BACKGROUND
the vertebrate head skeleton is largely derived from cranial neural crest cells  which originate in the dorsal neural tube and migrate ventrally to populate the pharyngeal arches   <cit> .
the final skeletal pattern depends on intrinsic positional information acquired by cncc prior to migration  <cit> , instructive signals encountered by cncc during migration  <cit> , and morphogenetic cues that cncc receive from surrounding tissues upon completing migration  <cit> .
there are seven paired skeletal elements that arise on either side of the midline.
the cncc of the first pa  forms the jaw, while those in the second pa  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  <cit> .
however, the genes that regulate the production of cartilage from cncc are shared regardless of location  <cit> .
genetic studies have revealed conserved requirements for hedgehog -signaling in craniofacial development.
abrogation of hh-signaling through inactivation of the hh-receptor smoothened  leads to a complete loss of cranial skeletal elements in zebrafish and mouse  <cit> .
it has been pointed out that these animal models represent extreme phenotypes, with complete suppression of signaling, and often result in embryonic lethality  <cit> .
however, mutations within the key hh-family member, sonic hedgehog  in humans can lead to the spectral disorder of holoprosencephaly , often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia  <cit> .
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  <cit> .
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  <cit> .
thus, it is becoming apparent that the range of phenotypes that are apparent in hpe may be in part due to the affected tissue 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  <cit> .
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  <cit> .
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 , a pharmacological antagonist of hh-signaling, administered to whole embryos during the gastrulation period, prior to cncc migration  <cit> .
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  <cit> .
in addition to this, wada et al.,  <dig> 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  <cit> .
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 disp <dig> gene by using the chameleon  mutant zebrafish.
disp <dig> acts within hh-synthesizing cells to disperse hh-ligands into the extracellular field where they are received by hh-target cells  <cit> .
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 disp <dig> mutations  <cit> .
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.
methods
animals
zebrafish  embryos were obtained from natural crosses and staged as previously described  <cit> .
homozygous con/disp1tm15aandb <dig> were identified among their siblings by pcr genotyping prior to  <dig> hours post fertilization   or by somite morphology at later stages  <cit> .
genotyping primers: contm15a_forward, 5'ccagctgtggtggtcttaca3', contm15a_reverse, 5'cagaggcttcttcgcagtct3'; conb392_forward, 5'ccaaagatcattacgatcgaaagaatcga3', conb392_reverse, 5'ccatgtggtctcccatagtcatta <dig> ' next, restriction enzyme reactions were performed to differentiate pcr products, for contm15a we used pvuii  and for b <dig> we used clai  which both digest mutant alleles .
fli1:gfp transgenic zebrafish expressing the gfp-transgene under the control of the fli <dig> promoter was previously characterized  <cit> .
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  <dig> hpf and treated with alcian blue solution dissolved in 80% ethanol/20%glacial acetic acid  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  <cit> .
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  <cit> .
embryos fixed at a stage older than  <dig> hpf were raised in  <dig> 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 cs <dig>
immunohistochemistry was performed as previously described  <cit> .
the zn <dig> antibody  was used at a 1: <dig> dilution in goat serum blocking solution .
confocal stacks of the embryos were generated on a zeiss lsm <dig>
for histological preperations, gelatin-embedded embryos were sectioned at  <dig> μm on a leica cryostat and subjected to gill's 2× hematoxylin and eosin .
embryos were visualized on a leica dmr-hc upright microscope.
tunel staining
for cell death studies, embryos fixed for analysis at a stage older than  <dig> hpf were incubated in  <dig> μg/ml proteinase k solution to solubilize tissues and washed twice in pbs-tween .
embryos were then incubated in the labeling and enzymatic solutions that are provided by the manufacturer of the in situ cell death detection kit  for  <dig> hour at 37°celcius.
enzyme and label solution consists of tdt  and fluorescein-conjugated deoxynucleotides in buffer.
after three washes in pbst to remove non-specific labeling, embryos were bathed in blocking solution  for  <dig> hour at room temperature, followed by incubation in blocking solution containing an anti-fluorescein-conjugated alkaline phosphatase  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  to visualize dying cells.
stained embryos were visualized on a leica mz16f.
cyclopamine treatments
cya  was solubilized in 95% ethanol to obtain a stock solution of  <dig> mm, which was further diluted in zebrafish embryo water to a working stock concentration of  <dig> μm.
embryos were bathed in the cya working stock for 4- <dig> 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- <dig> hpf, wherein they were analyzed by in situ hybridization or cartilage staining.
qpcr analysis
total rna extraction was performed using rna tissue extraction and dnase kits .
cdna was synthesized using superscript iii and random primers .
pcr reactions were performed using platinum sybr green qpcr super mix-udg  on a mj research chroma- <dig> pcr machine and primers: gli1_forward: 5'cagacgtcctctcgccttac <dig> '
gli1_reverse: 5'agtagcgctgtccttgcatt <dig> ' disp1_forward: 5'gctgtagggctttctgtgga <dig> '
disp1_reverse: 5'gccactgttggtaggagcat <dig> ' gapdh_forward: 5'gaaggtgggaaactggtcat <dig> ' gapdh_reverse: 5'ttgcaccacccttaatgtga <dig> ' 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  <cit> , displaying data as fold difference in mutants relative to wild type.
rna microinjections
plasmids to generate full-length shh and flag-tagged shhn , which encodes unmodified shh, have been previously described  <cit> .
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.
~ <dig> nanoliter  of 100- <dig> pg/nl mrna, suspended in  <dig>  m kcl, was pressure-injected at 1- <dig> cell stage.
embryos were then allowed to develop to  <dig> hpf, wherein they were analyzed by cartilage staining.
RESULTS
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  <cit> .
to better understand the role of hh-signaling in this process, we studied cartilage development within two con/disp <dig> mutant alleles: the con/disp1tm15a  and con/disp1b <dig> .
con/disp <dig> mutant phenotypes are less severe than slow-muscle-omitted  mutant phenotypes, making them more amenable for detection of subtle deficits  <cit> .
con/disp1tm15a and con/disp1b <dig> alleles displayed similar penetrance in their craniofacial phenotype, and also shared similar gene expression reductions in gli <dig>  a hh-signaling pathway transcription factor that is sensitive to the pathway's attenuation .
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/disp1tm15a allele, which we will herein refer to as con/disp <dig>
<dig> day old con/disp <dig> mutants were distinguished by an overall reduction in head size and displayed micro- or agnathia due to reduced jaw outgrowth  and significant heart edema when compared to wild type larvae .
a closer examination of cartilage formation at  <dig> hpf using alcian blue staining revealed the mandibular arch cartilages  in wild type larvae to be completely patterned, including the ventral meckel's cartilage  and dorsal palatoquadrate  .
however, con/disp <dig> mc and pq elements were significantly reduced .
hyoid arch cartilages, which support the lower jaw, contain the dorsal hyosymplectics  and ventral ceratohyals .
con/disp <dig> hs and ch cartilages were smaller and dysmorphic, with the ch often inverted posteriorly .
inversion of midline arch elements is a common developmental byproduct of severe posterior arch cartilage disruptions  <cit>  and strikingly the ceratobranchial  cartilages of the five posterior arches  were consistently absent in the con/disp <dig> larvae .
closer examination of the remaining con/disp <dig> jaw elements revealed a complete loss of dorsal cartilage elements comprising the pterygoid process  of the mandibular arch and the symplectic  of the hyoid arch .
additionally, lateral views of cartilage preparations revealed malformed joints in both anterior arches
disp <dig> regulates the release of lipid-modified hh-molecules from their site of synthesis  <cit> .
to determine if disp <dig> action is conserved in the craniofacial skeleton, we compared the outcome on con/disp <dig> cartilages upon ectopic mrna expression of lipid-modified shh  or the n-terminal domain of shh , which retains hh-signaling capacity but fails to become lipid-modified.
injecting offspring from an incross mating of con/disp <dig> heterozygotes with  <dig> pg shhfl mrna at the 1-cell stage yielded no discernable differences in cartilage phenotypes  from their uninjected siblings  , likely due to the inability of lipid-modified-shhfl protein to be secreted from disp1-/-mutant cells.
in contrast, injecting con/disp <dig> heterozygote offspring with  <dig> pg of shhn mrna reproducibly decreased the number of larvae displaying pa-derived cartilage defects  compared to uninjected siblings .
