weak, and in fact was not reported in recent analysis of zebrafish wnt9a gene expression (Cox et al., 2010). We performed sections to more precisely localize the wnt9a expressing cells at 3 and 4 dpf. We found specific

expression in epithelial cells lining the oropharynx, and in cells lining the ventral surface that overlie the lower jaw structures. At the level of the ethmoid plate, there is staining in the epithelia lining the roof of the mouth that continues in

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Fig. 2 – Expression of wnt9a in pharyngeal arch and oropharyngeal epithelium. Whole-mount RNA in situ hybridization of wnt9a at 36 hpf (a), 48 hpf (b), 3 dpf (c, d, g and h), and 4 dpf (e, f and i). Embryos are oriented with head toward the left in lateral views (a–c and e) and head toward the top in ventral views (d and f) and sections (g–i). Sections are shown in 10· (g), and 40· (h and i) magnification. The levels of the sections shown in panels (g) and (h) are indicated in panel (d), and the level in panel (i) is indicated in (e). The wnt9a transcripts can be detected in the developing pharyngeal arches (arrow, a) as paired cell clusters. At 48 hpf, wnt9a expression is detected in the first pharyngeal arch, as it splits to dorsal (b, arrow) and ventral (b, arrow with asterisk) regions. There is also wnt9a expression along the posterior pharyngeal arch cells (c, bracket), and the otic vesicle (ov). By 3 dpf, facial skeletal structures have formed, where wnt9a expression can be detected surrounding the ethmoid plate (ep), trabeculae (tr), and lower jaw structures, including the five paired ceratobrachials (cb, 1–5). To better visualize this weak whole-mount staining, plastic sections were performed (g and h). Cross-section analysis reveals wnt9a expression in the oral epithelium along the roof and the floor of the oropharynx (g, arrow), and along the surface epithelium ventral to the lower jaw (g, arrow with asterisk). The ethmoid plate is surrounded by wnt9a expressing cells, bordered by the roof of the oropharynx below, and a single cell-layer of wnt9a expressing cells above (h, arrows). There are also clusters of wnt9a expressing cells laterally that may represent ocular muscle or optic nerve structure (h, arrowheads). Expression of wnt9a persists into 4 dpf along the oropharynx and continues into the foregut (e), detected in the epithelium that lines the oropharynx circumferentially (i). There is also weak wnt9a expression outlining the skeletal structures, such as Meckel’s cartilage (m), palatoquadrate (pq) and the ceratohyal (ch). Abbreviations: cb, ceratobrachial; ch, ceratohyal; ep, ethmoid plate; m, Meckel’s cartilage; ov, otic vesicle; pq, palatoquadrate; tr, trabeculae.

a single cell layer to circumscribe the ethmoid plate structure. At 4 dpf, the weak but specific wnt9a gene expression persists in the oropharynx and surface epithelium associated with the jaw structures (Fig. 2e, f and i). Distinct expression of wnt9a is also detected along the entire length of the paired pronephric tubules, extending into the cloaca (Fig. S2). In contrast to the prominent expression of wnt9a in craniofacial skeleton and oropharyngeal epithelia, wnt9b expression

occurs briefly around 48 hpf and is isolated in bilateral linear groups of cells that correspond to the first 2 pharyngeal arches at 48 hpf, as previously reported (Fig. S3; Jezewski et al., 2008). Based on these observations, it appears that in zebrafish it is wnt9a, and not wnt9b, that plays a more prominent role in facial morphogenesis, in contrast to mammals where wnt9b is implicated in cleft palate formation (Carroll et al., 2005; Juriloff et al., 2006).

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2.3. Disruption of wnt9a results in loss of craniofacial skeletal structures To determine whether wnt9a expression in the epithelium intimately associated with the adjacent skeletal structures holds functional importance, we performed targeted knockdown of wnt9a via morpholino injection. Given the limited but specific pharyngeal arch expression of wnt9b, we also performed wnt9b morpholino knockdown, individually and in combination with wnt9a morpholino. Morpholino knockdown of wnt9b failed to disrupt normal development and specific craniofacial phenotypes were not appreciated, even at high morpholino concentrations (data not shown). When wnt9b morpholino was injected with wnt9a morpholino, the resulting phenotype did not differ from that observed with wnt9a morpholino alone. In contrast, wnt9a knockdown reliably produced specific disruption of craniofacial development (Figs. 3 and 4). The wnt9a morphant develops normally with elongation of body axis, indistinguishable from mismatch morpholino injected controls during first 36–48 h of embryogenesis. However, by 3 dpf, wnt9a morphants can be distinguished by the hypoplastic head, where structures anterior to the eyes are deficient, with absence of lower jaw. Alcian blue staining of the wnt9a morphant reveals significant ablation of lower jaw structures (Fig. 3). The anteromedial portion of the parachordals of the skull base forms, but the lateral and caudal portions are absent. The upper jaw forms up to the level where the paired trabeculae join in the midline, but the anterior segment that contains the ethmoid plate is absent (Fig. 3h, k and n). To determine whether the cartilage template is present in the wnt9a morphant but failed to stain with Alcian blue, we examined the embryos under 40· magnification where cell morphology can be clearly discerned (Fig. 3m–o). High magnification examination of the leading edge of the truncated trabelculae shows that there are only 1–2 layers of cells found in this area that constitutes trabeculae midline union, and no cells more anteriorly (Fig. 3n, asterisk). The chondrocytes that do form in the wnt9a morphant trabeculae appear dysmorphic, as they appear rounded instead of elongated, and do

