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."     

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.     

Moz-dependent Hox expression controls segment-specific fate maps of skeletal precursors in the face

Development of the facial skeleton depends on interactions between intrinsic factors in the skeletal precursors and extrinsic signals in the facial environment. Hox genes have been proposed to act cell-intrinsically in skeletogenic cranial neural crest cells (CNC) for skeletal pattern. However, Hox genes are also expressed in other facial tissues, such as the ectoderm and endoderm, suggesting that Hox genes could also regulate extrinsic signalling from non-CNC tissues. Here we study moz mutant zebrafish in which hoxa2b and hoxb2a expression is lost and the support skeleton of the second pharyngeal segment is transformed into a duplicate of the first-segment-derived jaw skeleton. By performing tissue mosaic experiments between moz(-) and wild-type embryos, we show that Moz and Hox genes function in CNC, but not in the ectoderm or endoderm, to specify the support skeleton. How then does Hox expression within CNC specify a support skeleton at the cellular level? Our fate map analysis of skeletal precursors reveals that Moz specifies a second-segment fate map in part by regulating the interaction of CNC with the first endodermal pouch (p1). Removal of p1, either by laser ablation or in the itga5(b926) mutant, reveals that p1 epithelium is required for development of the wild-type support but not the moz(-) duplicate jaw-like skeleton. We present a model in which Moz-dependent Hox expression in CNC shapes the normal support skeleton by instructing second-segment CNC to undergo skeletogenesis in response to local extrinsic signals.     

Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest

The anterior-posterior identities of cells in the hindbrain and cranial neural crest are thought to be determined by their Hox gene expression status, but how and when cells become committed to these identities remain unclear. Here we address this in zebrafish by cell transplantation, to test plasticity in hox expression in single cells. We transplanted cells alone, or in small groups, between hindbrain rhombomeres or between the neural crest primordia of pharyngeal arches. We found that transplanted cells regulated hox expression according to their new environments. The degree of plasticity, however, depended on both the timing and the size of the transplant. At later stages transplanted cells were more likely to be irreversibly committed and maintain their hox expression, demonstrating a progressive loss of responsiveness to the environmental signals that specify segmental identities. Individual transplanted cells also showed greater plasticity than those lying within the center of larger groups, suggesting that a community effect normally maintains hox expression within segments. We also raised experimental embryos to larval stages to analyze transplanted cells after differentiation and found that neural crest cells contributed to pharyngeal cartilages appropriate to the anterior-posterior level of the new cellular environment. Thus, consistent with models implicating hox expression in control of segmental identity, plasticity in hox expression correlates with plasticity in final cell fate.     

lazarus is a novel pbx gene that globally mediates hox gene function in zebrafish

Individual vertebrate Hox genes specify aspects of segment identity along the anterior-posterior axis. The exquisite in vivo specificity of Hox proteins is thought to result from their interactions with members of the Pbx/Exd family of homeodomain proteins. Here, we report the identification and cloning of a zebrafish gene, lazarus, which is required globally for segmental patterning in the hindbrain and anterior trunk. We show that lazarus is a novel pbx gene and provide evidence that it is the primary pbx gene required for the functions of multiple hox genes during zebrafish development. lazarus plays a critical role in orchestrating the corresponding segmentation of the hindbrain and the pharyngeal arches, a key step in the development of the vertebrate body plan.     

Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development

Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region. Egr2, which is used in this study as an early developmental marker of rhombomeres 3 and 5, is expressed in two distinct bands with a location and spacing typical for these two rhombomeres in other species. Within the pharyngeal arches, hoxa2a is expressed at higher levels in the second pharyngeal arch, while hoxa2b is more strongly expressed in the posterior arches. Further, hoxa2b expression within the arches becomes undetectable at 60hpf, while hoxa2a expression is maintained at least up until the beginning of chondrogenesis. Comparison of the striped bass HoxA cluster paralog group 2 (PG2) genes to their orthologs and trans-orthologs shows that the striped bass hoxa2a gene expression pattern is similar to the overall expression pattern described for the hoxa2 genes in the lobe-finned fish lineage and for the hoxa2b gene from zebrafish. It is notable that the pharyngeal arch expression pattern of the striped bass hoxa2a gene is more divergent from its sister paralog, hoxa2b, than from the zebrafish hoxa2b gene. Overall, our results suggest that differences in the Hox PG2 gene complement of striped bass and zebrafish affects both their rhombomeric and pharyngeal arch expression patterns and may account for the similarities in pharyngeal arch expression between striped bass hoxa2a and zebrafish hoxa2b.     

Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.     

Hoxb-5 is expressed in gill arch 5 during pharyngeal arch development of flounder Paralichthys olivaceus embryos.

