Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development
© Schwend and Ahlgren; licensee BioMed Central Ltd. 2009
Received: 3 June 2009
Accepted: 30 November 2009
Published: 30 November 2009
The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1, 2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).
con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to inhibit Hh-signaling at different developmental stages, show that Hh-signaling is required during gastrulation for normal patterning of CNCC in the first PA, and then during the late pharyngula stage, to promote CNCC chondrogenesis within the posterior arches. Further, loss of disp1 disrupted normal expression of bapx1 and gdf5, markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp1 mutant animals.
This study reveals novel requirements for Hh-signaling in the zebrafish PA skeleton and highlights the functional diversity and differential sensitivity of craniofacial tissues to Hh-signaling throughout the face, a finding that may help to explain the spectrum of human facial phenotypes characteristic of HPE.
The vertebrate head skeleton is largely derived from cranial neural crest cells (CNCC) which originate in the dorsal neural tube and migrate ventrally to populate the pharyngeal arches (PA) . The final skeletal pattern depends on intrinsic positional information acquired by CNCC prior to migration [4–6], instructive signals encountered by CNCC during migration [7–9], and morphogenetic cues that CNCC receive from surrounding tissues upon completing migration [10, 11]. There are seven paired skeletal elements that arise on either side of the midline. The CNCC of the first PA (mandibular) forms the jaw, while those in the second PA (hyoid) add support to the jaw structure. Posterior to these two PA, the CNCC populate five posterior PA to form bilateral gill support structures. Although all of these elements derive from CNCC, the neural crest arise from different anterior-posterior regions of the neural axis, travel in different streams to arrive at the final destination, express different Hox genes, and encounter different local environments at the point of condensation [6, 12]. However, the genes that regulate the production of cartilage from CNCC are shared regardless of location .
Genetic studies have revealed conserved requirements for Hedgehog (Hh)-signaling in craniofacial development. Abrogation of Hh-signaling through inactivation of the Hh-receptor Smoothened (Smo) leads to a complete loss of cranial skeletal elements in zebrafish and mouse [14, 15]. It has been pointed out that these animal models represent extreme phenotypes, with complete suppression of signaling, and often result in embryonic lethality . However, mutations within the key Hh-family member, Sonic Hedgehog (SHH) in humans can lead to the spectral disorder of Holoprosencephaly (HPE), often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia . It has been further suggested that HPE can be found in patients with significant impairment of the Hh-signaling pathway rather than its complete elimination .
Genetic and pharmacological vertebrate studies have revealed that Hh-signaling is required by multiple facial tissues, including the neural crest, and the pathway's action is needed at multiple times during embryonic development in order for the face to grow and pattern normally [16–23]. Thus, it is becoming apparent that the range of phenotypes that are apparent in HPE may be in part due to the affected tissue(s) and the timing of the Hh-pathway disruption.
The precise role for Hh-signaling in the zebrafish PA skeleton has yet to be elucidated. In the dorsal neurocranium, Hh-signaling has been demonstrated to control the placement and condensation of CNCC upon facial ectoderm prior to their chondrification . This CNCC patterning defect is followed by a delay in the expression of the chondrogenic marker sox9a, or a complete absence of gene expression in more severe genetic backgrounds where smo is disrupted [18, 23]. Neurocranial and upper jaw cartilage elements, including the ethmoid plate, paired trabeculae and pterygoid process of the palatoquadrate, are lost or mislocalized with treatments of Cyclopamine (Cya), a pharmacological antagonist of Hh-signaling, administered to whole embryos during the gastrulation period, prior to CNCC migration [18, 23]. This requirement is likely cell non-autonomous as the oral ectoderm requires early Hh-signals in order to promote CNCC condensation and the expression of growth factors as revealed by genetic mosaic analysis . In addition to this, Wada et al., 2005 has proposed a second, cell-autonomous requirement for Hh-signaling wherein the Hh-signal is secreted, likely by the oral ectoderm, and is required by post-migratory CNCC that will form the neurocranium to undergo chondrogenesis. This finding is supported in mice and zebrafish with a tissue-specific loss of Smo function in CNCC, as these cells become properly patterned in the face but rarely differentiate into cartilage tissue [18, 22]. Despite this, these studies do not explain what happens to the CNCC after they fail to condense.
This study seeks to expand our understanding of Hh-signaling in craniofacial development by presenting a detailed characterization of ventral cartilage development in the PA upon genetic inactivation of the Disp1 gene by using the chameleon (con/disp1) mutant zebrafish. Disp1 acts within Hh-synthesizing cells to disperse Hh-ligands into the extracellular field where they are received by Hh-target cells . We found that these mutant fish demonstrate a significant, but not complete, reduction in Hh-signaling, similar to what has been proposed for HPE patients with DISP1 mutations . Furthermore, we pinpoint precisely during development when Hh-signals are required for ventral cartilage development through pharmacological inhibition using Cya. Collectively we report findings that provide insight into Hh-signaling function during jaw and gill cartilage formation in the developing zebrafish pharyngeal arch skeleton.