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not stack in an organized intercalated pattern, as observed in the mismatch control (Fig. 3m and n). Co-injection of wnt9a mRNA with the translation-blocking wnt9a morpholino resulted in rescue of most of the craniofacial skeletal structures and chondrocyte morphology (Figs. 3 and 4), demonstrating the specificity of the wnt9a knockdown phenotype. Over-expression of wnt9a mRNA did not disrupt embryogenesis and resulted in toxicity in higher concentrations (data not shown).

2.4. cells

Analysis of wnt9a requirement in cranial neural crest

In order to examine the role of wnt9a in the development of cranial neural crest cells (CNCC), we examined the migration of CNCC in the sox10:eGFP transgenic embryos following wnt9a MM and MO injection. Early during embryogenesis, premigratory CNCC at the somite stages is not affected by wnt9a knockdown (data not shown). The earliest time point where wnt9a MO phenotype is definitively different from the MM control occurs around 42–48 h, when the jaw structures begin to emerge. In the MM controls, dorsal bilateral linear groups of CNCC extend and move toward the midline at 48 h (Fig. 5a). These CNCC differentiate into chondrocytes that form the trabeculae and ethmoid plate. However, in the wnt9a MO injected embryos, these dorsal CNCC are distinct from the ventral cells that give rise to the lower jaw, but they do not migrate toward the midline (Fig. 5b). By 3 dpf, the ethmoid plate and the trabeculae are well-formed in the MM control embryos. In the wnt9a morphants, the trabeculae appear to form but the overall orientation is not oblique as in the wildtype, and the trabeculae are not joined in the midline (Fig. 5d). As time progresses, the morphant phenotype becomes more pronounced, as the lower jaw is not formed, and the only skeletal structures formed by the CNCC are the medial portions of the parachordals and the trabeculae. Here, the dysmorphic trabeculae meet in the midline, but the ethmoid plate remains absent. This analysis also confirms that CNCC-derived chondrogenic progenitors are in fact absent due to wnt9a knockdown, distinguishing it from the possibil-

c Fig. 3 – Knockdown of wnt9a profoundly disrupts craniofacial development. Live embryo morphology (a–c) and Alcian blue cartilage stain (d–o). Lateral view (a–f), ventral view (g–i), ventral view with lower jaw dissected away (j–l), taken at 10· magnification. View of the ethmoid plate of embryos in (g–i) at 40· magnification (m–o). Anterior is to the left in all views. Embryos injected with wnt9a mismatch control (MM) (a, d, g, j and m), translation-blocking morpholino (MO) (b, e, h, k and n), and wnt9a MO with wnt9a full-length capped mRNA rescue at 4 dpf (c, f, i, l and o). Normal craniofacial morphology (a) and jaw structures are seen in embryos injected with mismatch-control morpholino, with normal lower jaw (g) and ethmoid plate (j, arrow) and lower jaw (arrow in d, j). Knockdown of wnt9a produced loss of jaw structure apparent by facial morphology (b). In the wnt9a morphant, Alcian blue staining detects formation of the medial parachordals but the lateral parachordal structures are absent (h and k). In the wnt9a morphant, the proximal trabeculae form and converge in midline but fail to elongate, and the ethmoid plate does not form (arrow, k, n), and the lower jaw is absent. When wnt9a MO is co-injected with wnt9a mRNA, the morphant phenotype is rescued, albeit not entirely wild-type (c, f, i, l and o). In the rescued animals, the ethmoid plate forms but the trabelculae fail to flair out in the distal leading edge (arrow, l). The lower jaw and the parachordals form in the rescued embryos, but the vertex of the ceratohyal points posteriorly, and the ceratobrachials are not formed. Higher magnification comparison of the trabeculae between the wnt9a morphant and the controls show that the morphant chondrocytes fail to adopt the elongated morphology and remain rounded, with abrupt cutoff anteriorly, as the trabeculae end in a stump (arrow, n). There are no cells found anterior to the cutoff (asterisk).

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ity that the cells are there but not detected by Alcian blue staining.