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  in genotype-confirmed con/disp <dig> mutants .
failure of shhn to fully restore con/disp <dig> cartilage defects could be attributed to a loss of potency by non-lipid-modified versions of shh to activate hh-target genes  <cit> .
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  <cit> .
tracking con/disp <dig> cncc development with the fli1:gfp transgene
pa cartilages are derived from cncc in zebrafish, consistent with higher vertebrates  <cit> .
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  <cit> .
like these reports, we found that con/disp <dig> mutants were identical to wild type embryos in cncc specification  and early migration  into the pa as visualized by neural crest markers foxd <dig> and crestin   <cit> .
we tracked late-migrating and postmigratory-cncc by crossing con/disp1+/- fish to a transgenic line in which the fli <dig> promoter drives gfp in cncc   <cit> .
by  <dig> hpf, fli1gfp:con/disp <dig> cncc have completed migration into the arch primordia and are broadly distributed in the future jaw and gills, similar to their fli1gfp:wild type siblings .
by  <dig> hpf, interactions between the post-migratory cncc and surrounding epithelia take place to sequentially pattern the cncc within the seven pa .
ventral views at this stage revealed patterning differences along the anterioposterior  and mediolateral  axis' in fli1gfp:con/disp <dig> mutants .
in wild type embryos, the anterior-most cncc population migrated medial to the eyefield and resided anterior to the stomodeum  .
contrarily, upon migrating between the eyefield, the anterior-most cncc in fli1gfp:con/disp <dig> consistently resided posterior to the stomodeum, where they remained aberrantly patterned in mutant embryos at  <dig> hpf , similar to what has been shown for cncc contributing to the dorsal neurocranium  <cit> .
previous fate-mapping studies of cncc have shown that the anterior-most cncc populations contribute to the dorsal neurocranium and mandibular arch  cartilages  <cit> .
this suggests that the mispatterned population of post-migratory cncc in fli1gfp:con/disp <dig> are at least partly fated to become the ventral jaw  cartilages and thus may likely account for the later patterning defects seen in alcian blue-stained larvae.
fli1gfp:con/disp <dig> cncc patterning within pa <dig> was basically similar to wild type embryos , with the exception that they were located slightly more medial than those in wild type embryos .
viewing fli1gfp:con/disp <dig> at later stages  revealed that con/disp <dig> cncc resided in pa <dig>  yet overall they were less organized and may have defects in stacking.
interestingly, despite the complete absence of con/disp <dig> posterior arch cartilages, we detected no significant disruptions to cncc patterning within con/disp <dig> mutant pa3- <dig> .
further, by  <dig> hpf we clearly visualized demarcations between fli1gfp:con/disp <dig> gfp+ cncc and presumptive gfp- endodermal cells  upon which the cncc that have migrated into pa2- <dig> condense, indicating proper arch segmentation .
by staining embryos with the endodermal pouch-labeling zn <dig> antibody  <cit> , we confirmed that gfp- cells were endodermal and con/disp <dig> pharyngeal pouches were capable of forming .
furthermore, fli1gfp:con/disp <dig> cncc continued to express the fli1gfp-transgene and remained properly interdigitated within pharyngeal pouches up to  <dig> hpf .
despite this,  <dig> hpf alcian blue staining revealed that cncc within the fli1gfp con/disp <dig> posterior arches do not differentiate into cartilage .
taken in full, this data reveals that only the subset of cncc that have migrated into the pa <dig> 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- <dig> as this phenotype has not been studied in more severely affected alleles.
pharyngeal arch chondrocyte differentiation is perturbed in con/disp <dig> mutants
to investigate the role of hh-signaling in cartilage development we examined molecular markers that control chondrogenic and osteogenic differentiation in con/disp <dig> mutants.
we studied the expression patterns of sox9a and sox9b, two transcription factors necessary for chondrogenesis  <cit> , runx2b, a marker of chondrocyte and osteogenic differentiation  <cit> , and type ii collagen , the predominant extracellular matrix protein in cartilage  <cit> .
at  <dig> hpf, sox9a and sox9b expression marked four cncc mesenchymal condensations  in con/disp <dig> mutants and wild type siblings alike .
this indicated that both genes become properly induced in con/disp <dig> cncc.
however, by  <dig> hpf the sox9a transcript was absent in condensations within pa3- <dig> in con/disp <dig> mutants .
in contrast, sox9a expression remained detectable in pa 1-2-residing condensations in con/disp1mutants .
despite the gene expression reductions of sox9a, sox9b expression at  <dig> hpf yielded no detectable differences at this stage between con/disp <dig> and their wild type siblings .
by  <dig> 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 .
it has been suggested that organization of cncc into these dorsoventral  domains likely precedes their later chondrification into dorsal  and ventral  cartilage structures  <cit> .
at this stage, we detected a reduction in the number of sox9a and sox9b expressing cells residing in dorsal domains of pa1- <dig> in con/disp <dig> mutants compared to wild type siblings .
despite this, expression of both genes was retained at normal levels in cncc residing within the pa1- <dig> ventral domains .
expression of the sox <dig> genes in the posterior arches at  <dig> hpf revealed that con/disp <dig> cncc in these pa continued to express sox9b, while sox9a remained absent in these cells .
col2a <dig> is essential for chondrogenesis  <cit> , and is believed to be dependent on sox9a in cncc  <cit> .
consistent with this, col2a <dig> expression was absent within con/disp <dig> posterior arch-residing cncc at  <dig> hpf, while remaining robustly expressed in the ventral domains of anterior arches
to further study the role of hh-signaling in chondrocyte development we assessed runx2b expression in con/disp <dig> mutants.
mammalian runx <dig> genes act as major regulators for osteoblastogenesis  <cit> .
runx2b is expressed in mesenchymal condensations within the pa at 2- <dig> dpf in zebrafish and controls chondrocyte differentiation  <cit> .
at  <dig> hpf, we found the gene transcript was present in con/disp <dig> mesenchyme within each pa condensation, albeit at a reduced level in pa <dig> .
additionally, run2b was robustly expressed in the opercle  and cleithrum tissue groups in con/disp <dig> mutants, while its expression was consistently absent in con/disp <dig> dorsal neurocranial precursor cells .
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.
disp <dig> is differentially required for postmigratory-cncc development gene expression
to continue to study the role of hh-signaling in ventral cartilage development, we next sought to determine the expression of the essential neural crest differentiation markers dlx2a, hand <dig>  barx <dig>  msxb and msxe genes, which are expressed in postmigratory-cncc and whose homologs are required for proper mammalian pa development  <cit> .
expression of all of these genes at  <dig> hpf, or earlier, in con/disp <dig> mutants was indistinguishable from wild type siblings .
however, by  <dig> hpf we detected a reduction in the number of dlx2a+ cells residing in the con/disp <dig> anterior arches , possibly corresponding to the dorsal prechondrogenic condensations , although it was difficult to specifically detect which anterior arch had reduced dlx2a staining within in the con/disp <dig> mutant .
furthermore, we clearly noticed a significant reduction in dlx2a gene expression in the con/disp <dig> posterior arches .
thus, as is the case for sox9a expression , hh-signaling is required to maintain dlx2a expression in the cncc residing in pa3- <dig>  additionally at  <dig> hpf, dlx2a was also detectable in the diencephalon .
simultaneous visualization of brain and cncc by dlx2a staining in con/disp <dig> embryos further underscored the ap and ml patterning defects of cncc within the pa of these mutants .
unlike dlx2a, the remaining genes studied were maintained in con/disp <dig> cncc mesenchyme , despite being slightly downregulated in the dorsal domains of the hypoplastic pa <dig> and pa <dig>  suggesting that these genes may be regulated independently of hh-signaling in the pa.
fate of cncc within the posterior arches in con/disp <dig> mutants
the persistence of general neural crest markers in the con/disp <dig> mesenchymal condensations within the posterior arches  suggests that cncc survive in these arches.
this hypothesis is consistent with our findings, and those by eberhart et al.,  <dig> and wada et al.,  <dig>  which revealed that cell proliferation and cell death levels within the facial primordia of hh-mutants  are no different than wild type siblings  <cit> .
despite these three corroborating reports, a separate study by teraoka et al.,  <dig> attributed zebrafish jaw outgrowth defects in cya treated animals to changes in cell death and cell proliferation, which they observed at  <dig> hpf  <cit> .
our exhaustive efforts to show differences in cell death  and cell proliferation in the con/disp <dig> mutant at stages precluding, preceding or including this timepoint were unable to find any significant changes between con/disp <dig> mutants and wild type embryos.