Hox genes are expressed in domains with clear anterior borders exhibiting 3'-->5' hierarchy in hindbrain and in the pharyngeal area commonly in vertebrate embryos. Teleost embryos form seven pharyngeal arches, the mandibular arch, hyoid arch and the gill arches 1-5. We previously reported that, in Japanese flounder (Paralichthys olivaceus) embryos, Hoxd-4 is expressed from rhombomere 7 to the spinal cord in the central nervous system and at gill arches 2-5. At present, the hierarchy of Hox genes at gill arches 3-5 of teleost fish is unclear. Here, we investigated the expression domains of Hoxb-5 in the flounder embryo by whole-mount in situ hybridization to gain insight into the Hox code at gill arches. The initial signal indicating Hoxb-5 expression was identified in the spinal cord at hatching, corresponding with the prim-5 stage of zebrafish. Then, intense signals were detected from the anterior part of the spinal cord and from the posterior part of the pharyngeal area at 36 h after hatching. By serially sectioning the hybridized embryos, it was found that signal in the pharyngeal area came from the most posterior gill arch 5. Therefore, it is speculated that Hoxb-5 functions in regional identification of gill arch 5 in this teleost.     

Role of Hox PG2 genes in Nile tilapia pharyngeal arch specification: implications for gnathostome pharyngeal arch evolution

SUMMARY Phylogenetic reconstructions suggest that the ancestral osteichthyan Hox paralog group 2 gene complement was composed of two genes, Hoxa2 and b2 , both of which have been retained in tetrapods, but only one of which functions as a selector gene of second pharyngeal arch identity (PA2). Genome duplication at the inception of the teleosts likely generated four Hox PG2 genes, only two of which, hoxa2b and b2a , have been preserved in zebrafish, where they serve as functionally redundant PA2 selector genes. Evidence from our laboratory has shown that other telelosts, specifically striped bass and Nile tilapia, harbor three transcribed Hox PG2 genes, hoxa2a, a2b , and b2a , with unspecified function(s). We have focused on characterizing the function of the three Nile tilapia Hox PG2 genes as a model to examine the effects of postgenome duplication gene loss on the evolution of developmental gene function. We studied Hox PG2 gene function in tilapia by examining the effects of independent morpholino oligonucleotide (MO)-induced knockdowns on pharyngeal arch morphology and Hox gene expression patterns. Morphological defects resulting from independent MO-induced knockdowns of tilapia hoxa2a, a2b , and b2a included the expected PA2 to PA1 homeotic transformations previously observed in tetrapods and zebrafish, as well as concordant and unexpected morphological changes in posterior arch-derived cartilages. Of particular interest, was the observation of a MO-induced supernumerary arch between PA6 and PA7, which occurred concomitantly with other MO-induced pharyngeal arch defects. Beyond these previously unreported morphant-induced transformations, a comparison of Hox PG2 gene expression patterns in tilapia Hox PG2 morphants were indicative of arch-specific auto- and cross-regulatory activities as well as a Hox paralog group 2 interdependent regulatory network for control of pharyngeal arch specification.     

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.     

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.     

Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs

Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh-/- mutants, the pharyngeal pouches and arches formed by E9.5 and marker expression showed that initial patterning was normal. However, by E10.5-E11.0, the first arch had atrophied and the first pouch was missing. Although small, the second, third, and fourth arches and pouches were present. The expression patterns of Fgf8, Pax1, and Bmp4 suggested that pouch identity was abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers were expressed normally. Our data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh-/- mutants suggested that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. Our data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs.     

Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.     

Xenopus Teashirt1 regulates posterior identity in brain and cranial neural crest

We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.     

Japanese medaka Hox paralog group 2: insights into the evolution of Hox PG2 gene composition and expression in the Osteichthyes

Hox paralog group 2 (PG2) genes function to specify the development of the hindbrain and pharyngeal arch-derived structures in the Osteichthyes. In this article, we describe the cDNA cloning and embryonic expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. We show that there are only two functional canonical Hox genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, psihoxa2b. The functional genes, hoxa2a and b2a, were expressed in developing rhombomeres and pharyngeal arches in a manner that was relatively well conserved compared with zebrafish (Danio rerio) but differed significantly from orthologous striped bass (Morone saxatilis) and Nile tilapia (Oreochromis niloticus) genes, which, we suggest, may be owing to effects of post-genome duplication loss of a Hox PG2 gene in the medaka and zebrafish lineages. psihoxa2b was expressed at readily detectable levels in several noncanonical Hox expression domains, including the ventral aspect of the neural tube, the pectoral fin buds and caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatio-temporal expression have diverged from their ancestral counterparts. Comparative expression analyses showed medaka hoxa2a and b2a expression in the 2nd pharyngeal arch (PA2) beyond the onset of chondrogenesis, which, according to previous hypotheses, suggests these genes function redundantly as selector genes of PA2 identity. We conclude that Hox PG2 gene composition and expression have diverged significantly during osteichthyan evolution and that this divergence in teleosts may be related to lineage-dependent differential gene loss following an actinopterygian-specific whole genome duplication.