Fgf signaling is required for zebrafish tooth development

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.     

MAPK/ERK signalling is required for zebrafish cardiac regeneration

Objectives

To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks.

Results

After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling—mek and erk—demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1⁺ endothelial cells, without direct inhibition of cardiomyocyte proliferation.

Conclusion

Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.

     

Fgf19 is required for zebrafish lens and retina development

Fgf signaling plays crucial roles in morphogenesis. Fgf19 is required for zebrafish forebrain development. Here, we examined the roles of Fgf19 in the formation of the lens and retina in zebrafish. Knockdown of Fgf19 caused a size reduction of the lens and the retina, failure of closure of the choroids fissure, and a progressive expansion of the retinal tissue to the midline of the forebrain. Fgf19 expressed in the nasal retina and lens was involved in cell survival but not cell proliferation during embryonic lens and retina development. Fgf19 was essential for the differentiation of lens fiber cells in the lens but not for the neuronal differentiation and lamination in the retina. Loss of nasal fate in the retina caused by the knockdown of Fgf19, expansion of nasal fate in the retina caused by the overexpression of Fgf19 and eye transplantation indicated that Fgf19 in the retina was crucial for the nasal-temporal patterning of the retina that is critical for the guidance of retinal ganglion cell axons. Knockdown of Fgf19 also caused incorrect axon pathfinding. The present findings indicate that Fgf19 positively regulates the patterning and growth of the retina, and the differentiation and growth of the lens in zebrafish.     

HSPG synthesis by zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb development

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs.     

Fgf19 regulated by Hh signaling is required for zebrafish forebrain development

Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.     

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.     

Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.     

Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon

Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.     

Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling

ObjectivesTo explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model.MethodsMorpholino oligonucleotide was used to induce Dnmt1 deficiency. RNA sequencing, in situ hybridization (ISH), whole genomic bisulfide sequencing (WGBS) and immunostaining were used to investigate the morphologic alterations and mechanisms.ResultsWe found that downregulation of Dnmt1 induced decreased number of neuromasts and repressed cell proliferation of primordium in the developing posterior lateral line system of zebrafish. The ISH data uncovered that Fgf signalling pathway was inhibited and the expression of chemokine members cxcr4b, cxcr7b and cxcl12a were interfered, while lef1 expression was increased after inhibiting Dnmt1. Additionally, Dnmt1 downregulation led to malformed otoliths and deformed semicircular canals, and hair cell differentiation in utricle and saccule was inhibited severely. The in situ staining of otic placode markers pax2/5 and fgf 3/8/10 was decreased when Dnmt1 downregulated. The WGBS analysis demonstrated that the global methylation status was markedly downregulated, and cell cycle genes were among those most differently expressed between Dnmt1 morphants and the controls. Further ISH analysis confirmed the findings by RNA‐seq and WGBS assay that cdkn1a and tp53 were both upregulated after knockdown of Dnmt1.ConclusionOur results revealed that Dnmt1 is essential for the development of zebrafish auditory organ through regulating cell cycle genes together with Wnt and Fgf signalling pathways.

     

Fgf/MAPK signalling is a crucial positional cue in somite boundary formation.

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signalling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, we have found a novel component involved in this process. We report that the level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation.     

Hedgehog signalling is required for perichondral osteoblast differentiation in zebrafish

In tetrapod long bones, Hedgehog signalling is required for osteoblast differentiation in the perichondrium. In this work we analyse skeletogenesis in zebrafish larvae treated with the Hedgehog signalling inhibitor cyclopamine. We show that cyclopamine treatment leads to the loss of perichondral ossification of two bones in the head. We find that the Hedgehog co-receptors patched1 and patched2 are expressed in regions of the perichondrium that will form bone before the onset of ossification. We also show that cyclopamine treatment strongly reduces the expression of osteoblast markers in the perichondrium and that perichondral ossification is enhanced in patched1 mutant fish. This data suggests a conserved role for Hedgehog signalling in promoting perichondral osteoblast differentiation during vertebrate skeletal development. However, unlike what is seen during long bone development, we did not observe ectopic chondrocytes in the perichondrium when Hedgehog signalling is blocked. This result may point to subtle differences between the development of the skeleton in the skull and limb.     

Fgf8 is required for anterior heart field development

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8neo) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.     

Connexin 48.5 is required for normal cardiovascular function and lens development in zebrafish embryos

Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.     

The expression of novel membrane-type matrix metalloproteinase isoforms is required for normal development of zebrafish embryos.

Matrix metalloproteinases (MMPs) play important roles in the turnover of components of extracellular matrix (ECM) and in the processing of active and latent-signaling molecules bound to the ECM or associated with the cell surface. Through such actions, MMPs regulate a variety of cellular and developmental processes. Membrane-type matrix metalloproteinases (MT-MMPs) are of particular importance because they function in the immediate pericellular environment that modulates both cell-cell and cell-ECM interactions. In this study, we utilized zebrafish as a developmental model to study the role of MT-MMPs during early embryogenesis. We successfully isolated two isoforms of a MT-MMP homologue that are structurally similar to MT1-MMP. They have been named zebrafish MT-MMPalpha and beta. Zebrafish MT-MMPbeta is unique among vertebrate MT-MMPs in that it contains an Arg-Glu-Asp (RED) multiple-repeat motif in its linker region. Whole mount in situ analysis, RT-PCR, immunofluorescence, reporter analysis, Western blot analysis, and zymography indicated that MT-MMPalpha and beta were expressed through at least the first 72 h of development and that this expression was targeted to the cell surface. Functional studies using injection of either mRNA or morpholino antisense oligonucleotides resulted in a truncation of the cranial to caudal axis as monitored through 72 h post fertilization, indicating that zebrafish MT-MMPalpha and beta had an important role in embryonic development. Axis markers indicated that these effects likely involved processes occurring later than 10 h of embryogenesis.     

HrT is required for cardiovascular development in zebrafish

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.     

Fgf21 is essential for haematopoiesis in zebrafish

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21-knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo-erythroid progenitor cell fate in vivo.     

Insulin-like growth factor (IGF) signalling is required for early dorso-anterior development of the zebrafish embryo

The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.     

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.