Zebrafish (Danio rerio) embryos were obtained from natural crosses and staged as previously described . Homozygous con/disp1tm15aandb392 were identified among their siblings by PCR genotyping prior to 24 hours post fertilization (hpf) (detailed genotyping strategy for con/disp1tm15awas reported in Nakano et al., 2004, and the genotyping strategy for con/disp1b392 is reported in this manuscript) or by somite morphology at later stages . Genotyping primers: contm15a_Forward, 5'CCAGCTGTGGTGGTCTTACA3', contm15a_Reverse, 5'CAGAGGCTTCTTCGCAGTCT3'; conb392_Forward, 5'CCAAAGATCATTACGATCGAAAGAATCGA3', conb392_Reverse, 5'CCATGTGGTCTCCCATAGTCATTA3.' Next, restriction enzyme reactions were performed to differentiate PCR products, for contm15a we used PvuII (New England Biolabs) and for b392 we used ClaI (New England Biolabs) which both digest mutant alleles (see additional file 1 for further explanation). fli1:gfp transgenic zebrafish expressing the gfp-transgene under the control of the Fli1 promoter was previously characterized . Wild type refers to con/disp1+/+,+/- siblings, except in Cya-treatment experiments wherein the Tübingen strain was utilized.
Alcian blue staining
Cartilage analysis was performed as previously described, zebrafish larvae were fixed at 96 hpf and treated with Alcian blue solution dissolved in 80% ethanol/20%glacial acetic acid (acid alcohol) for several hours or overnight. Larvae were destained in several washes of acid alcohol before being transferred to a 1% KOH:3% hydrogen peroxide solution for further clearing of pigment cells. Larval tissue was then digested in trypsin, followed by dissection of cartilages which were then flat mounted as previously described . Cartilage preparations were visualized on a Leica MZ16F microscope with camera.
Whole mount in situ hybridization, immunohistochemistry and histology
Whole-mount in situ hybridization was conducted essentially as previously described . Embryos fixed at a stage older than 25 hpf were raised in 2 mM 1-phenyl-2-thiourea in embryo medium to prevent pigmentation. Embryos were visualized on a Leica MZ16F microscope with camera. Images were processed using Adobe Photoshop CS2.
Immunohistochemistry was performed as previously described . The Zn5 antibody (Zebrafish International Resource Center [ZIRC], Oregon) was used at a 1:500 dilution in goat serum blocking solution (ZIRC, Oregon). Confocal stacks of the embryos were generated on a Zeiss LSM510.
For histological preperations, gelatin-embedded embryos were sectioned at 10 μm on a Leica cryostat and subjected to Gill's 2× Hematoxylin and Eosin (Fischer Scientific). Embryos were visualized on a Leica DMR-HC upright microscope.
For cell death studies, embryos fixed for analysis at a stage older than 18 hpf were incubated in 1 μg/mL Proteinase K solution to solubilize tissues and washed twice in PBS-Tween (PBST). Embryos were then incubated in the labeling and enzymatic solutions that are provided by the manufacturer of the In Situ Cell Death Detection Kit (Roche) for 1 hour at 37°Celcius. Enzyme and label solution consists of TdT (terminal deoxynucleotide transferase-mediated dUTP) and fluorescein-conjugated deoxynucleotides in buffer. After three washes in PBST to remove non-specific labeling, embryos were bathed in blocking solution (2% BSA, 1% DMSO in PBST) for 1 hour at room temperature, followed by incubation in blocking solution containing an anti-fluorescein-conjugated alkaline phosphatase (Roche) overnight at 4°Celcius. The following day, embryos were washed multiple times with PBST and incubated at room temperature in an NBT/BCIP developing solution (Promega) to visualize dying cells. Stained embryos were visualized on a Leica MZ16F.
Cya (LC Laboratories) was solubilized in 95% ethanol to obtain a stock solution of 50 mM, which was further diluted in zebrafish embryo water to a working stock concentration of 100 μM. Embryos were bathed in the Cya working stock for 4-24 hours at a time. Immediately following treatment, the Cya solution was removed, embryos were washed in multiple rounds of zebrafish embryo water, and then allowed to develop further until 48-96 hpf, wherein they were analyzed by in situ hybridization or cartilage staining.
Total RNA extraction was performed using RNA Tissue Extraction and DNase kits (Versagene). cDNA was synthesized using SuperScript III and random primers (Invitrogen). PCR reactions were performed using Platinum SYBR Green qPCR Super Mix-UDG (Invitrogen) on a MJ Research Chroma-4 PCR machine and primers: gli1_Forward: 5'CAGACGTCCTCTCGCCTTAC3,'
gli1_Reverse: 5'AGTAGCGCTGTCCTTGCATT3,' disp1_Forward: 5'GCTGTAGGGCTTTCTGTGGA3,'
disp1_Reverse: 5'GCCACTGTTGGTAGGAGCAT3,' gapdh_Forward: 5'GAAGGTGGGAAACTGGTCAT3,' gapdh_Reverse: 5'TTGCACCACCCTTAATGTGA3.' Genes being analyzed were then normalized to gapdh levels and relative quantification of gene expression was calculated using the Pfaffl method variation on 2-ΔΔCt , displaying data as fold difference in mutants relative to wild type.
