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.     

Shh signalling restricts the expression of Gcm2 and controls the position of the developing parathyroids

The parathyroid glands originate from the endoderm of the caudal pharyngeal pouches. How these parathyroids are restricted to developing in the caudal pouches is unclear. In this paper we investigate the role of Shh signalling in patterning the vertebrate pharyngeal pouches, and show that Hh signalling may be involved in restricting the expression of the parathyroid marker Gcm2 in the pharyngeal epithelium. In the chick and mouse, Shh signalling is excluded or highly reduced in the posterior/caudal pouches, where the parathyroid marker Gcm2 is expressed, while remaining at high levels in the more anterior pouches. Moreover, though the block of Shh signalling at early developmental stages results in the loss of chick Gcm2 expression, at later stages, it induces ectopic Gcm2 expression domains in the second and first pharyngeal epithelium, suggesting that HH signalling prevents Gcm2 in those tissues. These ectopic domains go on to express other parathyroid markers but do not migrate and develop into ectopic parathyroids. Differences in the expression of Gcm2 in the chick, mouse and zebrafish, correlate with changing patterns of Shh signalling, indicating a conserved regulatory mechanism that acts to define pouch derivatives.     

Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm

Branchial arch development involves dynamic interactions between neural crest cells as well as ectodermal, endodermal and mesodermal cell populations. Despite their importance and evolutionary conservation, the intercellular interactions guiding the early development of the branchial arches are still poorly understood. We have here studied fibroblast growth factor (FGF) signalling in early pharyngeal development. In mice homozygous for a hypomorphic allele of Fgfr1, neural crest cells migrating from the hindbrain mostly fail to enter the second branchial arch. This defect is non-cell-autonomous suggesting that Fgfr1 provides a permissive environment for neural crest cell migration. Here we demonstrate localized down-regulation of the expression of the FGF responsive gene, Sprouty1 in the epithelium covering the presumptive second branchial arch of hypomorphic Fgfr1 mutants. This appears to result in a failure to establish an ectodermal signalling center expressing Fgf3 and Fgf15. We also studied differentiation of the ectoderm in the second branchial arch region. Development of the geniculate placode as well as the VIIth cranial ganglion is affected in Fgfr1 hypomorphs. Our results suggest that Fgfr1 is important for localized signalling in the pharyngeal ectoderm and consequently for normal tissue interactions in the developing second branchial arch.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

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.     

A new origin for the maxillary jaw

One conserved feature of craniofacial development is that the first pharyngeal arch has two components, the maxillary and mandibular, which then form the upper and lower jaws, respectively. However, until now, there have been no tests of whether the maxillary cells originate entirely within the first pharyngeal arch or whether they originate in a separate condensation, cranial to the first arch. We therefore constructed a fate map of the pharyngeal arches and environs with a series of dye injections into stage 13-17 chicken embryos. We found that from the earliest stage examined, the major contribution to the maxillary bud is from post-optic mesenchyme with a relatively minor contribution from the maxillo-mandibular cleft. Cells labeled within the first pharyngeal arch contributed exclusively to the mandibular prominence. Gene expression data showed that there were different molecular codes for the cranial and caudal maxillary prominence. Two of the genes examined, Rarbeta (retinoic acid receptor beta) and Bmp4 (bone morphogenetic protein) were expressed in the post-optic mesenchyme and epithelium prior to formation of the maxillary prominence and then were restricted to the cranial half of the maxillary prominence. In order to determine the derivatives of the maxillary prominence, we performed focal injections of CM-DiI into the stage 24 maxillary prominence. Labeled cells contributed to the maxillary, palatine, and jugal bones, but not the other elements of the upper beak, the premaxilla and prenasal cartilage. We also determined that the cranial cells give rise to more distal parts of the upper beak, whereas caudal cells form proximal structures. Grafts of stage 24 maxillary prominences were also analyzed to determine skeletal derivatives and these results concurred with the DiI maps. These early and later fate maps indicate that the maxillary prominence and its skeletal derivatives are not derived from the first pharyngeal arch but rather from a separate maxillary condensation that occurs between the eye and the maxillo-mandibular cleft. These data also suggest that during evolution, recession of the first pharyngeal arch-derived palatoquadrate cartilage to a more proximal position gave way to the bony upper jaw of amniotes.     

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."     

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.     

斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

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.     

Expression analysis of zebrafish membrane type-2 matrix metalloproteinases during embryonic development

Membrane tethered matrix metalloproteinases (MMPs) cleave a variety of extracellular matrix (ECM) and non-ECM targets and play important roles during embryonic development and tumor progression. Membrane tethered MMPs in particular are important regulators of both tissue invasion and morphogenesis. Much attention has been given to understanding the function of human and mouse MMP14 (also called membrane type-1 MMP, MT1-MMP) and our own data have linked zebrafish Mmp14 to the regulation of gastrulation cell movements. However, less is known regarding the expression and function of other membrane tethered MMPs. We report the cloning and gene expression analysis of zebrafish mmp15a and mmp15b (MT2-MMP) during early embryonic and larval development. Our data show that mmp15a exhibits limited expression prior to segmentation stages and is first detected in the tectum and posterior tailbud. At 24hours post-fertilization (hpf) mmp15a localizes to the caudal hematopoietic tissue, pectoral fin buds, and mandibular arch. By contrast, mmp15b is strongly expressed during gastrula stages before becoming restricted to the polster and anterior neural plate. From 24 to 48hpf, mmp15b expression is detected in the pharyngeal arches, fin buds, otic vesicle, pronephric ducts, proctodeum, tail epidermis, posterior lateral line primordia, and caudal notochord. During the larval period beginning at 72hpf, mmp15b expression becomes restricted to the brain ventricular zone, pharyngeal arches, pectoral fins, and the proctodeum. Many of the mmp15-expressing tissues have been shown to express genes encoding components of the ECM including collagens, fibronectin, and laminins. Our data thus provide a foundation for uncovering the role of Mmp15-dependent pericellular proteolysis during zebrafish embryonic development.     