an alternative hypothesis to cncc failing to proliferate or survive in the pa and thus accounting for reduced craniofacial cartilage, could be that cncc persist in the pa, but do so as a non-cartilage cncc derivative.
mesenchymal cncc that have migrated into the pa primordia are multipotent, able to give rise to fibrous-connective tissue such as smooth muscle cells and myofibroblasts, in addition to specialized-connective tissue such as facial cartilage  <cit> .
therefore, in the absence of pro-chondrogenic gene expression  we wondered if the persisting posterior arch-residing cncc gave rise to fibrous-connective tissue or an alternative cell type.
to address this, we stained  <dig> day-old wild type and con/disp <dig> larvae with hematoxylin and eosin in order to differentiate cell types.
wild type larvae revealed the presence of chondrocytes  in each pa .
viewing pa <dig> at higher magnification revealed that chondroctyes were neatly arranged into a circular rod, lying dorsal to the arch's muscle cell , and surrounded by a mass of fibrous-connective tissue  .
within con/disp <dig> larvae, chondrocytes were clearly present within the hyoid arch, however chondrocytes were not detectable in the posterior arches .
rather, these arches were populated fully by fibrous-connective tissue .
similar to the wild type pa <dig> organization, con/disp <dig> pa <dig> ectopic fibrous-connective tissue cells were organized within a small circular rod, lying just dorsal to the arch's muscle cell, surrounded by connective tissue in a highly ordered fashion .
horizontal sections through the con/disp <dig> pa confirmed the widespread presence of fibrous-connective tissue within pa3- <dig> .
thus, in the absence of hh-signaling cncc in the posterior arches become, or alternatively remain, fibrous-connective tissue cells.
this may potentially explain why this study and others have not found an increased number of dying cells in the heads of hh-signaling mutants despite the lack of cartilage elements in the pa  or the neurocranium  <cit>  of these animals.
hh-signaling is required for cranial joint formation
our analysis of the mandibular and hyoid arch cartilages in con/disp <dig> mutants revealed abnormal joints .
by  <dig> hpf, bilateral joints were present between the ventral mc and dorsal pq of the mandibular arch  and the ventral ch and the dorsal hs of the hyoid arch .
frequently in the con/disp <dig> mutant, the cartilaginous retroarticular process  that forms at the posterior extent of the mc element was reduced and the jaw joint between the mc and pq was difficult to visualize .
in the con/disp <dig> mutant hyoid arch, ectopic cartilage cells were often present between the ventral ch and dorsal hyomandibular  cartilage elements , where localized inhibition of chondrogenesis at the joint site normally produces a thin interhyal  element .
less frequently, dorsal and ventral pa <dig> cartilage elements failed to articulate with one another .
furthermore, midline joint formation was also disrupted in con/disp <dig> mutants, whereas ectopic cartilage condensed at the presumptive mandibular arch joint domain between bilateral mc elements .
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 bapx <dig> and the tgfβ signaling molecule gdf <dig>  both of which are expressed in early cartilage condensations and are required for vertebrate joint organization and growth  <cit> .
at  <dig> hpf, ventral views of con/disp <dig> mutants revealed the presence of two bilateral bapx <dig> condensations that prefigure the mandibular and hyoid arch lateral joints .
however, when compared to wild type siblings the numbers of bapx1+ cells in these bilateral domains were reduced .
those remaining bapx1-expressing cells may reside in the pa <dig> joint region where the mc and pq elements attempt to set up a joint between the poorly developed cartilage elements .
furthermore, bapx <dig> expression was completely reduced within the midline of the con/disp <dig> mutant .
at  <dig> hpf, gdf <dig> 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 .
however, these expression domains are significantly reduced in con/disp <dig> mutants, correlating with the reduced pa <dig> midline joint and basihyal cartilage phenotype .
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, bapx <dig> and gdf <dig>  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  which in more severely affected larvae leads to the loss of all cranial skeletal elements  <cit> .
disp <dig> and shh are co-expressed in brain and facial epithelia during development
to determine the cells that produce and secrete hh-ligands during craniofacial development we investigated the expression patterns for disp <dig> and shh.
we confirmed that both genes are co-expressed within the dorsal mesoderm during gastrulation and persist in their notochord and presomitic-mesoderm derivatives during somitogenesis  <cit> .
by 22- <dig> hpf both genes are localized to the ventral brain  .
by  <dig> hpf, shh expression is expanded to the oral ectoderm  and the pharyngeal endoderm  and by  <dig> hpf we also could visualize robust expression in the pharyngeal ectodermal margin   where shh remained up to  <dig> hpf .
disp <dig> rna transcripts appeared to be expressed in a similar spatiotemporal fashion to shh in neural and facial tissues , although specific tissue detection for this rna probe was made difficult due to a high signal-to-noise ratio in embryos older than  <dig> hpf.
despite this limitation, and based on the conserved functional role for disp <dig> in hh-expressing cells and our visualization of disp <dig> transcripts in facial tissue beginning at  <dig> hpf, we strongly believe that disp <dig> is localized to the oe, pem and pe tissues by  <dig> days post fertilization.
hh-signaling is temporally required early and late to direct ventral cartilage development
the dynamic expression pattern of shh, first in the brain and then in facial epithelia, coupled with previous findings from wada et al.,  <dig> that hh-signaling controls dorsal neurocranium chondrogenesis at timepoints from gastrulation up to  <dig> days post fertilization, prompted us to study the temporal requirement for hh-signaling in pa skeleton development.
we treated wild type embryos with cya, to antagonize hh-signaling activation by compromising smo action  <cit> , at different developmental stages from 4- <dig> hpf.
a  <dig> μm cya concentration was chosen as this concentration has been used by our laboratory and others to effectively block hh-signaling sensitive ptc <dig> or gli <dig> expression  <cit> .
embryos treated during gastrulation , then allowed to develop to  <dig> hpf for cartilage analysis by alcian blue staining, exhibited a general decrease in mc and pq chondrogenesis and jaw outgrowth when compared to vehicle-treated control embryos alone .
the mandibular arch cartilage defects of gastrulation-stage treated embryos were similar to those described for the con/disp <dig> mutant .
despite this, hyoid and posterior arch cartilages were only slightly reduced and fairly well patterned in gastrulation stage treated embryos .
cya-treatments administered during stages of early neurulation and neural crest specification  occasionally resulted in reduced mandibular arch cartilages but rarely affected other pa cartilages .
next, we found that in contrast to earlier treatments, cya-treatments during cncc migration  or during the timepoint critical for cncc condensation within facial epithelial  produced no negative effects on the development of the pa elements .
we next treated embryos with cya during the late pharyngula stage .
strikingly, embryos treated during this stage exhibited severe cb cartilage loss in the posterior arches .
despite this, cya treatment during the late pharyngula stage had only minor anterior arch deficits, including inverted mc and ch elements, a common developmental by-product in the pa when the gill cartilages are absent  <cit> .
finally, blocking hh-signaling in embryos after  <dig> hpf had no influence on pa cartilage development .
collectively, this data indicates two critical time periods during which hh-signaling is required for pa cartilage development, one early  to direct jaw outgrowth and chondrogenesis within portions of the anterior arches, and one later  to promote chondrogenesis in the posterior arches .
early and late hedgehog inhibition influences cncc development
in light of our cya results on ventral skeleton development, we wondered if cya treatments during gastrulation and the late pharyngula stage could together explain the cncc defects we identified in con/disp <dig> mutants.
we found that treating embryos with cya during gastrulation  sufficiently and reproducibly disrupted the patterning of pa1-localized fli1:gfp+cncc at  <dig> hpf , while vehicle-treated siblings never displayed patterning defects .
mispatterned cncc in pa <dig> were aberrantly localized posterior to the eyefield/presumptive mouth, similar to con/disp <dig> mutants .
later treatments, notably during the late pharyngula stage , did not result in pa <dig> cncc patterning defects .
thus, the crucial time for hh-signaling in promoting proper patterning of cncc within pa <dig> is during gastrulation .
along with pa <dig> patterning defects, we found that sox9a expression was significantly reduced in the pa <dig> dorsal domain  at  <dig> hpf in embryos treated with cya during gastrulation stages , correlating with the reduced dorsal cartilages in the anterior arches .
despite reduced sox9a gene reduction in the dorsal first arch domain, gastrulation-stage treated embryos displayed normal sox9a expression in the ventral domain  as well as small subset of dorsal-lying cells, likely correlating to the remaining pq cartilage elements , similar to the con/disp <dig> mutant cartilage phenotype .
unlike gastrulation-stage treated embryos, vehicle-treated siblings displayed normal sox9a expression in the pa <dig> , as did siblings treated after gastrulation, including the late pharyngula stage .
as shown previously, by  <dig> hpf, con/disp <dig> cncc in posterior arches no longer express sox9a, and dlx2a expression is significantly downregulated in the same cells by  <dig> hpf.
we found that cya-treatment during the late pharyngula stage eliminated sox9a expression  in mesenchymal condensations within the posterior arches at  <dig> hpf, and dlx2a by  <dig> hpf .
in contrast, vehicle treatments never influenced gene expression in the posterior arches , nor rarely did any earlier cya-treatments, including during gastrulation .
in our con/disp <dig> studies above we showed that sox9b and barx <dig> are regulated independently of hh-signaling in the majority of pa-derived mesenchyme .
similarly, sox9b and barx <dig> expression in mesenchymal condensations within the pa were not influenced by cya treatments during gastrulation or the late pharyngula stage .