2.5. Molecular characterization of wnt9a requirement during craniofacial morphogenesis To gain molecular insight into the wnt9a requirement for cranial neural crest migration, we examined the expression of dlx2a, sox10, gsc, as key markers of early cranial neural crest cells (Carney et al., 2006; Eberhart et al., 2006; Ferguson

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et al., 2000; Honore et al., 2003; Hu and Marcucio, 2009; McKeown et al., 2005; Wada et al., 2005). At the onset of cranial neural crest cephalad migration at 24 hpf, expression of dlx2a and sox10 appear unperturbed by wnt9a knockdown, and the neural crest cells express gsc appropriately in the first 2 pharyngeal arches at 42 hpf (Fig. 6). To examine the effect of wnt9a knockdown on chondrocyte differentiation, we examined the expression of sox9a and col2a. The transcription factor sox9a is necessary for neural crest chondrogenesis and col2a encodes collagen type II, the

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main extracellular matrix protein in cartilage (Vandenberg et al., 1991; Yan et al., 1995, 2005). At 3 dpf, sox9a expression is prominent in the ethmoid plate and the pharyngeal arches in the MM control. In contrast, wnt9a disruption resulted in aberrant sox9a expression in the dorsal domains, as ethmoid plate does not form. Further, sox9a expression is absent in the posterior pharyngeal arches (Fig. 7a and b). Therefore, wnt9a is required for the development of chondrogenic progenitors that form both the upper and lower jaws. The upper jaw phenotype is better illustrated by col2a expression. At 48 hpf as the upper jaw and skull base is starting to form, col2a expression can be detected in the chondrogenic cells that populate the ethmoid plate and the paired trabeculae (Fig. 7c, arrow). In contrast, col2a expression shows that the chondrogenic precursors are lateralized and do not meet in the midline at 48 hpf (Fig. 7d). This pattern of laterally displaced chondrogenic cells is confirms the observed aberrant CNCC location from sox10:eGFP transgenic analysis. Meanwhile, shh expression in the oropharyngeal epithelium is unaffected by wnt9a knockdown. The expression of shh in the stomodeum epithelium is comparable between the MM and MO injected embryos (Fig. 7e–h). However, the distribution of the shh expressing cells surrounding the stomodeum appears altered in the wnt9a morphant (Fig. 7h). Additional work is necessary to determine whether loss of wnt9a disrupts formation of facial features primarily, or that the soft tissue phenotype is secondary to altered skeletal development. These results suggest that wnt9a is dispensable for early cranial neural crest migration to pharyngeal arches. However, wnt9a is required for the development of chondrogenic subpopulations that form the ethmoid plate, lateral parachordals, and the lower jaw. The function of wnt9a appears to be specific to chondrocyte development, where development of the oropharyngeal epithelium appears unaffected. Therefore, although wnt9a is expressed in the epithelial sheath that surrounds chondrogenic structure, knockdown of wnt9a results in a chondrogenic but not intrinsic epithelial phenotype.

3.

Discussion

In all vertebrates, migratory streams of neural crest cells that originate from anterior of the hindbrain forms the neurocranium, whereas more posterior neural crest cells stream to form the viscerocranium (Lumsden and Guthrie, 1991; Osumi-Yamashita et al., 1994; Schilling, 1997; Schilling and Kimmel, 1994). Neural crest cell migration during embryogenesis is regulated by signals from the neuroectoderm, provided by sonic hedgehog (Shh). In amniotes, the neurocranial crest cells contribute to the skull as well as the frontonasal prominence, which forms the forehead, nose, and primary palate. In contrast, the viscerocranial neural crest cells populate the first pharyngeal arch, which divides into the maxillary and mandibular prominences, forming the maxilla and mandible. Formation of the palate occurs as the nasal prominence fuse with the maxillary prominences, which requires ectodermal-mesenchyme transition of the leading edge. Wnt signaling pathway is one of the major regulators of neural crest development, conserved across vertebrates

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Fig. 4 – Morpholino knockdown of wnt9a. Graph summarizes the representative phenotypes presented in Fig. 3. Mismatch control (MM), morpholino alone (MO) and rescue with wnt9a mRNA are presented, where the total number of embryos scored for the wildtype (blue bar) and morphant (red bar) phenotypes are shown, with 5% error bars. (Raible, 2006; Raible and Ragland, 2005; van Amerongen and Nusse, 2009). The Wnt family of proteins are secreted glycoproteins that participate in at least three different signaling pathways: (1) the canonical Wnt/b-catenin, (2) non-canonical planar cell polarity and (3) the Wnt Ca2+ pathways. Several genes in the Wnt signaling cascade are specifically expressed in the embryonic facial primordial, such as Tcf1 and Lef1 (Oosterwegel et al., 1993). Double homozygote lacking Lef1 and Tcf4 exhibits significant alterations in facial morphology, with hypertelorism and altered upper lip contour (Brugmann et al., 2007). The murine mutant clf1 harbors a retrotransposon insertion downstream of the Wnt9b gene and fails to complement targeted disruption of Wnt9b (Juriloff et al., 2006). Wnt3 and Wnt9b are expressed in the ectoderm of murine facial primordial during embryogenesis (Jiang et al., 2006). Further, Wnt5a is expressed in the developing palate of the mouse and regulates directional cell migration during palatogenesis (He et al., 2008). In humans, polymorphisms for several Wnt genes (Wnt3A, Wnt11, and Wnt5A) are associated with nonsyndromic cleft lip and palate (Chiquet et al., 2008). In zebrafish, embryonic requirement for canonical Wnt signaling via b-catenin was interrogated using a dominant inhibitor approach (Lewis et al., 2004). The dominant inhibitor of Wnt was engineered by replacing the N-terminal b-catenin binding domain of zebrafish Tcf3 with GFP, with the transgene controlled by heat shock promoter. Lewis and colleagues found that Wnt signaling is required for neural crest induction, and demonstrated that wnt8 is a candidate for mediating this early process. As a strategy to achieve tissue specificity, it was suggested that wnt8 or different Wnt members may be required for neural crest cell induction during different phases in vertebrate embryogenesis. This theme of sequential discrete spatiotemporal requirements of regulatory signaling during craniofacial development has been demonstrated for Hedgehog signaling (Eberhart et al., 2006; Schwend and Ahlgren, 2009; Wada et al., 2005). Embryonic requirement for Wnt signaling was also demonstrated in the Wnt11/silberblick mutant (Heisenberg et al.,