Plasmids to generate full-length shh and Flag-tagged shhN (N terminus of shh fused to C-terminal Flag epitope), which encodes unmodified shh, have been previously described [26, 32]. To synthesize capped mRNAs, the plasmids were linearized downstream of the insert, followed by transcription reactions using the Ambion mMESSAGE mMACHINE kit according to manufacturer's instructions. ~1 nanoliter (nl) of 100-150 pg/nl mRNA, suspended in 0.2 M KCl, was pressure-injected at 1-2 cell stage. Embryos were then allowed to develop to 96 hpf, wherein they were analyzed by cartilage staining.
Requirement for Hh-signaling in ventral cartilage development
Zebrafish lacking the Hh-signaling pathway, due to smo loss, display complete absence of PA-derived cartilages . To better understand the role of Hh-signaling in this process, we studied cartilage development within two con/disp1 mutant alleles: the con/disp1tm15a(genetic mutation characterized in ) and con/disp1b392 (genetic mutation described in this manuscript, see additional file 1). con/disp1 mutant phenotypes are less severe than slow-muscle-omitted (smu/smo) mutant phenotypes, making them more amenable for detection of subtle deficits . con/disp1tm15aand con/disp1b392 alleles displayed similar penetrance in their craniofacial phenotype, and also shared similar gene expression reductions in gli1, a Hh-signaling pathway transcription factor that is sensitive to the pathway's attenuation (additional file 1). Due to the similar nature of the mutant phenotypes, and in an effort to be concise, in this study we simply report our findings using the con/disp1tm15aallele, which we will herein refer to as con/disp1.
Disp1 regulates the release of lipid-modified Hh-molecules from their site of synthesis . To determine if Disp1 action is conserved in the craniofacial skeleton, we compared the outcome on con/disp1 cartilages upon ectopic mRNA expression of lipid-modified Shh (shhFL) or the N-terminal domain of Shh (shhN), which retains Hh-signaling capacity but fails to become lipid-modified. Injecting offspring from an incross mating of con/disp1 heterozygotes with 150 pg shhFL mRNA at the 1-cell stage yielded no discernable differences in cartilage phenotypes (11/45 [24%] larvae showing con/disp1-/- cartilage defects) from their uninjected siblings (15/55 [26%] larvae) (Figure 1H), likely due to the inability of lipid-modified-ShhFL protein to be secreted from disp1-/-mutant cells. In contrast, injecting con/disp1 heterozygote offspring with 150 pg of shhN mRNA reproducibly decreased the number of larvae displaying PA-derived cartilage defects (3/50 [6%]) compared to uninjected siblings (12/45 [25%]). We determined cartilage rescue in these studies to be a partial-to-full restoration of cartilage outgrowth in the mandibular arch coupled with partial restoration of cb cartilages (arrow in Figure 1G shows unilateral rescue in cb 4-5) in genotype-confirmed con/disp1 mutants (Figure 1G). Failure of shhN to fully restore con/disp1 cartilage defects could be attributed to a loss of potency by non-lipid-modified versions of Shh to activate Hh-target genes . Alternatively, because shhN completely restored cartilages in the anterior arches, loss of full cartilage restoration may be due to the observation that arch cartilages develop sequentially, with posterior elements forming last, thus allowing for ectopic mRNA dilution and degradation to occur prior to the time in which chondrogenesis has completed .
Tracking con/disp1 CNCC development with the fli1:gfptransgene
PA cartilages are derived from CNCC in zebrafish, consistent with higher vertebrates [37, 38]. Thus, cartilage defects could be due to either a failure of migrating CNCC to populate arch primordia or to a lack of postmigratory-CNCC differentiation or survival. Others have reported that zebrafish with Hh-signaling disruptions display no discernable disruptions to CNCC specification or migration into the arch primordia [18, 23]. Like these reports, we found that con/disp1 mutants were identical to wild type embryos in CNCC specification (12 hpf) and early migration (14-22 hpf) into the PA as visualized by neural crest markers foxd3 and crestin (additional file 2) [39, 40].
fli1GFP:con/disp1 CNCC patterning within PA2 was basically similar to wild type embryos (Figure 2B-K), with the exception that they were located slightly more medial than those in wild type embryos (Figure 2C-J). Viewing fli1GFP:con/disp1 at later stages (Figure 2H, K) revealed that con/disp1 CNCC resided in PA2, yet overall they were less organized and may have defects in stacking.