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 .
moreover, the crucial time for hh-signaling in promoting posterior arch chondrogenesis is the late pharyngula stage .
discussion
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  <cit> .
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  <cit> .
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  <cit> , or utilized severe loss-of-function alleles representing extreme cases of cranial skeleton loss  <cit> .
here we provide a detailed characterization for the role of hh-signaling in the pa skeleton by utilizing a hypomorphic allele of hh-signaling .
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  <cit> .
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 , and has also been shown to influence patterning and chondrogenesis in the zebrafish dorsal skeleton  <cit> .
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  <cit> .
despite impairing jaw outgrowth, multiple cranial structures remained in the lower jaw of con/disp <dig> larvae.
this not only revealed that some cranial structures were more sensitive to hh-attenuation , 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/disp <dig> 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  <cit>
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  <cit> .
fate mapping has revealed that dorsal cncc precursors  condense upon the oral ectoderm roof upon their arrival to the face, while ventral cncc precursors  migrate to, and condense upon, the oral ectoderm floor  <cit> .
the ability of cncc to condense upon the oral stomodeum is negatively affected by the loss of hh-signaling  <cit> , likely resulting in the aberrant localization of first arch cncc.
in our studies of fli1gfp:con/disp <dig> 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  <cit> .
by closely examining the cell differentiation defects in the con/disp <dig> mutants, we found significant variability in the expression of cncc markers between mesenchymal condensations in different dv domains within the lower jaw .
notably, neural crest-specific gene expression often appeared to be reduced in con/disp <dig> 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  mandibular arch and maxillary mesenchyme, while the more distal  mandibular arch regions are less affected  <cit> .
furthermore, a shortened, hypoplastic ventral, first arch mc element is the only remaining structure when hh-responsiveness is specifically removed in all cranial precursors  in the mouse  <cit> .
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  <cit> .
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   <cit> .
finally, our findings that hh-signaling is required during gastrulation  for proper ventral jaw development is similar to previous findings that this time period is required for proper neurocranium development  <cit> .
thus, patterning of all anterior craniofacial structures  is likely controlled by the same hh-signaling event that originates in the ventral brain primordium during gastrulation, as hypothesized by eberhart et al.,  <dig>  however, unlike dorsal neurocranium precursors that continue to require hh-signaling for subsequent chondrogenesis up to  <dig> dpf  <cit> , we show that jaw precursors require a single hh-signaling event during gastrulation for both patterning and chondrogenesis .
importantly, this early hh-signal also influences later gene expression in the oral ectoderm, such as pitx <dig>  <cit>  and shh itself , 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 disp <dig> 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  <cit> .
bapx <dig> 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 gdf <dig> expression which is required in mouse appendicular and axial joint formation  <cit> .
the joint specifying marker bapx <dig> was either reduced  or completely absent  in con/disp <dig> mesenchyme.
these bapx <dig> gene disruptions were sufficient to reduce gdf <dig> expression to barely detectable amounts in the con/disp <dig> 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/disp <dig> larvae and requires gdf <dig> function  <cit> .
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  and secondly in joint organization within cells that will eventually chondrify .
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 pa <dig> mutant phenotypes occurring on opposite sides of the same con/disp <dig> larvae .
hh-signaling controls chondrogenic differentiation of cncc in the posterior arches
unlike con/disp <dig> 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- <dig> 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- <dig> 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, 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  and the posterior arches   <cit> .
in chick embryos, grafts of facial ectoderm or foregut endoderm, into the pa <dig> region has no influence on the fate of this arch  <cit> .
rather, pa <dig> mesenchyme is thought to be influenced by the pharyngeal ectodermal margin, and shh from this tissue has been implicated  in hyoid outgrowth  <cit> .
the absence of cartilage elements in the posterior arches of  <dig> hpf con/disp <dig> 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/disp <dig> 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  <cit> .
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 hand <dig>  barx <dig>  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  mutant has revealed a critical function for this gene in cartilage development within the posterior arches, potentially by influencing normal col2a <dig> expression in a similar fashion to the con/disp <dig> mutant  <cit> .
the critical importance of sox9a in directing pa3- <dig> 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/disp <dig> mutants alike .
so what then happens to the mesenchyme within the posterior arches of con/disp <dig> mutants?
our histological sections on  <dig> dpf con/disp <dig> mutants suggest that cncc in pa3- <dig> 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  <cit> .
examining later skeletal development in the con/disp <dig> mutant pharyngeal arches is frustrated by early embryonic death by 6- <dig> 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 .
at this time, shh and disp <dig> 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  <cit> .
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  <cit> .
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/disp <dig> mutants resemble loss-of-function mutations to shh in mouse  <cit>  and treatments with shh-blocking-antibodies in chick embryos  <cit> , 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  <cit> , indicating a pro-survival role for hh-signaling in epithelial and mesenchymal facial tissue.
hh-signaling may mediate its pro-chondrogenic  or pro-survival  effects within cncc through its influence on sox <dig> gene activity.
we noticed a strong correlation between the loss of sox9a expression in con/disp <dig> cncc and the failure of those same cells to chondrify.
in shh-/- mouse there is a striking pattern of sox <dig> gene expression loss in streaming cncc just prior to cell death within the same regions  <cit> .
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 sox <dig> expression  <cit> .
in zebrafish, due to gene duplication there are two orthologs of sox <dig>  sox9a and sox9b, which are co-expressed in postmigratory-cncc and they control chondrocyte morphogenesis and survival, respectively  <cit> .
these roles are likely controlled by the single tetrapod sox <dig> 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  gene expression is independent of hh-signaling in cncc within the pa.
CONCLUSIONS
while it has been previously established that hh-signaling is required for craniofacial development, the characterization of the con/disp <dig> 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
ap: anterioposterior; bh: basihyal; cb: ceratobranchials; ch: ceratohyal; cl: cleithrum; cncc: cranial neural crest cells; cya: cyclopamine; dpf: days post fertilization; dispatched 1: disp1; hh: hedgehog; hpe: holoprosencephaly; hpf: hours post fertilization; hs: hyosymplectic; hy: hyomandibular element; m: prospective mouth/stomodeum; mc: meckel's cartilage; ml: mediolateral; ne: neuroectoderm; op: opercle; pa: pharyngeal arches; pq: palatoquadrate; ptp: ptyergoid process; oe: oral ectoderm; pe: pharyngeal endoderm; pem: posterior ectodermal margin; shh: sonic hedgehog; smo: smoothened; sy: symplectic; tr: trabeculae.
authors' contributions
ts participated in the design of the study, carried out all the experiments and drafted the manuscript.
sca participated in its design and coordination and revised and approved the manuscript.
supplementary material
additional file 1
con/disp1b <dig> allele encodes a prematurely truncated dispatched <dig>   ventral views of  <dig>  dpf alcian blue-stained cranial cartilage in con/disp1b <dig>  and con/disp1tm15a  larvae are phenotypically similar.
both con/disp <dig> mutant alleles reveal a similar decrease in whole-embryo gene transcript levels of hh-signaling responsive gene gli <dig> by qpcr.
in contrast, disp <dig> rna levels are similar between con/disp <dig> mutants and their wild type siblings, indicating that disp <dig> gene levels are not sensitive to hh-signaling in whole embryos.
sequence traces of disp <dig> from the con/disp1b <dig> allele reveal a premature stop codon in the sixth exon.
schematic representation of the disp <dig> protein.
arrows indicate sites of single base changes that introduce stop codons within disp <dig> gene as it corresponds to the mutant alleles used in this manuscript .
pcr genotyping strategy which utilizes a primer to introduce two unique nucleotides into disp <dig> amino acids  <dig> and  <dig>  these pcr generated mutations, when combined with the con/disp1b <dig> c to t transition in amino acid  <dig>  generates a unique clai recognition site.
contrary to this, the clai site is not created in a wild type allele .
the clai recognition site co-segregates with the con/disp1b <dig> mutant phenotype.
larvae were sorted at  <dig> hpf by characteristic mutant shh-signaling axis defects, then genotyped for the clai rflp by pcr and gel electrophoresis using primers within the genomic sequence of the sixth exon and subsequently digested by clai.
clai digestion of the con/disp1b392mutant allele generates both  <dig> base pair  and  <dig> bp fragments.
clai fails to digest wild type alleles which results in an  <dig> bp fragment .
the  <dig> bp fragments are not shown in  as they are hard to delineate within resulting primer dimers generated by the pcr and are unnecessary for our genotyping conclusions as the  <dig> bp fragment is sufficient.