Development of the pharyngeal arch skeleton in Catostomus commersonii (Teleostei: Cypriniformes)

Skeletal elements of the gill arches of adult cypriniform fishes vary widely in number, size, and shape and are important characters in morphologically based phylogenetic studies. Understanding the developmental basis for this variation is thus phylogenetically significant but also important in relation to the many developmental genetic and molecularly based studies of the early developing and hence experimentally tractable gill arches in the zebrafish, a cyprinid cypriniform. We describe the sequence of the chondrification and ossification of the pharyngeal arches and associated dermal bones from Catostomus commersonii (Catostomidae, Cypriniformes) and make selected comparisons to other similarly described pharyngeal arches. We noted shared spatial trends in arch development including the formation of ventral cartilages before dorsal and anterior cartilages before posterior. Qualitatively variable gill arch elements in Cypriniformes including pharyngobranchial 1, pharyngobranchial 4, and the sublingual are the last such elements to chondrify in C. commersonii. We show that the sublingual bone in C. commersonii has two cartilaginous precursors that fuse and ossify to form the single bone in adults. This indicates homology of the sublingual in catostomids to the two sublingual bones in the adults of cobitids and balitorids. Intriguing patterns of fusion and segmentation of the cartilages in the pharyngeal arches were discovered. These include the individuation of the basihyal and anterior copula through segmentation of a single cartilage rod, fusion of cartilaginous basibranchials 4 and 5, and fusion of hypobranchial 4 with ceratobranchial 4. Such “fluidity” in cartilage patterning may be widespread in fishes and requires further comparative developmental studies. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues

The morphology of skeletal tissues formed in each of the branchial arches of higher vertebrates is unique. In addition to these structures, which are derived from the neural crest, the crest-derived connective tissues and mesodermal muscles also form different patterns in each of the branchial arches. The objective of this study was to examine how these patterns arise during avian embryonic development. Presumptive second or third arch neural crest cells were excised from chick hosts and replaced with presumptive first arch crest cells. Both quail and chick embryos were used as donors; orthotopic crest grafts were performed as controls. Following heterotopic transplantation, the hosts developed several unexpected anomalies. Externally they were characterized by the appearance of ectopic, beak-like projections from the ventrolateral surface of the neck and also by the formation of supernumerary external auditory depressions located immediately caudal to the normal external ear. Internally, the grafted cells migrated in accordance with normal, second arch pathways but then formed a complete, duplicate first arch skeletal system in their new location. Squamosal, quadrate, pterygoid, Meckel's, and angular elements were present in most cases. In addition, anomalous first arch-type muscles were found associated with the ectopic skeletal tissues in the second arch. These results indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural crest population prior to its emigration from the neural epithelium, and not within the pharynx or pharyngeal pouches as had previously been suggested. Furthermore, the patterns of myogenesis by mesenchymal populations derived from paraxial mesoderm is dependent upon properties inherent to the neural crest.     

CXCL14 expression during chick embryonic development

Chemokines are small secreted signalling molecules best known for their roles as chemoattractants for cells of the immune system. CXCL12 and its receptor CXCR4 comprise one chemokine signalling pathway with essential functions in non-immune cell types during embryonic development. CXCL14, a chemokine-encoding gene related to CXCL12, is developmentally regulated in zebrafish and Xenopus embryos, but its role during embryogenesis remains unknown. Here we describe the embryonic expression pattern of CXCL14 in an amniote, the chick. Although expression in some regions is conserved with that of fish and frog, chick CXCL14 displays a complex pattern of expression in several novel sites. We analyse the expression pattern in the branchial arches, trigeminal placode and ganglion, inner ear, dorsal midline of the brain, somites, trunk neural tube and limb bud. Expression in several domains raises the possibility that CXCL14 may be involved in some of the same developmental events during which CXCL12-CXCR4 signalling is known to play a role.     

Aortic arch and pharyngeal phenotype in the absence of BMP-dependent neural crest in the mouse

Neural crest cells are essential for proper development of a variety of tissues and structures, including peripheral and autonomic nervous systems, facial skeleton, aortic arches and pharyngeal glands like the thymus and parathyroids. Previous work has shown that bone morphogenic protein (BMP) signalling is required for the production of migratory neural crest cells that contribute to the neurogenic and skeletogenic lineages. We show here that BMP-dependent neural crest cells are also required for development of the embryonic aortic arches and pharynx-derived glands. Blocking formation or migration of this crest cell population from the caudal hindbrain resulted in strong phenotypes in the cardiac outflow tract and the thymus. Thymic aplasia or hypoplasia occurs despite uncompromised gene induction in the pharyngeal endoderm. In addition, when hypoplastic thymic tissue is found, it is ectopically located, but functional in thymopoiesis. Our data indicate that thymic phenotypes produced by neural crest deficits result from aberrant formation of pharyngeal pouches and impaired migration of thymic primordia because the mesenchymal content in the branchial arches is below a threshold level.