Interestingly, despite the complete absence of con/disp1 posterior arch cartilages, we detected no significant disruptions to CNCC patterning within con/disp1 mutant PA3-7 (Figure 2B-K). Further, by 48 hpf we clearly visualized demarcations between fli1GFP:con/disp1 GFP+ CNCC and presumptive GFP- endodermal cells (dark) upon which the CNCC that have migrated into PA2-7 condense, indicating proper arch segmentation (Figure 2C, F). By staining embryos with the endodermal pouch-labeling Zn5 antibody [42, 43], we confirmed that GFP- cells were endodermal and con/disp1 pharyngeal pouches were capable of forming (Figure 2G, J). Furthermore, fli1GFP:con/disp1 CNCC continued to express the fli1GFP-transgene and remained properly interdigitated within pharyngeal pouches up to 80 hpf (Figure 2H, K). Despite this, 80 hpf Alcian blue staining revealed that CNCC within the fli1GFP con/disp1 posterior arches do not differentiate into cartilage (Figure 2I, L). Taken in full, this data reveals that only the subset of CNCC that have migrated into the PA1 primordium rely on Hh-signaling to become properly patterned. Alternatively, in our hypomorphic allele, it is possible that this CNCC subset is simply the most sensitive to Hh-attenuation and larger disruptions would also affect CNCC patterning in PA2-7 as this phenotype has not been studied in more severely affected alleles.
Pharyngeal arch chondrocyte differentiation is perturbed in con/disp1mutants
To investigate the role of Hh-signaling in cartilage development we examined molecular markers that control chondrogenic and osteogenic differentiation in con/disp1 mutants. We studied the expression patterns of sox9a and sox9b, two transcription factors necessary for chondrogenesis [13, 44], runx2b, a marker of chondrocyte and osteogenic differentiation [45, 46], and type II collagen (col2a1), the predominant extracellular matrix protein in cartilage [47, 48].
By 60 hpf, CNCC in the first two arches became organized into dorsal and ventral prechondrogenic cartilage condensations, which can be visualized by sox9a and sox9b gene expression (Figure 3E, G). It has been suggested that organization of CNCC into these dorsoventral (DV) domains likely precedes their later chondrification into dorsal (pq, ptp, hs) and ventral (Mc and ch) cartilage structures [49, 50]. At this stage, we detected a reduction in the number of sox9a and sox9b expressing cells residing in dorsal domains of PA1-2 in con/disp1 mutants compared to wild type siblings (Figure 3E-H). Despite this, expression of both genes was retained at normal levels in CNCC residing within the PA1-2 ventral domains (Figure 3E-H). Expression of the sox9 genes in the posterior arches at 60 hpf revealed that con/disp1 CNCC in these PA continued to express sox9b, while sox9a remained absent in these cells (Figure 3E-H).
col2a1 is essential for chondrogenesis [47, 48], and is believed to be dependent on sox9a in CNCC . Consistent with this, col2a1 expression was absent within con/disp1 posterior arch-residing CNCC at 72 hpf, while remaining robustly expressed in the ventral domains of anterior arches (Figure 3I, J)
To further study the role of Hh-signaling in chondrocyte development we assessed runx2b expression in con/disp1 mutants. Mammalian Runx2 genes act as major regulators for osteoblastogenesis . runx2b is expressed in mesenchymal condensations within the PA at 2-3 dpf in zebrafish and controls chondrocyte differentiation [45, 46]. At 72 hpf, we found the gene transcript was present in con/disp1 mesenchyme within each PA condensation, albeit at a reduced level in PA1 (Figure 3K, L). Additionally, run2b was robustly expressed in the opercle (Figure 3K', L' insets) and cleithrum tissue groups in con/disp1 mutants, while its expression was consistently absent in con/disp1 dorsal neurocranial precursor cells (Figure 3K, L). Thus, runx2b expression in PA mesenchyme does not appear to be directly dependent on Hh-signaling.
In full, this expression data suggests that the Hh-pathway likely controls chondrocyte development in the posterior PA through the transcription factor sox9a, although the pathway may be required for broader transcription factor expression in the dorsal jaw precursors.
Disp1 is differentially required for postmigratory-CNCC development gene expression
Fate of CNCC within the posterior arches in con/disp1mutants
The persistence of general neural crest markers in the con/disp1 mesenchymal condensations within the posterior arches (PA3-7) suggests that CNCC survive in these arches. This hypothesis is consistent with our findings, and those by Eberhart et al., 2006 and Wada et al., 2005, which revealed that cell proliferation and cell death levels within the facial primordia of Hh-mutants (severe loss-of-function as well as hypomorphic alleles) are no different than wild type siblings [18, 23]. Despite these three corroborating reports, a separate study by Teraoka et al., 2006 attributed zebrafish jaw outgrowth defects in Cya treated animals to changes in cell death and cell proliferation, which they observed at 65 hpf . Our exhaustive efforts to show differences in cell death (additional file 3) and cell proliferation in the con/disp1 mutant at stages precluding, preceding or including this timepoint were unable to find any significant changes between con/disp1 mutants and wild type embryos.
Hh-signaling is required for cranial joint formation
To provide a potential genetic mechanism for the role of Hh-signaling in cranial joint formation we studied the expression of the bagpipe-related transcription factor bapx1 and the TGFβ signaling molecule gdf5, both of which are expressed in early cartilage condensations and are required for vertebrate joint organization and growth [62–67]. At 63 hpf, ventral views of con/disp1 mutants revealed the presence of two bilateral bapx1 condensations that prefigure the mandibular and hyoid arch lateral joints (Figure 6A). However, when compared to wild type siblings the numbers of bapx1+ cells in these bilateral domains were reduced (Figure 6C). Those remaining bapx1-expressing cells may reside in the PA1 joint region where the Mc and pq elements attempt to set up a joint between the poorly developed cartilage elements (Figure 6H). Furthermore, bapx1 expression was completely reduced within the midline of the con/disp1 mutant (Figure 6C). At 74 hpf, gdf5 expression can be detected in a bilateral population of cells that prefigures the jaw joint, as well as in a cell population at the midline that prefigures the unpaired ventral basihyal (Figure 6B). However, these expression domains are significantly reduced in con/disp1 mutants, correlating with the reduced PA1 midline joint and basihyal cartilage phenotype (Figure 6I and Figure 1D). Together this data reveals novel requirements for Hh-signaling in the development of jaw joints, organization of the first arch RAP cartilage element and the establishment of jaw joint domains during the second day of development, which may be partly attributed to a reduction in early gene expression of two genes, bapx1 and gdf5. This phenotype is likely hidden in many Hh-signaling mutants due to the additional requirement for Hh-signaling on anterior arch chondrogenesis near the joint regions (Figure 6K, findings in this study and ) which in more severely affected larvae leads to the loss of all cranial skeletal elements .
disp1 and shhare co-expressed in brain and facial epithelia during development
Hh-signaling is temporally required early and late to direct ventral cartilage development
Early and late Hedgehog inhibition influences CNCC development
As shown previously, by 48 hpf, con/disp1 CNCC in posterior arches no longer express sox9a, and dlx2a expression is significantly downregulated in the same cells by 60 hpf. We found that Cya-treatment during the late pharyngula stage eliminated sox9a expression (9/12 treated embryos displayed defect, Figure 9K) in mesenchymal condensations within the posterior arches at 48 hpf, and dlx2a by 60 hpf (8/10 treated embryos displayed defect, Figure 9L). In contrast, vehicle treatments never influenced gene expression in the posterior arches (0/10 treated embryos for sox9a and 0/10 treated embryos for dlx2a displayed defects, Figure 9C, D), nor rarely did any earlier Cya-treatments, including during gastrulation (1/11 treated embryos for sox9a, 0/10 treated embryos for dlx2a displayed defects, Figure 9G, H). In our con/disp1 studies above we showed that sox9b and barx1 are regulated independently of Hh-signaling in the majority of PA-derived mesenchyme (an exception is sox9b in dorsal subset of anterior arch mesenchyme, see Figures 3 and 4). Similarly, sox9b and barx1 expression in mesenchymal condensations within the PA were not influenced by Cya treatments during gastrulation or the late pharyngula stage (additional file 4).
Collectively, this data shows that CNCC patterning and chondrogenesis defects in the first arch of Hh-signaling mutants can be attributed to a loss of Hh-signaling during gastrulation stages (4-10 hpf). Moreover, the crucial time for Hh-signaling in promoting posterior arch chondrogenesis is the late pharyngula stage (32-48 hpf).
HPE is a common malformation of the face in humans. Its etiology is highly heterogeneous, likely due to the involvement of multiple genetic factors that are required in a variety of craniofacial tissues and at various times during facial growth and patterning [17, 73]. Disruption of the Hh-signaling pathway is the most frequently reported deficit underlying HPE, or its closely related clinical microforms of the disorder, in humans . For this reason, multiple studies have sought to experimentally demonstrate the requirements for the Hh-signaling pathway in craniofacial tissues and facial precursors. In the zebrafish, these previous studies have either focused solely on the development of the skull and upper jaw [18, 23, 33], or utilized severe loss-of-function alleles representing extreme cases of cranial skeleton loss . Here we provide a detailed characterization for the role of Hh-signaling in the PA skeleton by utilizing a hypomorphic allele of Hh-signaling (con/disp1). Indeed, it has been suggested by others that these less-severe alleles may provide very useful insights into the etiology of Hh-associated HPE as they display characteristic craniofacial abnormalities in the absence of more debilitating phenotypes, such as cyclopia and nervous system deficits [2, 74].
