Mutations in cdon and boc affect trunk neural crest cell migration and slow-twitch muscle development in zebrafish.

The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon and boc may play additional functions in regulating directed cell movements. We use newly generated and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects in the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a-containing extracellular matrix, suggesting that neural crest defects may be a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and suggest that the zebrafish can be used to study the function of hedgehog receptor paralogs.

PRDM paralogs antagonistically balance Wnt/ß-catenin activity during craniofacial chondrocyte differentiation.

Cranial neural crest cell (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either paralog results in hypoplastic and disorganized chondrocytes due to impaired cellular orientation and polarity. We show that these proteins regulate cartilage differentiation by controlling the timing of Wnt/ß-catenin activity in strikingly different ways: Prdm3 represses whereas Prdm16 activates global gene expression, although both act by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/ß-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/ß-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.

The power of zebrafish models for understanding the co-occurrence of craniofacial and limb disorders.

Craniofacial and limb defects are two of the most common congenital anomalies in the general population. Interestingly, these defects are not mutually exclusive. Many patients with craniofacial phenotypes, such as orofacial clefting and craniosynostosis, also present with limb defects, including polydactyly, syndactyly, brachydactyly, or ectrodactyly. The gene regulatory networks governing craniofacial and limb development initially seem distinct from one another, and yet these birth defects frequently occur together. Both developmental processes are highly conserved among vertebrates, and zebrafish have emerged as an advantageous model due to their high fecundity, relative ease of genetic manipulation, and transparency during development. Here we summarize studies that have used zebrafish models to study human syndromes that present with both craniofacial and limb phenotypes. We discuss the highly conserved processes of craniofacial and limb/fin development and describe recent zebrafish studies that have explored the function of genes associated with human syndromes with phenotypes in both structures. We attempt to identify commonalities between the two to help explain why craniofacial and limb anomalies often occur together.

GCN5 acetylation is required for craniofacial chondrocyte maturation.

Development of the craniofacial structures requires the precise differentiation of cranial neural crest cells into osteoblasts or chondrocytes. Here, we explore the epigenetic and non-epigenetic mechanisms that are required for the development of craniofacial chondrocytes. We previously demonstrated that the acetyltransferase activity of the highly conserved acetyltransferase GCN5, or KAT2A, is required for murine craniofacial development. We show that Gcn5 is required cell autonomously in the cranial neural crest. Moreover, GCN5 is required for chondrocyte development following the arrival of the cranial neural crest within the pharyngeal arches. Using a combination of in vivo and in vitro inhibition of GCN5 acetyltransferase activity, we demonstrate that GCN5 is a potent activator of chondrocyte maturation, acting to control chondrocyte maturation and size increase during pre-hypertrophic maturation to hypertrophic chondrocytes. Rather than acting as an epigenetic regulator of histone H3K9 acetylation, our findings suggest GCN5 primarily acts as a non-histone acetyltransferase to regulate chondrocyte development. Here, we investigate the contribution of GCN5 acetylation to the activity of the mTORC1 pathway. Our findings indicate that GCN5 acetylation is required for activation of this pathway, either via direct activation of mTORC1 or through indirect mechanisms. We also investigate one possibility of how mTORC1 activity is regulated through RAPTOR acetylation, which is hypothesized to enhance mTORC1 downstream phosphorylation. This study contributes to our understanding of the specificity of acetyltransferases, and the cell type specific roles in which these enzymes function.

The conserved and divergent roles of Prdm3 and Prdm16 in zebrafish and mouse craniofacial development.

The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.

Loss of prdm1a accelerates melanoma onset and progression.

Melanoma is an aggressive, deadly skin cancer derived from melanocytes, a neural crest cell derivative. Melanoma cells mirror the developmental program of neural crest cells in that they exhibit the same gene expression patterns and utilize similar cellular mechanisms, including increased cell proliferation, epithelial-mesenchymal transition, and migration. Here we studied the role of neural crest regulator PRDM1 in melanoma onset and progression. In development, Prdm1a functions to promote neural crest progenitor fate, and in melanoma, we found that PRDM1 has reduced copy number and is recurrently deleted in both zebrafish and humans. When examining expression of neural crest and melanocyte development genes, we show that sox10 progenitor expression is high in prdm1a-/- mutants, while more differentiated melanocyte markers are reduced, suggesting that normally Prdm1a is required for differentiation. Data mining of human melanoma datasets indicates that high PRDM1 expression in human melanoma is correlated with better patient survival and decreased PRDM1 expression is common in metastatic tumors. When one copy of prdm1a is lost in the zebrafish melanoma model Tg(mitfa:BRAFV600E );p53-/- ;prdm1a+/- , melanoma onset occurs more quickly, and the tumors that form have a larger area with increased expression of sox10. These data demonstrate a novel role for PRDM1 as a tumor suppressor in melanoma.

Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway.

The old and new face of craniofacial research: How animal models inform human craniofacial genetic and clinical data.

The craniofacial skeletal structures that comprise the human head develop from multiple tissues that converge to form the bones and cartilage of the face. Because of their complex development and morphogenesis, many human birth defects arise due to disruptions in these cellular populations. Thus, determining how these structures normally develop is vital if we are to gain a deeper understanding of craniofacial birth defects and devise treatment and prevention options. In this review, we will focus on how animal model systems have been used historically and in an ongoing context to enhance our understanding of human craniofacial development. We do this by first highlighting "animal to man" approaches; that is, how animal models are being utilized to understand fundamental mechanisms of craniofacial development. We discuss emerging technologies, including high throughput sequencing and genome editing, and new animal repository resources, and how their application can revolutionize the future of animal models in craniofacial research. Secondly, we highlight "man to animal" approaches, including the current use of animal models to test the function of candidate human disease variants. Specifically, we outline a common workflow deployed after discovery of a potentially disease causing variant based on a select set of recent examples in which human mutations are investigated in vivo using animal models. Collectively, these topics will provide a pipeline for the use of animal models in understanding human craniofacial development and disease for clinical geneticist and basic researchers alike.

Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development.

Kabuki syndrome (KS) is a rare multiple congenital anomaly syndrome characterized by distinctive facial features, global developmental delay, intellectual disability and cardiovascular and musculoskeletal abnormalities. While mutations in KMT2D have been identified in a majority of KS patients, a few patients have mutations in KDM6A. We analyzed 40 individuals clinically diagnosed with KS for mutations in KMT2D and KDM6A. Mutations were detected in KMT2D in 12 and KDM6A in 4 cases, respectively. Observed mutations included single-nucleotide variations and indels leading to frame shifts, nonsense, missense or splice-site alterations. In two cases, we discovered overlapping chromosome X microdeletions containing KDM6A. To further elucidate the functional roles of KMT2D and KDM6A, we knocked down the expression of their orthologs in zebrafish. Following knockdown of kmt2d and the two zebrafish paralogs kdm6a and kdm6al, we analyzed morphants for developmental abnormalities in tissues that are affected in individuals with KS, including craniofacial structures, heart and brain. The kmt2d morphants exhibited severe abnormalities in all tissues examined. Although the kdm6a and kdm6al morphants had similar brain abnormalities, kdm6a morphants exhibited craniofacial phenotypes, whereas kdm6al morphants had prominent defects in heart development. Our results provide further support for the similar roles of KMT2D and KDM6A in the etiology of KS by using a vertebrate model organism to provide direct evidence of their roles in the development of organs and tissues affected in KS patients.

Cdon promotes neural crest migration by regulating N-cadherin localization.

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

MicroRNA-30a regulates zebrafish myogenesis through targeting the transcription factor Six1.

Precise spatiotemporal regulation of the SIX1 homeoprotein is required to coordinate vital tissue development, including myogenesis. Whereas SIX1 is downregulated in most tissues following embryogenesis, it is re-expressed in numerous cancers, including tumors derived from muscle progenitors. Despite crucial roles in development and disease, the upstream regulation of SIX1 expression has remained elusive. Here, we identify the first direct mechanism for Six1 regulation in embryogenesis, through microRNA30a (miR30a)-mediated repression. In zebrafish somites, we show that miR30a and six1a and six1b (hereafter six1a/b) are expressed in an inverse temporal pattern. Overexpression of miR30a leads to a reduction in six1a/b levels, and results in increased apoptosis and altered somite morphology, which phenocopies six1a/b knockdown. Conversely, miR30a inhibition leads to increased Six1 expression and abnormal somite morphology, revealing a role for endogenous miR30a as a muscle-specific miRNA (myomiR). Importantly, restoration of six1a in miR30a-overexpressing embryos restores proper myogenesis. These data demonstrate a new role for miR30a at a key node in the myogenic regulatory gene network through controlling Six1 expression.

Prdm1a directly activates foxd3 and tfap2a during zebrafish neural crest specification.

The neural crest comprises multipotent precursor cells that are induced at the neural plate border by a series of complex signaling and genetic interactions. Several transcription factors, termed neural crest specifiers, are necessary for early neural crest development; however, the nature of their interactions and regulation is not well understood. Here, we have established that the PR/SET domain-containing transcription factor Prdm1a is co-expressed with two essential neural crest specifiers, foxd3 and tfap2a, at the neural plate border. Through rescue experiments, chromatin immunoprecipitation and reporter assays, we have determined that Prdm1a directly binds to and transcriptionally activates enhancers for foxd3 and tfap2a and that they are functional, direct targets of Prdm1a at the neural plate border. Additionally, analysis of dominant activator and dominant repressor Prdm1a constructs suggests that Prdm1a is required both as a transcriptional activator and transcriptional repressor for neural crest development in zebrafish embryos.

prdm1a functions upstream of itga5 in zebrafish craniofacial development.

Cranial neural crest cells are specified and migrate into the pharyngeal arches where they subsequently interact with the surrounding environment. Signaling and transcription factors, such as prdm1a regulate this interaction, but it remains unclear which specific factors are required for posterior pharyngeal arch development. Previous analysis suggests that prdm1a is required for posterior ceratobranchial cartilages in zebrafish and microarray analysis between wildtype and prdm1a mutants at 25 h post fertilization demonstrated that integrin α5 (itga5) is differentially expressed in prdm1a mutants. Here, we further investigate the interaction between prdm1a and itga5 in zebrafish craniofacial development. In situ hybridization for itga5 demonstrates that expression of itga5 is decreased in prdm1a mutants between 18 and 31 h post fertilization and itga5 expression overlaps with prdm1a in the posterior arches, suggesting a temporal window for interaction. Double mutants for prdm1a;itga5 have an additive viscerocranium phenotype more similar to prdm1a mutants, suggesting that prdm1a acts upstream of itga5. Consistent with this, loss of posterior pharyngeal arch expression of dlx2a, ceratobranchial cartilages 2-5, and cell proliferation in prdm1a mutants can be rescued with itga5 mRNA injection. Taken together, these data suggest that prdm1a acts upstream of itga5 and are both necessary for posterior pharyngeal arch development in zebrafish.

Hcfc1b, a zebrafish ortholog of HCFC1, regulates craniofacial development by modulating mmachc expression.

Mutations in HCFC1 (MIM300019), have been recently associated with cblX (MIM309541), an X-linked, recessive disorder characterized by multiple congenital anomalies including craniofacial abnormalities. HCFC1 is a transcriptional co-regulator that modulates the expression of numerous downstream target genes including MMACHC, but it is not clear how these HCFC1 targets play a role in the clinical manifestations of cblX. To begin to elucidate the mechanism by which HCFC1 modulates disease phenotypes, we have carried out loss of function analyses in the developing zebrafish. Of the two HCFC1 orthologs in zebrafish, hcfc1a and hcfc1b, the loss of hcfc1b specifically results in defects in craniofacial development. Subsequent analysis revealed that hcfc1b regulates cranial neural crest cell differentiation and proliferation within the posterior pharyngeal arches. Further, the hcfc1b-mediated craniofacial abnormalities were rescued by expression of human MMACHC, a downstream target of HCFC1 that is aberrantly expressed in cblX. Furthermore, we tested distinct human HCFC1 mutations for their role in craniofacial development and demonstrated variable effects on MMACHC expression in humans and craniofacial development in zebrafish. Notably, several individuals with mutations in either HCFC1 or MMACHC have been reported to have mild to moderate facial dysmorphia. Thus, our data demonstrates that HCFC1 plays a role in craniofacial development, which is in part mediated through the regulation of MMACHC expression.

The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migration.