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Fig. 5 – Analysis of cranial neural crest cells after wnt9a knockdown in sox10:eGFP transgenics. Embryos injected with MM control (a, c and e) and translation-blocking MO (b, d and f), where the cranial neural crest cells (CNCC) are followed over embryogenesis. Embryos are shown in ventral views at 48 hpf (a and b), 72 hpf (c and d) and 96 hpf (e and f). Initial CNCC delamination from the neural plate occurs normally in the MO injected embryos (not shown) and the first distinct phenotype is observed with when the dorsal group of CNCC that differentiate into the trabeculae become obliquely oriented and the distal leading edge migrate toward the midline (arrows, a). In the MO injected embryos, the dorsal CNCC fail to move medially, resulting in a wide separation between the CNCC groups. At 72 hpf, the ethmoid plate and the trabeculae are formed (c). In the morphant, the trabeculae is formed but does not join in the midline, and the ethmoid plate is absent (d). By 96 hpf, the distal edges of the morphant trabeculae come into the midline and is joined by 1–2 cell layers of tissue, with absent ethmoid plate (f). Abbreviations: ep (ethmoid plate), tr (trabeculae).

2000). Wnt11 is necessary for proper convergent extension movements during gastrulation. In the absence of wnt11, the mutant homozygotes exhibited midline defects including cyclopia. Interestingly, over-expression of a truncated form of disheveled containing protein interaction PDZ and DEP domains was sufficient to rescue the mutant phenotype, suggesting that dsh acts permissively downstream of wnt11. Here, we show that wnt9a has the proper spatiotemporal expression pattern to play a regulatory role in craniofacial development during zebrafish embryogenesis (Fig. 2). We demonstrate that wnt9a exhibits prominent expression in pharyngeal arches from 36 to 48 hpf, a critical time period for chondorgenic progenitor differentiation (Wada et al., 2005). Subsequently, wnt9a expression is detected in the ectodermal sheath the surrounds the ethmoid plate, analogous to sox9b expression in the ectoderm that surrounds sox9a expressing chondrogenic cells (Yan et al., 2005). Disruption of wnt9a function resulted in loss of the lower jaw, lateral and posterior portions of the parachordals, and absence of ethmoid plate. Using sox10:eGFP transgenic analysis, we show that CNCC localization is disrupted by wnt9a knockdown, and the CNCC-derived structures are not prop-

erly formed. Therefore, wnt9a is a signal provided by ectoderm that regulates differentiation of CNCC derived mesenchyme. Consistent with the CNCC analysis, we show that early migration of neural crest cells to the first two pharyngeal arches was not affected by wnt9a knockdown, evident by unperturbed dlx2a and gsc expression in these areas (Fig. 5). In contrast, knockdown of wnt9a caused segregation of cranial neural crest cells to a lateral position, as delineated by col2a expression (Fig. 6). Additionally, chondrogenic precursors of the upper jaw and posterior pharyngeal arches are aberrant or absent, evidenced by altered sox9a expression. Despite significant effects on chondrogenic development, shh expression in the stomodeum and oral epithelium is largely unaffected by wnt9a knockdown. Therefore, we present evidence for a late stage requirement for wnt9a signaling, provided by the oropharyneal epithelium to support chondrogenic development. The genetic tractability and developmental advantages of zebrafish provides a powerful model to address mechanism of wnt9a regulation of pharyngeal neural crest and chondrogenic development. Our current work is focused on characterizing the inductive signal from the oral ectoderm that regulates palate and jaw development.

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Fig. 6 – Knockdown of wnt9a does not alter early cranial neural crest migration. Embryos injected with wnt9a mismatch control (MM) (a–c), and translation-blocking morpholino (MO) (e–h). Timepoints are 24 hpf (a, b, d and e), and 42 hpf (c and f). Embryos are mounted in lateral view with head toward left detected with dlx2a riboprobe (a and d), detected with sox10 riboprobe in dorsal view with head toward top (b and e), and gsc riboprobe in ventral view (c and f). These neural crest markers all demonstrate normal early migration to appropriate pharyngeal arch positions, in mismatch (MM) and translation-blocking (MO) morpholino injected embryos. No developmental delay was observed.