Our study revealed multiple requirements for Hh-signaling during CNCC development that influences the final facial skeletal pattern. First, Hh-signaling controls jaw outgrowth by influencing both patterning and chondrogenic differentiation of postmigratory-CNCC that reside in the anterior arches. Using the Hh-inhibitor Cya we revealed that Hh-signaling is needed during gastrulation for proper jaw formation. Hh-inhibition during gastrulation in zebrafish and chick embryos is also associated with cyclopia and mid-facial dysplasia (data not shown, ), and has also been shown to influence patterning and chondrogenesis in the zebrafish dorsal skeleton [18, 23]. These phenotypes mimic severe loss-of-function mutations within human SHH in HPE, which is characterized by facial anomalies including cyclopia, cleft lip, and underdeveloped jaw . Despite impairing jaw outgrowth, multiple cranial structures remained in the lower jaw of con/disp1 larvae. This not only revealed that some cranial structures were more sensitive to Hh-attenuation (i.e. the dorsal jaw and jaw support elements), but also allowed for detection of deficits in the jaw joint.
Our work further revealed a requirement for Hh-signaling in cartilage development within the posterior gill-forming PA. con/disp1 mutant posterior arch-residing CNCC fail to chondrify, despite becoming properly patterned within well-formed endodermal pouches. Cya treatments revealed the single and crucial time for Hh-signaling in this event is during the late-pharyngula stage, well beyond the time when CNCC have migrated into the PA primordia. It is not surprising that Hh-signaling is required by postmigratory-CNCC in the posterior arches as Hh-ligands become widely expressed throughout the facial epithelial tissues shortly after the time in which CNCC migrate into the face. By examining CNCC differentiation in these arches, we found that certain genes are specifically downregulated in the absence of Hh-signaling, and histological preparation of the gill arches suggest that CNCC are transfated to fibrous-connective tissue as a result. It is clear from these findings that multiple signaling interactions involving the Hh-pathway must occur for proper craniofacial development, and this likely reflects the variability in human phenotypes associated with disruptions to the pathway 
Hh-signaling is required for mandibular arch patterning and chondrogenesis
Frontonasal development and the outgrowth of anterior craniofacial structures are strongly influenced by the facial oral ectoderm [21, 23, 75]. Fate mapping has revealed that dorsal CNCC precursors (neurocranium and pq) condense upon the oral ectoderm roof upon their arrival to the face, while ventral CNCC precursors (Mc and a subset of pq) migrate to, and condense upon, the oral ectoderm floor [18, 41]. The ability of CNCC to condense upon the oral stomodeum is negatively affected by the loss of Hh-signaling , likely resulting in the aberrant localization of first arch CNCC. In our studies of fli1GFP:con/disp1 mutants, CNCC that have migrated into the first arch were conspicuously absent just anterior to the stomodeum, and rather these cells preferentially localized posterior to the stomodeum. Interestingly, the anterior-most CNCC in the fli1GFP:wild type siblings never localized posterior to the stomodeum, suggesting that Hh-signaling may normally act to restrict CNCC from this region, either through promoting CNCC-stomodeum adhesion and/or by inducing midline tissue expansion posterior to the stomodeum that would serve as a physical barrier.
In addition to patterning defects, our studies revealed cell differentiation defects in the anterior-most CNCC. Local morphogenetic signals from the oral ectoderm, such as shh, are likely involved in promoting mesenchymal differentiation and skeletal outgrowth in the jaw and neurocranium. In chick embryos, Shh from the oral ectoderm influences expression of critical CNCC markers to promote maxillary and frontonasal outgrowth [21, 76, 77]. By closely examining the cell differentiation defects in the con/disp1 mutants, we found significant variability in the expression of CNCC markers between mesenchymal condensations in different DV domains within the lower jaw (PA1-2). Notably, neural crest-specific gene expression often appeared to be reduced in con/disp1 dorsal condensations within anterior PA, while expression in ventral condensations were less affected. This is similar to the anterior craniofacial development of Shh-/- mouse embryos wherein severe hypoplasia is seen in the proximal (dorsal) mandibular arch and maxillary mesenchyme, while the more distal (ventral) mandibular arch regions are less affected . Furthermore, a shortened, hypoplastic ventral, first arch Mc element is the only remaining structure when Hh-responsiveness is specifically removed in all cranial precursors (CNCC) in the mouse . Collectively, this suggests that the dorsal jaw, dorsal jaw support and dorsal neurocranium are more sensitive to Hh-attenuation than the ventral jaw, a hypothesis that is supported by the suggestion that dorsal and ventral mandibular identities are controlled by specific sets of transcription factors and signaling molecules . This has been validated by the existence of a number of zebrafish jaw mutants where the dorsal and ventral structures within the first two arches are affected to differing degrees (chameleon, sucker, schmerle) .
Finally, our findings that Hh-signaling is required during gastrulation (4-10 hpf) for proper ventral jaw development is similar to previous findings that this time period is required for proper neurocranium development . Thus, patterning of all anterior craniofacial structures (both dorsal and ventral) is likely controlled by the same Hh-signaling event that originates in the ventral brain primordium during gastrulation, as hypothesized by Eberhart et al., 2006. However, unlike dorsal neurocranium precursors that continue to require Hh-signaling for subsequent chondrogenesis up to 2 dpf , we show that jaw precursors require a single Hh-signaling event during gastrulation for both patterning and chondrogenesis (see Figures 8 and 9). Importantly, this early Hh-signal also influences later gene expression in the oral ectoderm, such as pitx2  and shh itself (data not shown), which are good candidate genes for signals required by jaw precursors for CNCC chondrogenesis. However, more work will be required in order to determine the specific Hh-dependent signals for the proper development of both the neurocranium and jaw in the zebrafish.
Hh-signaling is required for patterning the jaw joint region in the mandibular and hyoid arch
Our studies here reveal multiple requirements for disp1 and Hh-signaling in the development of the jaw joint region. Larval cartilage preparations show absence of the jaw joints, both bilaterally in the anterior arches and within the midline of the mandibular arch. Additional morphological defects included the loss of the RAP extension of the Mc element in the mandibular arch. These phenotypes were reminiscent of bapx1-morphant larvae . bapx1 is expressed in arch mesenchyme and prospective joint domains within the first and second arch and its absence results in a failure of the joints to become properly specified, potentially through influencing gdf5 expression which is required in mouse appendicular and axial joint formation [63, 66, 67]. The joint specifying marker bapx1 was either reduced (bilaterally) or completely absent (midline) in con/disp1 mesenchyme. These bapx1 gene disruptions were sufficient to reduce gdf5 expression to barely detectable amounts in the con/disp1 embryos. Collectively, this data provides a genetic mechanism for Hh-signaling in both the development of jaw joint regions as well as the ventral midline bh element, which is also lost in con/disp1 larvae and requires gdf5 function .
Furthermore, careful analysis of the bilateral joints in the hyoid arch revealed two consistent, varying phenotypes: either ectopic cartilage cells forming an aberrant joint between the hs and ch elements or the complete absence of cartilage cells at the would-be joint site, leading to the failure of hs and ch elements to properly articulate. The presence of these two phenotypes in the mutant hyoid arch cartilages likely reflects two separate requirements for Hh-signaling in CNCC and neighboring cells that reside within future joint sites; first, in CNCC chondrogenesis to promote cartilage formation (as described in Figures 1 and 3) and secondly in joint organization within cells that will eventually chondrify (as described above). The different mutant phenotypes may reflect very slight differences in the amount of Hh-signaling present in the joint regions, as we occasionally visualized both of the described PA2 mutant phenotypes occurring on opposite sides of the same con/disp1 larvae (left and right sides of single larvae shown in Figure 6J, K).
Hh-signaling controls chondrogenic differentiation of CNCC in the posterior arches
Unlike con/disp1 CNCC that have migrated into the mandibular arch, CNCC migrating into the hyoid and posterior arches condense normally. These crest cells become segmentally patterned within well-formed endodermal pouches, indicating that Hh-signaling is not involved in endodermal pouch morphogenesis nor is it required for invading CNCC to adhere to the PA2-7 epithelia. However, our studies reveal that those cells residing in the posterior arches require Hh-signaling to undergo chondrogenic differentiation. This is not the case in the hyoid arch, however, wherein normal CNCC differentiation and subsequent chondrification occurs. Variations in the potential of PA2-7 CNCC to differentiate may be due to intrinsic differences in the ability of these cell groups to respond to Hh-signals, or Hh-dependent co-factor(s), in the facial primordia. CNCC-responsiveness to local facial patterning cues depends in part on their Hox gene expression, which is determined by the original location of these cells in the hindbrain. Moreover, Hox status differs between CNCC in the second arch (hoxa2) and the posterior arches (hoxa2/hoxb3) . In chick embryos, grafts of facial ectoderm or foregut endoderm, into the PA2 region has no influence on the fate of this arch [4, 21]. Rather, PA2 mesenchyme is thought to be influenced by the pharyngeal ectodermal margin, and shh from this tissue has been implicated (directly or indirectly) in hyoid outgrowth [82, 83].
The absence of cartilage elements in the posterior arches of 96 hpf con/disp1 mutant larvae is not due to changes in cell survival or proliferation as we were unable to find any variation in cell death or cell proliferation in the craniofacial region between con/disp1 mutants and their wild type siblings. This is consistent with zebrafish studies utilizing both smu/smo mutants and Shha;Shhb double morphant embryos, which lack dorsal cartilages yet display no changes in cell death or proliferation [18, 23]. Rather, we found that Hh-signaling is involved in maintaining the chondrogenic differentiation potential of posterior arch mesenchyme. Specifically, critical transcription factors, sox9a and dlx2a, become down-regulated in these cells during the second day of development. Notably, other critical transcription factors such as hand2, barx1, msxb, msxe were not downregulated in these cells indicating that these cells persist in the posterior arches in a differentiated state that still resembles their neural crest origin.
Genetic analysis on the sox9a/jellyfish (jef) mutant has revealed a critical function for this gene in cartilage development within the posterior arches, potentially by influencing normal col2a1 expression in a similar fashion to the con/disp1 mutant . The critical importance of sox9a in directing PA3-7 prechondrogenic condensations to develop as chondrocytes is underscored by the findings that other chondrogenic factors, like runx2b, are expressed at normal levels in the sox9a/jef and con/disp1 mutants alike [ and our studies].