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additional file 2
specification and migration of cncc occur normally in con/disp <dig> mutants.
dorsal , or lateral  views of  <dig> hpf ,  <dig> hpf , or  <dig> hpf  wild type  or con/disp <dig> mutants  embryos labeled with rna probe to foxd <dig> , crestin  or sox9a .
the patterns are indistinguishable between wild type and con/disp <dig> mutants at  <dig> hpf  and  <dig> hpf .
sox9a marks cnc condensations at  <dig> hpf in wild type embryos  and con/disp <dig> mutants alike .
click here for file
additional file 3
con/disp <dig> mutants do not display an increase in cell death within pa.  lateral views displaying tunel positive cells in  <dig> hpf wild type  or con/disp <dig>  embryos.
red arrows in  are showing a portion of the cells we counted positive to reflect a dying cell in the pharyngeal region.
the pharyngeal region was defined by us to include all tissue ventral to the eye and otic vesicle and dorsal to the heart and yolk.
graph displaying the mean number of dying cells  for  <dig> wild type embryos  and the mean number of dying cells  for  <dig> con/disp <dig> mutants .
this was not considered significant  by a two-tailed student t-test.
these results reflect a lack of cell death at  <dig> hpf, which is consistent with what we saw at earlier and later stages .
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additional file 4
sox9b and barx <dig> expression following early and late cya treatments.
lateral views of  <dig> hpf  or ventral views of  <dig> hpf  embryos that were untreated  or treated with cya from 4- <dig> hpf  or 32- <dig> hpf  that are labeled with rna probe for sox9b  or barx <dig> .
cya treatments at these stages did not disrupt sox9b or barx <dig> expression patterns within mesenchymal condensation in pa.
click here for file
acknowledgements
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 , for fish and reagents used in the manuscript.
zirc is supported by grant p <dig> rr <dig> .
this work was supported by a ruth l. kirschtein predoctoral nrsa f31de <dig>  to t.s.
as well as grants from n.i.h  and the state of illinois to s.c.a.

 the vertebrate head skeleton is largely derived from cranial neural crest cells which originate in the dorsal neural tube and migrate ventrally to populate the pharyngeal arches. the final skeletal pattern depends on intrinsic positional information acquired by cncc prior to migration, instructive signals encountered by cncc during migration, and morphogenetic cues that cncc receive from surrounding tissues upon completing migration. there are seven paired skeletal elements that arise on either side of the midline. the cncc of the first pa forms the jaw, while those in the second pa 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. however, the genes that regulate the production of cartilage from cncc are shared regardless of location. genetic studies have revealed conserved requirements for hedgehog -signaling in craniofacial development. abrogation of hh-signaling through inactivation of the hh-receptor smoothened leads to a complete loss of cranial skeletal elements in zebrafish and mouse. 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 in humans can lead to the spectral disorder of holoprosencephaly, 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. thus, it is becoming apparent that the range of phenotypes that are apparent in hpe may be in part due to the affected tissue 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. 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, a pharmacological antagonist of hh-signaling, administered to whole embryos during the gastrulation period, prior to cncc migration. 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., 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. 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 disp gene by using the chameleon mutant zebrafish. disp 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 disp 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. methods animals zebrafish embryos were obtained from natural crosses and staged as previously described. homozygous con/disp1tm15aandb were identified among their siblings by pcr genotyping prior to hours post fertilization or by somite morphology at later stages. genotyping primers: contm15a_forward, 5'ccagctgtggtggtcttaca3', contm15a_reverse, 5'cagaggcttcttcgcagtct3'; conb392_forward, 5'ccaaagatcattacgatcgaaagaatcga3', conb392_reverse, 5'ccatgtggtctcccatagtcatta' next, restriction enzyme reactions were performed to differentiate pcr products, for contm15a we used pvuii and for b we used clai which both digest mutant alleles. fli1:gfp transgenic zebrafish expressing the gfp-transgene under the control of the fli 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 hpf and treated with alcian blue solution dissolved in 80% ethanol/20%glacial acetic acid 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 hpf were raised in 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 cs immunohistochemistry was performed as previously described. the zn antibody was used at a 1: dilution in goat serum blocking solution. confocal stacks of the embryos were generated on a zeiss lsm for histological preperations, gelatin-embedded embryos were sectioned at μm on a leica cryostat and subjected to gill's 2× hematoxylin and eosin. embryos were visualized on a leica dmr-hc upright microscope. tunel staining for cell death studies, embryos fixed for analysis at a stage older than hpf were incubated in μg/ml proteinase k solution to solubilize tissues and washed twice in pbs-tween. embryos were then incubated in the labeling and enzymatic solutions that are provided by the manufacturer of the in situ cell death detection kit for hour at 37°celcius. enzyme and label solution consists of tdt and fluorescein-conjugated deoxynucleotides in buffer. after three washes in pbst to remove non-specific labeling, embryos were bathed in blocking solution for hour at room temperature, followed by incubation in blocking solution containing an anti-fluorescein-conjugated alkaline phosphatase 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 to visualize dying cells. stained embryos were visualized on a leica mz16f. cyclopamine treatments cya was solubilized in 95% ethanol to obtain a stock solution of mm, which was further diluted in zebrafish embryo water to a working stock concentration of μm. embryos were bathed in the cya working stock for 4- 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- hpf, wherein they were analyzed by in situ hybridization or cartilage staining. qpcr analysis total rna extraction was performed using rna tissue extraction and dnase kits. cdna was synthesized using superscript iii and random primers. pcr reactions were performed using platinum sybr green qpcr super mix-udg on a mj research chroma- pcr machine and primers: gli1_forward: 5'cagacgtcctctcgccttac' gli1_reverse: 5'agtagcgctgtccttgcatt' disp1_forward: 5'gctgtagggctttctgtgga' disp1_reverse: 5'gccactgttggtaggagcat' gapdh_forward: 5'gaaggtgggaaactggtcat' gapdh_reverse: 5'ttgcaccacccttaatgtga' 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. rna microinjections plasmids to generate full-length shh and flag-tagged shhn, which encodes unmodified shh, have been previously described. 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. ~ nanoliter of 100- pg/nl mrna, suspended in m kcl, was pressure-injected at 1- cell stage. embryos were then allowed to develop to 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/disp mutant alleles: the con/disp1tm15a and con/disp1b. con/disp mutant phenotypes are less severe than slow-muscle-omitted mutant phenotypes, making them more amenable for detection of subtle deficits. con/disp1tm15a and con/disp1b alleles displayed similar penetrance in their craniofacial phenotype, and also shared similar gene expression reductions in gli a hh-signaling pathway transcription factor that is sensitive to the pathway's attenuation. 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/disp1tm15a allele, which we will herein refer to as con/disp day old con/disp mutants were distinguished by an overall reduction in head size and displayed micro- or agnathia due to reduced jaw outgrowth and significant heart edema when compared to wild type larvae. a closer examination of cartilage formation at hpf using alcian blue staining revealed the mandibular arch cartilages in wild type larvae to be completely patterned, including the ventral meckel's cartilage and dorsal palatoquadrate. however, con/disp mc and pq elements were significantly reduced. hyoid arch cartilages, which support the lower jaw, contain the dorsal hyosymplectics and ventral ceratohyals. con/disp hs and ch cartilages were smaller and dysmorphic, with the ch often inverted posteriorly. inversion of midline arch elements is a common developmental byproduct of severe posterior arch cartilage disruptions and strikingly the ceratobranchial cartilages of the five posterior arches were consistently absent in the con/disp larvae. closer examination of the remaining con/disp jaw elements revealed a complete loss of dorsal cartilage elements comprising the pterygoid process of the mandibular arch and the symplectic of the hyoid arch. additionally, lateral views of cartilage preparations revealed malformed joints in both anterior arches disp regulates the release of lipid-modified hh-molecules from their site of synthesis. to determine if disp action is conserved in the craniofacial skeleton, we compared the outcome on con/disp cartilages upon ectopic mrna expression of lipid-modified shh or the n-terminal domain of shh, which retains hh-signaling capacity but fails to become lipid-modified. injecting offspring from an incross mating of con/disp heterozygotes with pg shhfl mrna at the 1-cell stage yielded no discernable differences in cartilage phenotypes from their uninjected siblings, likely due to the inability of lipid-modified-shhfl protein to be secreted from disp1-/-mutant cells. in contrast, injecting con/disp heterozygote offspring with pg of shhn mrna reproducibly decreased the number of larvae displaying pa-derived cartilage defects compared to uninjected siblings. 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 in genotype-confirmed con/disp mutants. failure of shhn to fully restore con/disp 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/disp cncc development with the fli1:gfp transgene pa cartilages are derived from cncc in zebrafish, consistent with higher vertebrates. 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. like these reports, we found that con/disp mutants were identical to wild type embryos in cncc specification and early migration into the pa as visualized by neural crest markers foxd and crestin. we tracked late-migrating and postmigratory-cncc by crossing con/disp1+/- fish to a transgenic line in which the fli promoter drives gfp in cncc. by hpf, fli1gfp:con/disp cncc have completed migration into the arch primordia and are broadly distributed in the future jaw and gills, similar to their fli1gfp:wild type siblings. by hpf, interactions between the post-migratory cncc and surrounding epithelia take place to sequentially pattern the cncc within the seven pa. ventral views at this stage revealed patterning differences along the anterioposterior and mediolateral axis' in fli1gfp:con/disp mutants. in wild type embryos, the anterior-most cncc population migrated medial to the eyefield and resided anterior to the stomodeum. contrarily, upon migrating between the eyefield, the anterior-most cncc in fli1gfp:con/disp consistently resided posterior to the stomodeum, where they remained aberrantly patterned in mutant embryos at hpf, similar to what has been shown for cncc contributing to the dorsal neurocranium. previous fate-mapping studies of cncc have shown that the anterior-most cncc populations contribute to the dorsal neurocranium and mandibular arch cartilages. this suggests that the mispatterned population of post-migratory cncc in fli1gfp:con/disp are at least partly fated to become the ventral jaw cartilages and thus may likely account for the later patterning defects seen in alcian blue-stained larvae. fli1gfp:con/disp cncc patterning within pa was basically similar to wild type embryos, with the exception that they were located slightly more medial than those in wild type embryos. viewing fli1gfp:con/disp at later stages revealed that con/disp cncc resided in pa yet overall they were less organized and may have defects in stacking. interestingly, despite the complete absence of con/disp posterior arch cartilages, we detected no significant disruptions to cncc patterning within con/disp mutant pa3-. further, by hpf we clearly visualized demarcations between fli1gfp:con/disp gfp+ cncc and presumptive gfp- endodermal cells upon which the cncc that have migrated into pa2- condense, indicating proper arch segmentation. by staining embryos with the endodermal pouch-labeling zn antibody, we confirmed that gfp- cells were endodermal and con/disp pharyngeal pouches were capable of forming. furthermore, fli1gfp:con/disp cncc continued to express the fli1gfp-transgene and remained properly interdigitated within pharyngeal pouches up to hpf. despite this, hpf alcian blue staining revealed that cncc within the fli1gfp con/disp posterior arches do not differentiate into cartilage. taken in full, this data reveals that only the subset of cncc that have migrated into the pa 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- as this phenotype has not been studied in more severely affected alleles. pharyngeal arch chondrocyte differentiation is perturbed in con/disp mutants to investigate the role of hh-signaling in cartilage development we examined molecular markers that control chondrogenic and osteogenic differentiation in con/disp mutants. we studied the expression patterns of sox9a and sox9b, two transcription factors necessary for chondrogenesis, runx2b, a marker of chondrocyte and osteogenic differentiation, and type ii collagen, the predominant extracellular matrix protein in cartilage. at hpf, sox9a and sox9b expression marked four cncc mesenchymal condensations in con/disp mutants and wild type siblings alike. this indicated that both genes become properly induced in con/disp cncc. however, by hpf the sox9a transcript was absent in condensations within pa3- in con/disp mutants. in contrast, sox9a expression remained detectable in pa 1-2-residing condensations in con/disp1mutants. despite the gene expression reductions of sox9a, sox9b expression at hpf yielded no detectable differences at this stage between con/disp and their wild type siblings. by 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. it has been suggested that organization of cncc into these dorsoventral domains likely precedes their later chondrification into dorsal and ventral cartilage structures. at this stage, we detected a reduction in the number of sox9a and sox9b expressing cells residing in dorsal domains of pa1- in con/disp mutants compared to wild type siblings. despite this, expression of both genes was retained at normal levels in cncc residing within the pa1- ventral domains. expression of the sox genes in the posterior arches at hpf revealed that con/disp cncc in these pa continued to express sox9b, while sox9a remained absent in these cells. col2a is essential for chondrogenesis, and is believed to be dependent on sox9a in cncc. consistent with this, col2a expression was absent within con/disp posterior arch-residing cncc at hpf, while remaining robustly expressed in the ventral domains of anterior arches to further study the role of hh-signaling in chondrocyte development we assessed runx2b expression in con/disp mutants. mammalian runx genes act as major regulators for osteoblastogenesis. runx2b is expressed in mesenchymal condensations within the pa at 2- dpf in zebrafish and controls chondrocyte differentiation. at hpf, we found the gene transcript was present in con/disp mesenchyme within each pa condensation, albeit at a reduced level in pa. additionally, run2b was robustly expressed in the opercle and cleithrum tissue groups in con/disp mutants, while its expression was consistently absent in con/disp dorsal neurocranial precursor cells. 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. disp is differentially required for postmigratory-cncc development gene expression to continue to study the role of hh-signaling in ventral cartilage development, we next sought to determine the expression of the essential neural crest differentiation markers dlx2a, hand barx msxb and msxe genes, which are expressed in postmigratory-cncc and whose homologs are required for proper mammalian pa development. expression of all of these genes at hpf, or earlier, in con/disp mutants was indistinguishable from wild type siblings. however, by hpf we detected a reduction in the number of dlx2a+ cells residing in the con/disp anterior arches, possibly corresponding to the dorsal prechondrogenic condensations, although it was difficult to specifically detect which anterior arch had reduced dlx2a staining within in the con/disp mutant. furthermore, we clearly noticed a significant reduction in dlx2a gene expression in the con/disp posterior arches. thus, as is the case for sox9a expression, hh-signaling is required to maintain dlx2a expression in the cncc residing in pa3- additionally at hpf, dlx2a was also detectable in the diencephalon. simultaneous visualization of brain and cncc by dlx2a staining in con/disp embryos further underscored the ap and ml patterning defects of cncc within the pa of these mutants. unlike dlx2a, the remaining genes studied were maintained in con/disp cncc mesenchyme, despite being slightly downregulated in the dorsal domains of the hypoplastic pa and pa suggesting that these genes may be regulated independently of hh-signaling in the pa. fate of cncc within the posterior arches in con/disp mutants the persistence of general neural crest markers in the con/disp mesenchymal condensations within the posterior arches suggests that cncc survive in these arches. this hypothesis is consistent with our findings, and those by eberhart et al., and wada et al., which revealed that cell proliferation and cell death levels within the facial primordia of hh-mutants are no different than wild type siblings. despite these three corroborating reports, a separate study by teraoka et al., attributed zebrafish jaw outgrowth defects in cya treated animals to changes in cell death and cell proliferation, which they observed at hpf. our exhaustive efforts to show differences in cell death and cell proliferation in the con/disp mutant at stages precluding, preceding or including this timepoint were unable to find any significant changes between con/disp mutants and wild type embryos. an alternative hypothesis to cncc failing to proliferate or survive in the pa and thus accounting for reduced craniofacial cartilage, could be that cncc persist in the pa, but do so as a non-cartilage cncc derivative. mesenchymal cncc that have migrated into the pa primordia are multipotent, able to give rise to fibrous-connective tissue such as smooth muscle cells and myofibroblasts, in addition to specialized-connective tissue such as facial cartilage. therefore, in the absence of pro-chondrogenic gene expression we wondered if the persisting posterior arch-residing cncc gave rise to fibrous-connective tissue or an alternative cell type. to address this, we stained day-old wild type and con/disp larvae with hematoxylin and eosin in order to differentiate cell types. wild type larvae revealed the presence of chondrocytes in each pa. viewing pa at higher magnification revealed that chondroctyes were neatly arranged into a circular rod, lying dorsal to the arch's muscle cell, and surrounded by a mass of fibrous-connective tissue. within con/disp larvae, chondrocytes were clearly present within the hyoid arch, however chondrocytes were not detectable in the posterior arches. rather, these arches were populated fully by fibrous-connective tissue. similar to the wild type pa organization, con/disp pa ectopic fibrous-connective tissue cells were organized within a small circular rod, lying just dorsal to the arch's muscle cell, surrounded by connective tissue in a highly ordered fashion. horizontal sections through the con/disp pa confirmed the widespread presence of fibrous-connective tissue within pa3-. thus, in the absence of hh-signaling cncc in the posterior arches become, or alternatively remain, fibrous-connective tissue cells. this may potentially explain why this study and others have not found an increased number of dying cells in the heads of hh-signaling mutants despite the lack of cartilage elements in the pa or the neurocranium of these animals. hh-signaling is required for cranial joint formation our analysis of the mandibular and hyoid arch cartilages in con/disp mutants revealed abnormal joints. by hpf, bilateral joints were present between the ventral mc and dorsal pq of the mandibular arch and the ventral ch and the dorsal hs of the hyoid arch. frequently in the con/disp mutant, the cartilaginous retroarticular process that forms at the posterior extent of the mc element was reduced and the jaw joint between the mc and pq was difficult to visualize. in the con/disp mutant hyoid arch, ectopic cartilage cells were often present between the ventral ch and dorsal hyomandibular cartilage elements, where localized inhibition of chondrogenesis at the joint site normally produces a thin interhyal element. less frequently, dorsal and ventral pa cartilage elements failed to articulate with one another. furthermore, midline joint formation was also disrupted in con/disp mutants, whereas ectopic cartilage condensed at the presumptive mandibular arch joint domain between bilateral mc elements. 