Fig. 7 – Knockdown of wnt9a results in late craniofacial phenotypes. Embryos injected with wnt9a mismatch control (MM) (a, c, e and g), and translation-blocking morpholino (MO) (b, d, f and h). Embryos are mounted in lateral view with head toward left (a, b, e–h) and in ventral view with head toward top (c and d). Transcripts of sox9a (a and b) at 3 dpf, col2a (c and d) at 2 dpf, and shh (e–h) at 2 dpf are detected by whole mount RNA in situ hybridization. The truncated trabeculae and absent ethmoid plate are confirmed by comparison of sox9a expression between MO (b) and MM control (a). In the wnt9a morphant, sox9a expressing cells do not elongate to form the trabeculae, and there is no ethmoid plate (b, arrow). The expression of sox9a in the posterior pharyngeal arches is also absent (b, bar). The expression of col2a is present but aberrantly located in the wnt9a morphant, distinguishable from MM control as early as 48 hpf. In the MM control, col2a expression prefigures the trabeculae and the ethmoid plate (c), whereas in the morphant, the cranial neural crest cells are lateralized in linear groups (d). Oral epithelium express shh in a wildtype pattern in the MM control and the morphant, outlining the stomodeum (g, arrowheads). The stomodeum forms in the wnt9a morphant, although the shape is altered (f, arrowheads), and is continuous to the foregut, with shh expression outlining the roof (e, f, arrows) and floor (e, f, arrowheads) of the oropharynx.

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4.

Experimental procedures

4.5.

4.1.

Zebrafish strains and maintenance

Total RNA was isolated from whole embryo lysates from different time points and purified using InvitrogenTM SuperScript First-Strand Synthesis System for RT-PCR. Primers designed to separate exons of wnt9a gene were used to amplify from the cDNA library by RT-PCR, with ef1a as positive control, genomic DNA as negative control. Gel image was captured using Alpha Innotech ChemilmagerTM 5500 and AlphaEase FC (FluorChem 5500) software for Windows XP. Primer sequences are as follows

All embryos and fish were raised and cared for using established protocols as per Subcommittee on Research Animal Care, Massachusetts General Hospital, essentially as described (Westerfield, 1993). Zebrafish were maintained and bred at 28 C on a 14 h light, 10 h dark cycle. Embryos were generated and collected by natural spawning, and also kept at 28 C. Tubingen fish were used as wild type in our experiments.

4.2.

Gene cloning and sequence analysis

wnt9a cDNA was cloned by RT-PCR from cDNA prepared from 24 hpf embryos and subcloned into pCS7 vector. Sequence analysis was performed with Lasergene 8.0 program suite, CLUSTAL W alignment algorithm. Species and accession numbers are Hs: Homo sapiens, NM_003395; Mm: Mus musculus, NM_139298; Gg: Gallus gallus, NM_204981; Xt: Xenopus tropicalis, DQ658159; Gg: Gallus gallus, NM_204981, and Dr: Danio rerio, FJ231750. Additional zebrafish wnt9a sequence has also been deposited in Genbank, NM_001045363, FJ231750, and BC164356 (Crotwell and Mabee, 2007; Strausberg et al., 2002).

• • • •

RT-PCR

wnt9a exon 2 sense primer 5 0 -gggaggctgtggagacaacctcaa-3 0 wnt9a exon 3 anti-sense primer 5 0 -ctcgcagttcttgtccttgtagc30 ef1a sense primer 5 0 -atctacaaatgcggtggaat-3 0 ef1a anti-sense primer 5 0 -ataccagcctcaaactcacc-3 0

4.6.

Morpholino and RNA injection

Gene Tools, LLC supplied morpholino oligonucleotides. These were designed according to manufacturer’s recommendations, i.e. 21–25 bp antisense oligonucleotides with approximately 50% G/C and A/T content and no predicted internal hairpins. Sequences were as follows: wnt9a MO, 5 0 -CCAGGAGAAGGTGTCCATCCAGCAT-3 0 ; wnt9a MM, 5 0 -CCAGcAcAAGGTcTCCATCgAcCAT-3 0 , wnt9b MO, 5 0 -CAGTCCTCGGAAGCCCGGTGCACAT-3 0

4.3. Whole mount RNA in situ hybridization and cartilage staining

• • •

RNA in situ hybridization and riboprobe synthesis were performed as described (Schulte-Merker et al., 1992), Anti-sense riboprobes of wnt9a, wnt9b, col2a dlx2a, gsc, shh, and sox9a were all synthesized from plasmid preparations using T7 or Sp6 polymerase, or from PCR products containing T3 promoter. Digoxigenin or fluorescein labeled riboprobes were detected with alkaline phosphatase conjugated anti-digoxigenin and BCIP/NBT (Vector Laboratories, Inc.). Alcian blue staining was performed essentially as described, using acid-free technique to decrease background staining, trypsinized and photographed (Walker and Kimmel, 2007). Images were captured using Nikon SMZ1000 and Nikon Eclipse 80i microscopes, mounted with Nikon Coolpix 4500 and Hamamatsu Orca-ER C4742-80 digital cameras.