So what then happens to the mesenchyme within the posterior arches of con/disp1 mutants? Our histological sections on 5 dpf con/disp1 mutants suggest that CNCC in PA3-7 give rise to fibrous connective tissue, a common derivative of pluripotent mesenchymal neural crest cells. We hypothesize that these ectopic fibrous-connective tissue cells are of CNC origin and have committed to a mesenchymal lineage, instead of a neural one, independently of Hh-signaling. These cells then require the Hh-pathway to adopt, or maintain, a chondrogenic fate. Consistent with this, exogenous Shh can induce multipotent CNCC and pluripotent mesenchymal stem cells to adopt a chondrogenic fate in vitro [84–87]. Examining later skeletal development in the con/disp1 mutant pharyngeal arches is frustrated by early embryonic death by 6-7 dpf in these animals.
Our stage-specific Hh-signaling inhibition studies revealed that the critical time for Hh-signaling to maintain chondrogenic differentiation in posterior arch-residing CNCC is the late pharyngula stage (32-48 hpf). At this time, shh and disp1 are coexpressed in facial ectoderm and endoderm, as well as the ventral brain, making it difficult to interpret the crucial Hh-source for posterior arch cartilage formation. An intriguing tissue source of shh in this process is the pharyngeal endoderm. In casanova mutants, which fail to make endoderm, all PA-derived cartilages are lost . Further, surgical removal of foregut endoderm in chick embryo prior to CNCC migration results in a loss of ventral arch structures, yet arch identity is restored when Shh-soaked heparin beads are placed in the region of the excised tissue . More studies will be required to determine the cell autonomy of Hh-ligands in posterior arch cartilage formation, as well as whether the signal must be interpreted by mesenchymal-neural crest and/or the surrounding epithelia.
Conserved and non-conserved roles for Hh-signaling in PA development
Zebrafish PA skeletal defects in con/disp1 mutants resemble loss-of-function mutations to Shh in mouse [89, 90] and treatments with Shh-blocking-antibodies in chick embryos , indicating a conserved role for Hh-signaling in PA development. However, unlike in fish, the primary defect attributing to the loss of mouse and chick PA skeleton is a high degree of cell death in CNCC as they migrate into the pharyngeal pouches. Additionally in Shh-/- mouse, further cell death is seen in the pharyngeal pouch endoderm , indicating a pro-survival role for Hh-signaling in epithelial and mesenchymal facial tissue.
Hh-signaling may mediate its pro-chondrogenic (zebrafish) or pro-survival (mouse, chick) effects within CNCC through its influence on Sox9 gene activity. We noticed a strong correlation between the loss of sox9a expression in con/disp1 CNCC and the failure of those same cells to chondrify. In Shh-/- mouse there is a striking pattern of Sox9 gene expression loss in streaming CNCC just prior to cell death within the same regions . Inverse experiments, where ectopic Shh was used to stimulate mouse or human chondrocytes revealed that increased levels of Shh and downstream target genes in chondrocytes correlated with higher levels of Sox9 expression [91, 92]. In zebrafish, due to gene duplication there are two orthologs of Sox9, sox9a and sox9b, which are co-expressed in postmigratory-CNCC and they control chondrocyte morphogenesis and survival, respectively [13, 44, 93–95]. These roles are likely controlled by the single tetrapod Sox9 in higher vertebrates and can account for the cell death seen in these animals upon gene loss, whereas we show here that, for the most part, sox9b (survival) gene expression is independent of Hh-signaling in CNCC within the PA.
While it has been previously established that Hh-signaling is required for craniofacial development, the characterization of the con/disp1 mutant has been invaluable for helping us determine precisely the requirements of Hh-signaling in ventral arch development. In full, we found that Hh-signaling is required for jaw size and outgrowth, jaw joint formation and for chondrogenesis within the posterior gill arches. The Hh-pathway controls CNCC patterning and differentiation in jaw precursors, but simply differentiation in gill cartilage precursors. We showed that multiple Hh-signaling events are required and these events are temporally separated and partly unique from those previously described for the neurocranium skeleton.
List of abbreviations
cranial neural crest cells
days post fertilization
- Dispatched 1:
hours post fertilization
posterior ectodermal margin
We thank members of the Ahlgren, Morris, and Topczewski laboratories for discussions and comments on the manuscript. We would like to thank the CMRC fish facility staff for their outstanding fish care. We also thank members of the Henion, Ingham, Mason, Schilling, Topczewski, and Westerfield laboratories for plasmids used in the manuscript. We are indebted to the members of the Raible, Schilling and Whitfield laboratories, as well as the Zebrafish International Resource Center (ZIRC), for fish and reagents used in the manuscript. ZIRC is supported by grant P40 RR012546 (NIH-NCRR). This work was supported by a Ruth L. Kirschtein predoctoral NRSA F31DE019604 (NIH-NIDCR) to T.S. as well as grants from N.I.H (AA13596) and the State of Illinois to S.C.A.
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