The homeodomain transcription factor Nkx2.2 is essential for pancreatic development and islet cell type differentiation. We have identified Tm4sf4, an L6 domain tetraspanin family member, as a transcriptional target of Nkx2.2 that is greatly upregulated during pancreas development in Nkx2.2(-/-) mice. Tetraspanins and L6 domain proteins recruit other membrane receptors to form active signaling centers that coordinate processes such as cell adhesion, migration and differentiation. In this study, we determined that Tm4sf4 is localized to the ductal epithelial compartment and is prominent in the Ngn3(+) islet progenitor cells. We also established that pancreatic tm4sf4 expression and regulation by Nkx2.2 is conserved during zebrafish development. Loss-of-function studies in zebrafish revealed that tm4sf4 inhibits α and ß cell specification, but is necessary for ε cell fates. Thus, Tm4sf4 functional output opposes that of Nkx2.2. Further investigation of how Tm4sf4 functions at the cellular level in vitro showed that Tm4sf4 inhibits Rho-activated cell migration and actin organization in a ROCK-independent fashion. We propose that the primary role of Nkx2.2 is to inhibit Tm4sf4 in endocrine progenitor cells, allowing for delamination, migration and/or appropriate cell fate decisions. Identification of a role for Tm4sf4 during endocrine differentiation provides insight into islet progenitor cell behaviors and potential targetable regenerative mechanisms.

Redundant roles of PRDM family members in zebrafish craniofacial development.

BACKGROUND: PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. RESULTS: Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. CONCLUSIONS: Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish.

A morpholino-based screen to identify novel genes involved in craniofacial morphogenesis.

BACKGROUND: The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS: This set of ∼80 genes was used for a high-throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53-dependent and -independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS: This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human.

Epigenetic regulation of craniofacial development and disease.

BACKGROUND: The formation of the craniofacial complex relies on proper neural crest development. The gene regulatory networks (GRNs) and signaling pathways orchestrating this process have been extensively studied. These GRNs and signaling cascades are tightly regulated as alterations to any stage of neural crest development can lead to common congenital birth defects, including multiple syndromes affecting facial morphology as well as nonsyndromic facial defects, such as cleft lip with or without cleft palate. Epigenetic factors add a hierarchy to the regulation of transcriptional networks and influence the spatiotemporal activation or repression of specific gene regulatory cascades; however less is known about their exact mechanisms in controlling precise gene regulation. AIMS: In this review, we discuss the role of epigenetic factors during neural crest development, specifically during craniofacial development and how compromised activities of these regulators contribute to congenital defects that affect the craniofacial complex.

Human split hand/foot variants are not as functional as wildtype human PRDM1 in the rescue of craniofacial defects.

BACKGROUND: Split hand/foot malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor, PRDM1, that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants in PRDM1 had not been associated with craniofacial anomalies. METHODS: Using transient mRNA overexpression assays in prdm1a-/- mutant zebrafish, we tested whether the PRDM1 SHFM variants were functional and could lead to a rescue of the craniofacial defects observed in prdm1a-/- mutants. We also mined previously published CUT&RUN and RNA-seq datasets that sorted EGFP-positive cells from a Tg(Mmu:Prx1-EGFP) transgenic line that labels the pectoral fin, pharyngeal arches, and dorsal part of the head to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes. RESULTS: The prdm1a-/- mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtype (WT) hPRDM1 in prdm1a-/- mutants partially rescues the palatoquadrate phenotype. However, injection of each of the three SHFM variants fails to rescue this skeletal defect. Loss of prdm1a leads to a decreased expression of important craniofacial genes by RNA-seq, including emilin3a, confirmed by hybridization chain reaction expression. Other genes including dlx5a/dlx6a, hand2, sox9b, col2a1a, and hoxb genes are also reduced. Validation by real-time quantitative PCR in the anterior half of zebrafish embryos failed to confirm the expression changes suggesting that the differences are enriched in prx1 expressing cells. CONCLUSION: These data suggest that the three SHFM variants are likely not functional and may be associated with the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate genes involved in craniofacial development.

Identification and characterization of the zebrafish pharyngeal arch-specific enhancer for the basic helix-loop-helix transcription factor Hand2.

The development of the vertebrate jaw relies on a network of transcription factors that patterns the dorsal-ventral axis of the pharyngeal arches. Recent findings in both mouse and zebrafish illustrate that the basic-helix-loop-helix transcription factor, Hand2, is crucial in this patterning process. While Hand2 has functionally similar roles in these two species, little is known about the regulatory sequences controlling hand2 expression in zebrafish. Using bioinformatics and Tol2-mediated transgenesis, we have generated zebrafish transgenic reporter lines in which either the mouse or zebrafish arch-specific hand2 enhancer direct expression of a fluorescent reporter. We find that both the mouse and zebrafish enhancers drive early reporter expression in a hand2-specific pattern in the ventral pharyngeal arches of zebrafish embryos. These lines provide useful tools to follow ventral arch cells during vertebrate jaw development while also allowing dissection of hand2 transcriptional regulation during this process.