The oligonucleotides were solubilized in RNase-free water at a concentration of 1 mM. This stock solution was diluted to working concentrations of 0.1 mM and was stored at room temperature as per manufacturer’s instructions. Capped wnt9a mRNA was synthesized from pCS7:wnt9a plasmid using mMessage mMachine kit and made up to a concentration of 1 lg/ll, and stored at 80 C. This stock solution was diluted to 100 ng/ll for injection. For rescue injections, wnt9a MO at 848 ng/ll (0.1 mM) was co-injected with wnt9a mRNA at 50 ng/ll at the one-cell stage. We injected embryos with 5 nl of these solutions (4 ng wnt9a MO, 0.25 ng wnt9a mRNA). Higher concentrations led to non-specific toxicity. wnt9b MO concentration up to 1 mM was tested, leading to toxicity and brain necrosis, but no specific phenotypes.

4.4.

Acknowledgements

sox10:eGFP transgenic analysis

The sox10 7.2 kb promoter region was a generous gift from Robert Kelsh, previously described as psox104725 (Dutton et al., 2008). The sox10:eGFP transgene was generated using the Gateway Tol2 integration vector pDestTol2pA, as described (Kwan et al., 2007). The resulting transgenic animals were screened and bred for high signals in the cranial neural crest cells. The wnt9a MO and MM were injected into one-cell embryos from the sox10:eGFP transgenic line, followed and photographed at the timepoints presented on Nikon A1R confocal microscope, with images captured on NIS Elements Software v3.1, at 20· magnification.

This work was funded by the generous support of the Shriners Hospital for Children, Plastic Surgery Education Foundation Basic Science Grant, and the Amelia Peabody Charity Grant. E.C.L. is the recipient of the American Surgical Association Research Fellowship and the March of Dimes Basil O’Connor Starter Scholar Award.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2010.11.003.

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R E F E R E N C E S

Barrallo-Gimeno, A., Holzschuh, J., Driever, W., Knapik, E.W., 2004. Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function. Development 131, 1463– 1477. Brugmann, S.A., Goodnough, L.H., Gregorieff, A., Leucht, P., ten Berge, D., Fuerer, C., Clevers, H., Nusse, R., Helms, J.A., 2007. Wnt signaling mediates regional specification in the vertebrate face. Development 134, 3283–3295. Carney, T.J., Dutton, K.A., Greenhill, E., Delfino-Machin, M., Dufourcq, P., Blader, P., Kelsh, R.N., 2006. A direct role for Sox10 in specification of neural crest-derived sensory neurons. Development 133, 4619–4630. Carroll, T.J., Park, J.S., Hayashi, S., Majumdar, A., McMahon, A.P., 2005. Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev. Cell 9, 283–292. Chiquet, B.T., Blanton, S.H., Burt, A., Ma, D., Stal, S., Mulliken, J.B., Hecht, J.T., 2008. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum. Mol. Genet. 17, 2212–2218. Cox, A.A., Jezewski, P.A., Fang, P.K., Payne-Ferreira, T.L., 2010. Zebrafish Wnt9a,9b paralog comparisons suggest ancestral roles for Wnt9 in neural, oral-pharyngeal ectoderm and mesendoderm. Gene Expr. Patterns. Crotwell, P.L., Mabee, P.M., 2007. Gene expression patterns underlying proximal–distal skeletal segmentation in latestage zebrafish, Danio rerio. Dev. Dyn. 236, 3111–3128. Dutton, J.R., Antonellis, A., Carney, T.J., Rodrigues, F.S., Pavan, W.J., Ward, A., Kelsh, R.N., 2008. An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10. BMC Dev. Biol. 8, 105. Eberhart, J.K., Swartz, M.E., Crump, J.G., Kimmel, C.B., 2006. Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development. Development 133, 1069– 1077. Ferguson, C.A., Tucker, A.S., Sharpe, P.T., 2000. Temporospatial cell interactions regulating mandibular and maxillary arch patterning. Development 127, 403–412. Garriock, R.J., Warkman, A.S., Meadows, S.M., D’Agostino, S., Krieg, P.A., 2007. Census of vertebrate Wnt genes: isolation and developmental expression of Xenopus Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16. Dev. Dyn. 236, 1249–1258. He, F., Xiong, W., Yu, X., Espinoza-Lewis, R., Liu, C., Gu, S., Nishita, M., Suzuki, K., Yamada, G., Minami, Y., Chen, Y., 2008. Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development. Development 135, 3871–3879. Heisenberg, C.P., Tada, M., Rauch, G.J., Saude, L., Concha, M.L., Geisler, R., Stemple, D.L., Smith, J.C., Wilson, S.W., 2000. Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76–81. Hilliard, S.A., Yu, L., Gu, S., Zhang, Z., Chen, Y.P., 2005. Regional regulation of palatal growth and patterning along the anterior–posterior axis in mice. J. Anat. 207, 655–667. Honore, S.M., Aybar, M.J., Mayor, R., 2003. Sox10 is required for the early development of the prospective neural crest in Xenopus embryos. Dev. Biol. 260, 79–96. Hu, D., Marcucio, R.S., 2009. A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm. Development 136, 107–116. Jezewski, P.A., Fang, P.K., Payne-Ferreira, T.L., Yelick, P.C., 2008. Zebrafish Wnt9b synteny and expression during first and second arch, heart, and pectoral fin bud morphogenesis. Zebrafish 5, 169–177.