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 bapx and the tgfβ signaling molecule gdf both of which are expressed in early cartilage condensations and are required for vertebrate joint organization and growth. at hpf, ventral views of con/disp mutants revealed the presence of two bilateral bapx condensations that prefigure the mandibular and hyoid arch lateral joints. however, when compared to wild type siblings the numbers of bapx1+ cells in these bilateral domains were reduced. those remaining bapx1-expressing cells may reside in the pa joint region where the mc and pq elements attempt to set up a joint between the poorly developed cartilage elements. furthermore, bapx expression was completely reduced within the midline of the con/disp mutant. at hpf, gdf 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. however, these expression domains are significantly reduced in con/disp mutants, correlating with the reduced pa midline joint and basihyal cartilage phenotype. 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, bapx and gdf 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 which in more severely affected larvae leads to the loss of all cranial skeletal elements. disp and shh are co-expressed in brain and facial epithelia during development to determine the cells that produce and secrete hh-ligands during craniofacial development we investigated the expression patterns for disp and shh. we confirmed that both genes are co-expressed within the dorsal mesoderm during gastrulation and persist in their notochord and presomitic-mesoderm derivatives during somitogenesis. by 22- hpf both genes are localized to the ventral brain. by hpf, shh expression is expanded to the oral ectoderm and the pharyngeal endoderm and by hpf we also could visualize robust expression in the pharyngeal ectodermal margin where shh remained up to hpf. disp rna transcripts appeared to be expressed in a similar spatiotemporal fashion to shh in neural and facial tissues, although specific tissue detection for this rna probe was made difficult due to a high signal-to-noise ratio in embryos older than hpf. despite this limitation, and based on the conserved functional role for disp in hh-expressing cells and our visualization of disp transcripts in facial tissue beginning at hpf, we strongly believe that disp is localized to the oe, pem and pe tissues by days post fertilization. hh-signaling is temporally required early and late to direct ventral cartilage development the dynamic expression pattern of shh, first in the brain and then in facial epithelia, coupled with previous findings from wada et al., that hh-signaling controls dorsal neurocranium chondrogenesis at timepoints from gastrulation up to days post fertilization, prompted us to study the temporal requirement for hh-signaling in pa skeleton development. we treated wild type embryos with cya, to antagonize hh-signaling activation by compromising smo action, at different developmental stages from 4- hpf. a μm cya concentration was chosen as this concentration has been used by our laboratory and others to effectively block hh-signaling sensitive ptc or gli expression. embryos treated during gastrulation, then allowed to develop to hpf for cartilage analysis by alcian blue staining, exhibited a general decrease in mc and pq chondrogenesis and jaw outgrowth when compared to vehicle-treated control embryos alone. the mandibular arch cartilage defects of gastrulation-stage treated embryos were similar to those described for the con/disp mutant. despite this, hyoid and posterior arch cartilages were only slightly reduced and fairly well patterned in gastrulation stage treated embryos. cya-treatments administered during stages of early neurulation and neural crest specification occasionally resulted in reduced mandibular arch cartilages but rarely affected other pa cartilages. next, we found that in contrast to earlier treatments, cya-treatments during cncc migration or during the timepoint critical for cncc condensation within facial epithelial produced no negative effects on the development of the pa elements. we next treated embryos with cya during the late pharyngula stage. strikingly, embryos treated during this stage exhibited severe cb cartilage loss in the posterior arches. despite this, cya treatment during the late pharyngula stage had only minor anterior arch deficits, including inverted mc and ch elements, a common developmental by-product in the pa when the gill cartilages are absent. finally, blocking hh-signaling in embryos after hpf had no influence on pa cartilage development. collectively, this data indicates two critical time periods during which hh-signaling is required for pa cartilage development, one early to direct jaw outgrowth and chondrogenesis within portions of the anterior arches, and one later to promote chondrogenesis in the posterior arches. early and late hedgehog inhibition influences cncc development in light of our cya results on ventral skeleton development, we wondered if cya treatments during gastrulation and the late pharyngula stage could together explain the cncc defects we identified in con/disp mutants. we found that treating embryos with cya during gastrulation sufficiently and reproducibly disrupted the patterning of pa1-localized fli1:gfp+cncc at hpf, while vehicle-treated siblings never displayed patterning defects. mispatterned cncc in pa were aberrantly localized posterior to the eyefield/presumptive mouth, similar to con/disp mutants. later treatments, notably during the late pharyngula stage, did not result in pa cncc patterning defects. thus, the crucial time for hh-signaling in promoting proper patterning of cncc within pa is during gastrulation. along with pa patterning defects, we found that sox9a expression was significantly reduced in the pa dorsal domain at hpf in embryos treated with cya during gastrulation stages, correlating with the reduced dorsal cartilages in the anterior arches. despite reduced sox9a gene reduction in the dorsal first arch domain, gastrulation-stage treated embryos displayed normal sox9a expression in the ventral domain as well as small subset of dorsal-lying cells, likely correlating to the remaining pq cartilage elements, similar to the con/disp mutant cartilage phenotype. unlike gastrulation-stage treated embryos, vehicle-treated siblings displayed normal sox9a expression in the pa, as did siblings treated after gastrulation, including the late pharyngula stage. as shown previously, by hpf, con/disp cncc in posterior arches no longer express sox9a, and dlx2a expression is significantly downregulated in the same cells by hpf. we found that cya-treatment during the late pharyngula stage eliminated sox9a expression in mesenchymal condensations within the posterior arches at hpf, and dlx2a by hpf. in contrast, vehicle treatments never influenced gene expression in the posterior arches, nor rarely did any earlier cya-treatments, including during gastrulation. in our con/disp studies above we showed that sox9b and barx are regulated independently of hh-signaling in the majority of pa-derived mesenchyme. similarly, sox9b and barx expression in mesenchymal condensations within the pa were not influenced by cya treatments during gastrulation or the late pharyngula stage. 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. moreover, the crucial time for hh-signaling in promoting posterior arch chondrogenesis is the late pharyngula stage. discussion 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. 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, 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. 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. 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, and has also been shown to influence patterning and chondrogenesis in the zebrafish dorsal skeleton. 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/disp larvae. this not only revealed that some cranial structures were more sensitive to hh-attenuation, 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/disp 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. fate mapping has revealed that dorsal cncc precursors condense upon the oral ectoderm roof upon their arrival to the face, while ventral cncc precursors migrate to, and condense upon, the oral ectoderm floor. 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/disp 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. by closely examining the cell differentiation defects in the con/disp mutants, we found significant variability in the expression of cncc markers between mesenchymal condensations in different dv domains within the lower jaw. notably, neural crest-specific gene expression often appeared to be reduced in con/disp 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 mandibular arch and maxillary mesenchyme, while the more distal 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 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. finally, our findings that hh-signaling is required during gastrulation 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 is likely controlled by the same hh-signaling event that originates in the ventral brain primordium during gastrulation, as hypothesized by eberhart et al., however, unlike dorsal neurocranium precursors that continue to require hh-signaling for subsequent chondrogenesis up to dpf, we show that jaw precursors require a single hh-signaling event during gastrulation for both patterning and chondrogenesis. importantly, this early hh-signal also influences later gene expression in the oral ectoderm, such as pitx and shh itself, 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 disp 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. bapx 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 gdf expression which is required in mouse appendicular and axial joint formation. the joint specifying marker bapx was either reduced or completely absent in con/disp mesenchyme. these bapx gene disruptions were sufficient to reduce gdf expression to barely detectable amounts in the con/disp 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/disp larvae and requires gdf 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 and secondly in joint organization within cells that will eventually chondrify. 