moz regulates Hox expression and pharyngeal segmental identity in zebrafish

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, we screened for mutations affecting cartilage patterning in the zebrafish larval pharynx. We present the positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen. We show that a zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although we find early patterning of the moz mutant hindbrain to be normal, we find a late defect in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. We detect relatively minor defects in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, our results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch.     

edn1 and hand2 Interact in Early Regulation of Pharyngeal Arch Outgrowth during Zebrafish Development

Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic – reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific edn1-mediated proliferation of neural crest derived cells is required for DV expansion and outgrowth, and that this positive regulation is counterbalanced by a negative one exerted through a pivotal, ventrally expressed Edn1-target gene, hand2. We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.     

Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning

Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains.     

sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development

Mutation of sucker (suc) disrupts development of the lower jaw and other ventral cartilages in pharyngeal segments of the zebrafish head. Our sequencing, cosegregation and rescue results indicate that suc encodes an Endothelin-1 (Et-1). Like mouse and chick Et-1, suc/et-1 is expressed in a central core of arch paraxial mesoderm and in arch epithelia, both surface ectoderm and pharyngeal endoderm, but not in skeletogenic neural crest. Long before chondrogenesis, suc/et-1 mutant embryos have severe defects in ventral arch neural crest expression of dHAND, dlx2, msxE, gsc, dlx3 and EphA3 in the anterior arches. Dorsal expression patterns are unaffected. Later in development, suc/et-1 mutant embryos display defects in mesodermal and endodermal tissues of the pharynx. Ventral premyogenic condensations fail to express myoD, which correlates with a ventral muscle defect. Further, expression of shh in endoderm of the first pharyngeal pouch fails to extend as far laterally as in wild types. We use mosaic analyses to show that suc/et-1 functions nonautonomously in neural crest cells, and is thus required in the environment of postmigratory neural crest cells to specify ventral arch fates. Our mosaic analyses further show that suc/et-1 nonautonomously functions in mesendoderm for ventral arch muscle formation. Collectively our results support a model for dorsoventral patterning of the gnathostome pharyngeal arches in which Et-1 in the environment of the postmigratory cranial neural crest specifies the lower jaw and other ventral arch fates.     

Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning of skeletogenic neural crest cells in zebrafish

Genetic studies in mice and zebrafish have revealed conserved requirements for Endothelin 1 (Edn1) signaling in craniofacial development. Edn1 acts through its cognate type-A receptor (Ednra) to promote ventral skeletal fates and lower-jaw formation. Here, we describe the isolation and characterization of two zebrafish ednra genes - ednra1 and ednra2 - both of which are expressed in skeletal progenitors in the embryonic neural crest. We show that they play partially redundant roles in lower-jaw formation and development of the jaw joint. Knockdown of Ednra1 leads to fusions between upper- and lower-jaw cartilages, whereas the combined loss of Ednra1 and Ednra2 eliminates the lower jaw, similar to edn1-/- mutants. edn1 is expressed in pharyngeal arch ectoderm, mesoderm and endoderm. Tissue-mosaic studies indicate that, among these tissues, a crucial source of Edn1 is the surface ectoderm. This ectoderm also expresses ednrA1 in an edn1-dependent manner, suggesting that edn1 autoregulates its own expression. Collectively, our results indicate that Edn1 from the pharyngeal ectoderm signals through Ednra proteins to direct early dorsoventral patterning of the skeletogenic neural crest.     

Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated "knock-down" of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as "selector genes."     

Endothelin 1-mediated regulation of pharyngeal bone development in zebrafish

Endothelin 1 (Edn1), a secreted peptide expressed ventrally in the primordia of the zebrafish pharyngeal arches, is required for correct patterning of pharyngeal cartilage development. We have studied mutants and morpholino-injected larvae to examine the role of the Edn1 signal in patterning anterior pharyngeal arch bone development during the first week after fertilization. We observe a remarkable variety of phenotypic changes in dermal bones of the anterior arches after Edn1 reduction, including loss, size reduction and expansion, fusion and shape change. Notably, the changes that occur appear to relate to the level of residual Edn1. Mandibular arch dermal bone fusions occur with severe Edn1 loss. In the dorsal hyoid arch, the dermal opercle bone is usually absent when Edn1 is severely reduced and is usually enlarged when Edn1 is only mildly reduced, suggesting that the same signal can act both positively and negatively in controlling development of a single bone. Position also appears to influence the changes: a branchiostegal ray, a dermal hyoid bone normally ventral to the opercle, can be missing in the same arch where the opercle is enlarged. We propose that Edn1 acts as a morphogen; different levels pattern specific positions, shapes and sizes of bones along the dorso-ventral axis. Changes involving Edn1 may have occurred during actinopterygian evolution to produce the efficient gill-pumping opercular apparatus of teleosts.     

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.     

Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

Fgf and Bmp signals repress the expression of Bapx1 in the mandibular mesenchyme and control the position of the developing jaw joint

The development of the jaw joint between the palatoquadrate and proximal part Meckel's cartilage (articular) has recently been shown to involve the gene Bapx1. Bapx1 is expressed in the developing mandibular arch in two distinct caudal, proximal patches, one on either side of the head. These domains coincide later with the position of the developing jaw joint. The mechanisms that result in the restricted expression of Bapx1 in the mandibular arch were investigated, and two signaling factors that act as repressors were identified. Fibroblast growth factors (Fgfs) expressed in the oral epithelium restrict expression of Bapx1 to the caudal half of the mandibular arch, while bone morphogenetic proteins (Bmps) expressed in the distal mandibular arch restrict expression of Bapx1 to the proximal part of the mandible. Application of Fgf8 and Bmp4 beads to the proximal mesenchyme led to loss of Bapx1 expression and later fusion of the quadrate and articular as the jaw joint failed to form. In addition to fusion of the jaw joint, loss of Bapx1 lead to loss of the retroarticular process (RAP), phenocopying the defects seen after Bapx1 function was reduced in the zebrafish. By manipulating these signals, we were able to alter the expression domain of Bapx1, resulting in a new position of the jaw joint.     

barx1 is necessary for ectomesenchyme proliferation and osteochondroprogenitor condensation in the zebrafish pharyngeal arches

Barx1 modulates cellular adhesion molecule expression and participates in specification of tooth-types, but little is understood of its role in patterning the pharyngeal arches. We examined barx1 expression during zebrafish craniofacial development and performed a functional analysis using antisense morpholino oligonucleotides. Barx1 is expressed in the rhombencephalic neural crest, the pharyngeal arches, the pectoral fin buds and the gut in contrast to its paralogue barx2, which is most prominently expressed in the arch epithelium. Additionally, barx1 transient expression was observed in the posterior lateral line ganglia and developing trunk/tail. We show that Barx1 is necessary for proliferation of the arch osteochondrogenic progenitors, and that morphants exhibit diminished and dysmorphic arch cartilage elements due to reductions in chondrocyte differentiation and condensation. Attenuation of Barx1 results in lost arch expression of osteochondrogenic markers col2a1, runx2a and chondromodulin, as well as odontogenic marker dlx2b. Further, loss of barx1 positively influenced gdf5 and chordin, markers of jaw joint patterning. FGF signaling is required for maintaining barx1 expression, and that ectopic BMP4 induces expression of barx1 in the intermediate region of the second pharyngeal arch. Together, these results indicate an essential role for barx1 at early stages of chondrogenesis within the developing zebrafish viscerocranium.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.