1 2 8 ( 2 0 1 1 ) 1 0 4 –1 1 5

Jiang, R., Bush, J.O., Lidral, A.C., 2006. Development of the upper lip: morphogenetic and molecular mechanisms. Dev. Dyn. 235, 1152–1166. Juriloff, D.M., Harris, M.J., 2008. Mouse genetic models of cleft lip with or without cleft palate. Birth Defects Res. A. Clin. Mol. Teratol. 82, 63–77. Juriloff, D.M., Harris, M.J., McMahon, A.P., Carroll, T.J., Lidral, A.C., 2006. Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test. Birth Defects Res. A. Clin. Mol. Teratol. 76, 574–579. Kimmel, C.B., Miller, C.T., Moens, C.B., 2001. Specification and morphogenesis of the zebrafish larval head skeleton. Dev. Biol. 233, 239–257. Knight, R.D., Nair, S., Nelson, S.S., Afshar, A., Javidan, Y., Geisler, R., Rauch, G.J., Schilling, T.F., 2003. Lockjaw encodes a zebrafish tfap2a required for early neural crest development. Development 130, 5755–5768. Koillinen, H., Lahermo, P., Rautio, J., Hukki, J., Peyrard-Janvid, M., Kere, J., 2005. A genome-wide scan of non-syndromic cleft palate only (CPO) in Finnish multiplex families. J. Med. Genet. 42, 177–184. Kontges, G., Lumsden, A., 1996. Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development 122, 3229–3242. Koyama, E., Shibukawa, Y., Nagayama, M., Sugito, H., Young, B., Yuasa, T., Okabe, T., Ochiai, T., Kamiya, N., Rountree, R.B., Kingsley, D.M., Iwamoto, M., Enomoto-Iwamoto, M., Pacifici, M., 2008. A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Dev. Biol. 316, 62–73. Kwan, K.M., Fujimoto, E., Grabher, C., Mangum, B.D., Hardy, M.E., Campbell, D.S., Parant, J.M., Yost, H.J., Kanki, J.P., Chien, C.B., 2007. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099. Lan, Y., Ryan, R.C., Zhang, Z., Bullard, S.A., Bush, J.O., Maltby, K.M., Lidral, A.C., Jiang, R., 2006. Expression of Wnt9b and activation of canonical Wnt signaling during midfacial morphogenesis in mice. Dev. Dyn. 235, 1448–1454. Lewis, J.L., Bonner, J., Modrell, M., Ragland, J.W., Moon, R.T., Dorsky, R.I., Raible, D.W., 2004. Reiterated Wnt signaling during zebrafish neural crest development. Development 131, 1299– 1308. Lumsden, A., Guthrie, S., 1991. Alternating patterns of cell surface properties and neural crest cell migration during segmentation of the chick hindbrain. Development Suppl. 2, 9–15. McKeown, S.J., Newgreen, D.F., Farlie, P.G., 2005. Dlx2 overexpression regulates cell adhesion and mesenchymal condensation in ectomesenchyme. Dev. Biol. 281, 22–37. Murray, J.C., 2002. Gene/environment causes of cleft lip and/or palate. Clin. Genet. 61, 248–256. Oosterwegel, M., van de Wetering, M., Timmerman, J., Kruisbeek, A., Destree, O., Meijlink, F., Clevers, H., 1993. Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 118, 439–448. Osumi-Yamashita, N., Ninomiya, Y., Doi, H., Eto, K., 1994. The contribution of both forebrain and midbrain crest cells to the mesenchyme in the frontonasal mass of mouse embryos. Dev. Biol. 164, 409–419. Phillips, B.T., Kwon, H.J., Melton, C., Houghtaling, P., Fritz, A., Riley, B.B., 2006. Zebrafish msxB, msxC and msxE function together to refine the neural–nonneural border and regulate cranial placodes and neural crest development. Dev. Biol. 294, 376–390. Qian, J., Jiang, Z., Li, M., Heaphy, P., Liu, Y.H., Shackleford, G.M., 2003. Mouse Wnt9b transforming activity, tissue-specific expression, and evolution. Genomics 81, 34–46.