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 pa mutant phenotypes occurring on opposite sides of the same con/disp larvae. hh-signaling controls chondrogenic differentiation of cncc in the posterior arches unlike con/disp 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- 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- 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, 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 and the posterior arches. in chick embryos, grafts of facial ectoderm or foregut endoderm, into the pa region has no influence on the fate of this arch. rather, pa mesenchyme is thought to be influenced by the pharyngeal ectodermal margin, and shh from this tissue has been implicated in hyoid outgrowth. the absence of cartilage elements in the posterior arches of hpf con/disp 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/disp 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. 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 hand barx 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 mutant has revealed a critical function for this gene in cartilage development within the posterior arches, potentially by influencing normal col2a expression in a similar fashion to the con/disp mutant. the critical importance of sox9a in directing pa3- 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/disp mutants alike. so what then happens to the mesenchyme within the posterior arches of con/disp mutants? our histological sections on dpf con/disp mutants suggest that cncc in pa3- 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. examining later skeletal development in the con/disp mutant pharyngeal arches is frustrated by early embryonic death by 6- 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. at this time, shh and disp 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/disp mutants resemble loss-of-function mutations to shh in mouse 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 or pro-survival effects within cncc through its influence on sox gene activity. we noticed a strong correlation between the loss of sox9a expression in con/disp cncc and the failure of those same cells to chondrify. in shh-/- mouse there is a striking pattern of sox 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 sox expression. in zebrafish, due to gene duplication there are two orthologs of sox sox9a and sox9b, which are co-expressed in postmigratory-cncc and they control chondrocyte morphogenesis and survival, respectively. these roles are likely controlled by the single tetrapod sox 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 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/disp 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 ap: anterioposterior; bh: basihyal; cb: ceratobranchials; ch: ceratohyal; cl: cleithrum; cncc: cranial neural crest cells; cya: cyclopamine; dpf: days post fertilization; dispatched 1: disp1; hh: hedgehog; hpe: holoprosencephaly; hpf: hours post fertilization; hs: hyosymplectic; hy: hyomandibular element; m: prospective mouth/stomodeum; mc: meckel's cartilage; ml: mediolateral; ne: neuroectoderm; op: opercle; pa: pharyngeal arches; pq: palatoquadrate; ptp: ptyergoid process; oe: oral ectoderm; pe: pharyngeal endoderm; pem: posterior ectodermal margin; shh: sonic hedgehog; smo: smoothened; sy: symplectic; tr: trabeculae. authors' contributions ts participated in the design of the study, carried out all the experiments and drafted the manuscript. sca participated in its design and coordination and revised and approved the manuscript. supplementary material additional file 1 con/disp1b allele encodes a prematurely truncated dispatched ventral views of dpf alcian blue-stained cranial cartilage in con/disp1b and con/disp1tm15a larvae are phenotypically similar. both con/disp mutant alleles reveal a similar decrease in whole-embryo gene transcript levels of hh-signaling responsive gene gli by qpcr. in contrast, disp rna levels are similar between con/disp mutants and their wild type siblings, indicating that disp gene levels are not sensitive to hh-signaling in whole embryos. sequence traces of disp from the con/disp1b allele reveal a premature stop codon in the sixth exon. schematic representation of the disp protein. arrows indicate sites of single base changes that introduce stop codons within disp gene as it corresponds to the mutant alleles used in this manuscript. pcr genotyping strategy which utilizes a primer to introduce two unique nucleotides into disp amino acids and these pcr generated mutations, when combined with the con/disp1b c to t transition in amino acid generates a unique clai recognition site. contrary to this, the clai site is not created in a wild type allele. the clai recognition site co-segregates with the con/disp1b mutant phenotype. larvae were sorted at hpf by characteristic mutant shh-signaling axis defects, then genotyped for the clai rflp by pcr and gel electrophoresis using primers within the genomic sequence of the sixth exon and subsequently digested by clai. clai digestion of the con/disp1b392mutant allele generates both base pair and bp fragments. clai fails to digest wild type alleles which results in an bp fragment. the bp fragments are not shown in as they are hard to delineate within resulting primer dimers generated by the pcr and are unnecessary for our genotyping conclusions as the bp fragment is sufficient. click here for file additional file 2 specification and migration of cncc occur normally in con/disp mutants. dorsal, or lateral views of hpf, hpf, or hpf wild type or con/disp mutants embryos labeled with rna probe to foxd, crestin or sox9a. the patterns are indistinguishable between wild type and con/disp mutants at hpf and hpf. sox9a marks cnc condensations at hpf in wild type embryos and con/disp mutants alike. click here for file additional file 3 con/disp mutants do not display an increase in cell death within pa. lateral views displaying tunel positive cells in hpf wild type or con/disp embryos. red arrows in are showing a portion of the cells we counted positive to reflect a dying cell in the pharyngeal region. the pharyngeal region was defined by us to include all tissue ventral to the eye and otic vesicle and dorsal to the heart and yolk. graph displaying the mean number of dying cells for wild type embryos and the mean number of dying cells for con/disp mutants. this was not considered significant by a two-tailed student t-test. these results reflect a lack of cell death at hpf, which is consistent with what we saw at earlier and later stages. click here for file additional file 4 sox9b and barx expression following early and late cya treatments. lateral views of hpf or ventral views of hpf embryos that were untreated or treated with cya from 4- hpf or 32- hpf that are labeled with rna probe for sox9b or barx. cya treatments at these stages did not disrupt sox9b or barx expression patterns within mesenchymal condensation in pa. click here for file acknowledgements 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, for fish and reagents used in the manuscript. zirc is supported by grant p rr. this work was supported by a ruth l. kirschtein predoctoral nrsa f31de to t.s. as well as grants from n.i.h and the state of illinois to s.c.a.
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. genetic studies in zebrafish and mice have established that the hedgehog -signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in cncc development. 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 disp disrupted normal expression of bapx and gdf markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp mutant animals. cncc specification and migration into the pa primorida occurs normally in con/disp mutants, however disp is necessary for post-migratory cncc patterning and differentiation. we show that disp 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. con/disp mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. disruption of the hh-signaling pathway in humans can lead to the spectral disorder of holoprosencephaly, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia. 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. lipid-modified hh-ligands require the transmembrane-spanning receptor dispatched for proper secretion from hh-synthesizing cells to the extracellular field where they act on target cells. the vertebrate head skeleton is derived largely from cranial neural crest cells. histology reveals that posterior arch residing-cncc differentiate into fibrous-connective tissue, rather than becoming chondrocytes.
 the vertebrate head skeleton is derived largely from cranial neural crest cells. genetic studies in zebrafish and mice have established that the hedgehog -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, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia. 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 skeleton. lipid-modified hh-ligands require the transmembrane-spanning receptor dispatched 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 disp  con/disp 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/disp mutants, however disp is necessary for post-migratory cncc patterning and differentiation. we show that disp 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 disp disrupted normal expression of bapx and gdf markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp 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. 