tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Zebrafish tgfβ3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfβ3 in head skeletal formation, we knocked down tgfβ3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfβ3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfβ3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfβ3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfβ3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfβ3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfβ3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfβ3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfβ3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfβ3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfβ3 protein levels in zebrafish.     

Fgf signalling is required for formation of cartilage in the head

Characterisation of human craniofacial syndromes and studies in transgenic mice have demonstrated the requirement for Fgf signalling during morphogenesis of membrane bone of the cranium. Here, we report that Fgf activity is also required for development of the oro-pharyngeal skeleton, which develops first as cartilage with some elements subsequently becoming ossified. We show that inhibition of FGF receptor activity in the zebrafish embryo following neural crest emigration from the neural tube results in complete absence of neurocranial and pharyngeal cartilages. Moreover, this Fgf signal is required during a 6-h period soon after initiation of neural crest migration. The spatial and temporal expression of Fgf3 and Fgf8 in pharyngeal endoderm and ventral forebrain and its correlation with patterns of Fgf signalling activity in migrating neural crest makes them candidate regulators of cartilage development. Inhibition of Fgf3 results in the complete absence of cartilage elements that normally form in the third, fourth, fifth, and sixth pharyngeal arches, while those of the first, second, and seventh arches are largely unaffected. Inhibition of Fgf8 alone has variable, but mild, effects. However, inhibition of both Fgf3 and Fgf8 together causes a complete absence of pharyngeal cartilages and the near-complete loss of the neurocranial cartilage. These data implicate Fgf3 and Fgf8 as key regulators of cartilage formation in the vertebrate head.     

An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.     

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.     

Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.     

The relationship between migration and chondrogenic potential of trunk neural crest cells in Ambystoma mexicanum

Based on results of transplantation experiments, it has long been believed that trunk neural crest cells are incapable of chondrogenesis. When pigmented trunk neural crest cells of Ambystoma mexicanum are transplanted to cranial levels of albino (a/a) embryos, the graft cells ultimately produce ectopic fins, but are incapable of following the chondrogenic cranial neural crest pathways. Therefore, heterotopic transplantation does not expose these cells to the same environment experienced by cranial neural crest cells, and is neither an adequate nor a sufficient test of chondrogenic potential. However, in vitro culture of trunk neural crest cells with pharyngeal endoderm does provide a direct test of chondrogenic ability. That cartilage does not form under these conditions demonstrates conclusively that trunk neural crest cells possess no chondrogenic potential.     

Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo

Several syndromes characterized by defects in cardiovascular and craniofacial development are associated with a hemizygous deletion of chromosome 22q11 in humans and involve defects in pharyngeal arch and neural crest cell development. Recent efforts have focused on identifying 22q11 deletion syndrome modifying loci. In this study, we show that mouse embryos deficient for Gbx2 display aberrant neural crest cell patterning and defects in pharyngeal arch-derived structures. Gbx2(-/-) embryos exhibit cardiovascular defects associated with aberrant development of the fourth pharyngeal arch arteries including interrupted aortic arch type B, right aortic arch, and retroesophageal right subclavian artery. Other developmental abnormalities include overriding aorta, ventricular septal defects, cranial nerve, and craniofacial skeletal patterning defects. Recently, Fgf8 has been proposed as a candidate modifier for 22q11 deletion syndromes. Here, we demonstrate that Fgf8 and Gbx2 expression overlaps in regions of the developing pharyngeal arches and that they interact genetically during pharyngeal arch and cardiovascular development.     

Lysyl oxidase-like 3b is critical for cartilage maturation during zebrafish craniofacial development

Vertebrate craniofacial development requires coordinated morphogenetic interactions between the extracellular matrix (ECM) and the differentiating chondrocytes essential for cartilage formation. Recent studies reveal a critical role for specific lysyl oxidases in ECM integrity required for embryonic development. We now demonstrate that loxl3b is abundantly expressed within the head mesenchyme of the zebrafish and is critically important for maturation of neural crest derived cartilage elements. Histological and ultrastructural analyses of cartilage elements in loxl3b morphant embryos reveal abnormal maturation of cartilage and altered chondrocyte morphology. Spatiotemporal analysis of craniofacial markers in loxl3b morphant embryos shows that cranial neural crest cells migrate normally into the developing pharyngeal arches but that differentiation and condensation markers are aberrantly expressed. We further show that the loxl3b morphant phenotype is not due to P53 mediated cell death but likely to be due to reduced chondrogenic progenitor cell proliferation within the pharyngeal arches. Taken together, these data demonstrate a novel role for loxl3b in the maturation of craniofacial cartilage and can provide new insight into the specific genetic factors important in the pathogenesis of craniofacial birth defects.     

Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.     

inka1b expression in the head mesoderm is dispensable for facial cartilage development

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.     

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.