MECHANISMS OF DEVELOPMENT

Raible, D.W., 2006. Development of the neural crest: achieving specificity in regulatory pathways. Curr. Opin. Cell Biol. 18, 698–703. Raible, D.W., Ragland, J.W., 2005. Reiterated Wnt and BMP signals in neural crest development. Semin. Cell Dev. Biol. 16, 673–682. Roessler, E., Belloni, E., Gaudenz, K., Jay, P., Berta, P., Scherer, S.W., Tsui, L.C., Muenke, M., 1996. Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet. 14, 357– 360. Satokata, I., Maas, R., 1994. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348–356. Schilling, T.F., 1997. Genetic analysis of craniofacial development in the vertebrate embryo. Bioessays 19, 459–468. Schilling, T.F., Kimmel, C.B., 1994. Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo. Development 120, 483–494. Schorle, H., Meier, P., Buchert, M., Jaenisch, R., Mitchell, P.J., 1996. Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381, 235–238. Schulte-Merker, S., Ho, R.K., Herrmann, B.G., Nusslein-Volhard, C., 1992. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032. Schwend, T., Ahlgren, S.C., 2009. Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development. BMC Dev. Biol. 9, 59. Shi, M., Mostowska, A., Jugessur, A., Johnson, M.K., Mansilla, M.A., Christensen, K., Lie, R.T., Wilcox, A.J., Murray, J.C., 2009. Identification of microdeletions in candidate genes for cleft lip and/or palate. Birth Defects Res. A. Clin. Mol. Teratol. 85, 42– 51. Soriano, P., 1997. The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124, 2691–2700. Strausberg, R.L., Feingold, E.A., Grouse, L.H., Derge, J.G., Klausner, R.D., Collins, F.S., Wagner, L., Shenmen, C.M., Schuler, G.D., Altschul, S.F., Zeeberg, B., Buetow, K.H., Schaefer, C.F., Bhat, N.K., Hopkins, R.F., Jordan, H., Moore, T., Max, S.I., Wang, J., Hsieh, F., Diatchenko, L., Marusina, K., Farmer, A.A., Rubin, G.M., Hong, L., Stapleton, M., Soares, M.B., Bonaldo, M.F., Casavant, T.L., Scheetz, T.E., Brownstein, M.J., Usdin, T.B., Toshiyuki, S., Carninci, P., Prange, C., Raha, S.S., Loquellano, N.A., Peters, G.J., Abramson, R.D., Mullahy, S.J., Bosak, S.A.,

1 2 8 ( 2 0 1 1 ) 1 0 4 –1 1 5

115

McEwan, P.J., McKernan, K.J., Malek, J.A., Gunaratne, P.H., Richards, S., Worley, K.C., Hale, S., Garcia, A.M., Gay, L.J., Hulyk, S.W., Villalon, D.K., Muzny, D.M., Sodergren, E.J., Lu, X., Gibbs, R.A., Fahey, J., Helton, E., Ketteman, M., Madan, A., Rodrigues, S., Sanchez, A., Whiting, M., Young, A.C., Shevchenko, Y., Bouffard, G.G., Blakesley, R.W., Touchman, J.W., Green, E.D., Dickson, M.C., Rodriguez, A.C., Grimwood, J., Schmutz, J., Myers, R.M., Butterfield, Y.S., Krzywinski, M.I., Skalska, U., Smailus, D.E., Schnerch, A., Schein, J.E., Jones, S.J., Marra, M.A., 2002. Generation and initial analysis of more than 15,000 fulllength human and mouse cDNA sequences. Proc. Natl. Acad. Sci. USA 99, 16899–16903. Tallquist, M.D., Soriano, P., 2003. Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells. Development 130, 507–518. van Amerongen, R., Nusse, R., 2009. Towards an integrated view of Wnt signaling in development. Development 136, 3205–3214. Vandenberg, P., Khillan, J.S., Prockop, D.J., Helminen, H., Kontusaari, S., Ala-Kokko, L., 1991. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia. Proc. Natl. Acad. Sci. USA 88, 7640–7644. Wada, N., Javidan, Y., Nelson, S., Carney, T.J., Kelsh, R.N., Schilling, T.F., 2005. Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull. Development 132, 3977–3988. Walker, M.B., Kimmel, C.B., 2007. A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech. Histochem. 82, 23–28. Westerfield, M., 1993. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). University of Oregon Press, Eugene, Oregon. Yan, Y.L., Hatta, K., Riggleman, B., Postlethwait, J.H., 1995. Expression of a type II collagen gene in the zebrafish embryonic axis. Dev. Dyn. 203, 363–376. Yan, Y.L., Willoughby, J., Liu, D., Crump, J.G., Wilson, C., Miller, C.T., Singer, A., Kimmel, C., Westerfield, M., Postlethwait, J.H., 2005. A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development. Development 132, 1069–1083. Zhang, J., Hagopian-Donaldson, S., Serbedzija, G., Elsemore, J., Plehn-Dujowich, D., McMahon, A.P., Flavell, R.A., Williams, T., 1996. Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381, 238–241.