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.     

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.     

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.     

Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.     

Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction

The inner ear develops from an ectodermal placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor (Fgf)-19 is expressed in mesoderm underlying the presumptive otic placode, and human FGF19 induces expression of otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have otic abnormalities at E9.5-E10.5, suggesting that Fgf15 is not uniquely required for otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive otic placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse otic development.     

The homeobox gene irx1a is required for the propagation of the neurogenic waves in the zebrafish retina

Neurogenesis in the compound eyes of Drosophila and the camera eyes of vertebrates spreads in a wave-like fashion. In both phyla, waves of hedgehog expression are known to drive the wave of neuronal differentiation. The mechanism controlling the propagation of hedgehog expression during retinogenesis of the vertebrate eye is poorly understood. The Iroquois homeobox genes play important roles in Drosophila eye development; they are required for the up-regulation of hedgehog expression during propagation of the morphogenetic furrow. Here, we show that the zebrafish Iroquois homolog irx1a is expressed during retinogenesis and knockdown of irx1a results in a retinal phenotype strikingly similar to those of sonic hedgehog (shh) mutants. Analysis of shh-GFP transgene expression in irx1a knockdown retinas revealed that irx1a is required for the propagation of shh expression through the retina. Transplantation experiments illustrated that the effects of irx1a on shh expression are both cell-autonomous and non-cell-autonomous. Our results reveal a role for Iroquois genes in controlling hedgehog expression during vertebrate retinogenesis.     

Mummy encodes an UDP-N-acetylglucosamine-dipohosphorylase and is required during Drosophila dorsal closure and nervous system development

Throughout development cell-cell interactions are of pivotal importance. Cells bind to each other or share information via secreted signaling molecules. To a large degree, these processes are modulated by post-translational modifications of membrane proteins. Glycan-chains are frequently added to membrane proteins and assist their exact function at the cell surface. In addition, the glycosylation pathway is required to generate GPI-linkage in the endoplasmatic reticulum. Here, we describe the analysis of the cabrio/mummy gene, which encodes an UDP-N-acetylglucosamine diphosphorylase. This is a well-conserved and central enzyme in the glycosylation pathway. As expected from this central role in glycosylation, cabrio/mummy mutants show many phenotypic traits ranging from CNS fasciculation defects to defects in dorsal closure and eye development. These phenotypes correlate well with specific glycosylation and GPI-anchorage defects in mummy mutants.     

Endothelium is required for the promotion of interrenal morphogenetic movement during early zebrafish development

The adrenal cortex has a complex vasculature that is essential for growth, tissue maintenance, and access of secreted steroids to the bloodstream. However, the interaction between vasculature and adrenal cortex during early organogenesis remains largely unclear. In this study, we focused on the zebrafish counterpart of adrenal cortex, interrenal tissue, to explore the possible role of endothelium in the development of steroidogenic tissues. The ontogeny of interrenal tissue was found to be tightly associated with the endothelial cells (ECs) that constitute the axial vessels. The early interrenal primordia emerge as two clusters of cells that migrate centrally and converge at the midline, whereas the central convergence was abrogated in the avascular cloche (clo) mutant. Neither loss of blood circulation nor perturbations of vessel assembly could account for the interrenal convergence defect, implying a role of endothelial signaling prior to the formation of axial blood vessels. Moreover, as the absence of trunk endothelium in clo mutant was rescued by the forced expression of SCL, the interrenal fusion defect could be alleviated. We thus conclude that endothelial signaling is involved in the morphogenetic movement of early interrenal tissue.     

The anaphase-promoting complex is required in both dividing and quiescent cells during zebrafish development

The anaphase-promoting complex/cyclosome (APC/C) regulates multiple stages of the cell cycle, most prominently mitosis. We describe zebrafish with mutations in two APC/C subunits, Cdc16 and Cdc26, whose phenotypes reveal a multifaceted set of defects resulting from the gradual depletion of the APC/C. First, loss of the APC/C in dividing cells results in mitotic arrest, followed by apoptosis. This defect becomes detectable in different organs at different larval ages, because the subunits of the APC/C are maternally deposited, are unusually stable, and are depleted at uneven rates in different tissues. Second, loss of the APC/C in quiescent or differentiated cells results in improper re-entry into the cell cycle, again in an apparently tissue-specific manner. This study is the first demonstration of both functions of the APC/C in a vertebrate organism and also provides an illustration of the surprisingly complex effects that essential, maternally supplied factors can have on the growing animal over a period of 10 days or longer.     

Epibranchial and otic placodes are induced by a common Fgf signal, but their subsequent development is independent

The epibranchial placodes are cranial, ectodermal thickenings that give rise to sensory neurons of the peripheral nervous system. Despite their importance in the developing animal, the signals responsible for their induction remain unknown. Using the placodal marker, sox3, we have shown that the same Fgf signaling required for otic vesicle development is required for the development of the epibranchial placodes. Loss of both Fgf3 and Fgf8 is sufficient to block placode development. We further show that epibranchial sox3 expression is unaffected in mutants in which no otic placode forms, where dlx3b and dlx4b are knocked down, or deleted along with sox9a. However, the forkhead factor, Foxi1, is required for both otic and epibranchial placode development. Thus, both the otic and epibranchial placodes form in a common region of ectoderm under the influence of Fgfs, but these two structures subsequently develop independently. Although previous studies have investigated the signals that trigger neurogenesis from the epibranchial placodes, this represents the first demonstration of the signaling events that underlie the formation of the placodes themselves, and therefore, the process that determines which ectodermal cells will adopt a neural fate.     

Gdf6a is required for the initiation of dorsal–ventral retinal patterning and lens development

Dorsal–ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

nanos1 is required to maintain oocyte production in adult zebrafish

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

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.     

Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation

Cardiomelic or heart–hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb.