BMP signalling regulates anteroposterior endoderm patterning in zebrafish

In vertebrates, the embryonic dorsoventral asymmetry is regulated by the bone morphogenetic proteins (Bmp) activity gradient. In the present study, we have used dorsalized swirl (bmp2b) and ventralized chordino (chordin) zebrafish mutants to investigate the effects of dorsoventral signalling on endoderm patterning and on the differentiation and positioning of its derivatives. Alterations of dorsoventral Bmp signalling do not perturb the induction of endodermal precursors, as shown by normal amounts of cells expressing cas and sox17 in swirl and chordino gastrulae, but affect dramatically the expression pattern of her5, a regulator of endoderm anteroposterior patterning in zebrafish. In particular, increased levels of Bmp signalling in chordino gastrulae are associated with a markedly reduced her5 expression domain, that may be abolished by injecting bmp2b mRNA. Conversely, in swirl mutants, lacking Bmp2b, the her5 expression domain is expanded. Thus, a gradient of Bmp2b signalling defines the extension of the her5 expression domain at gastrulation and the allocation of anterior endodermal precursors. A balanced Bmp2b signalling is also required for the normal development of the pancreas, as shown by the sharp reduction of the pancreatic primordium in swirl embryos and its expansion in chordino mutants. In the latter, at 3 days post-fertilization, the increased Bmp signalling does not compromise the endocrine/exocrine pancreas compartmentalization, but the right/left positioning of the pancreas and liver is randomized. Our results suggest that by regulating the expression of her5, the Bmp2b/Chordin gradient directs the anteroposterior patterning of endoderm in zebrafish embryos.     

SRC-1 and Wnt signaling act together to specify endoderm and to control cleavage orientation in early C. elegans embryos

In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Here, we show that two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. Our findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell.     

Vegfc is required for vascular development and endoderm morphogenesis in zebrafish

During embryogenesis, complex morphogenetic events lead endodermal cells to coalesce at the midline and form the primitive gut tube and associated organs. While several genes have recently been implicated in endoderm differentiation, we know little about the genes that regulate endodermal morphogenesis. Here, we show that vascular endothelial growth factor C (Vegfc), an angiogenic as well as a lymphangiogenic factor, is unexpectedly involved in this process in zebrafish. Reducing Vegfc levels using morpholino antisense oligonucleotides, or through overexpression of a soluble form of the VEGFC receptor, VEGFR-3, affects the coalescence of endodermal cells in the anterior midline, leading to the formation of a forked gut tube and the duplication of the liver and pancreatic buds. Further analyses indicate that Vegfc is additionally required for the initial formation of the dorsal endoderm. We also demonstrate that Vegfc is required for vasculogenesis as well as angiogenesis in the zebrafish embryo. These data argue for a requirement of Vegfc in the developing vasculature and, more surprisingly, implicate Vegfc signalling in two distinct steps during endoderm development, first during the initial differentiation of the dorsal endoderm, and second in the coalescence of the anterior endoderm to the midline.     

Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish

Cranial sensory neurons largely derive from neurogenic placodes (epibranchial and dorsolateral), which are ectodermal thickenings that form the sensory ganglia associated with cranial nerves, but the molecular mechanisms of placodal development are unclear. Here, we show that the pharyngeal endoderm induces epibranchial neurogenesis in zebrafish, and that BMP signaling plays a crucial role in this process. Using a her5:egfp transgenic line to follow endodermal movements in living embryos, we show that contact between pharyngeal pouches and the surface ectoderm coincides with the onset of neurogenesis in epibranchial placodes. By genetic ablation and reintroduction of endoderm by cell transplantation, we show that these contacts promote neurogenesis. Using a genetic interference approach we further identify bmp2b and bmp5 as crucial components of the endodermal signals that induce epibranchial neurogenesis. Dorsolateral placodes (trigeminal, auditory, vestibular, lateral line) develop independently of the endoderm and BMP signaling, suggesting that these two sets of placodes are under separate genetic control. Our results show that the endoderm regulates the differentiation of cranial sensory ganglia, which coordinates the cranial nerves with the segments that they innervate.     

The zebrafish dyrk1b gene is important for endoderm formation

Nodal‐signaling is required for specification of mesoderm, endoderm, establishing left–right asymmetry, and craniofacial development. Wdr68 is a WD40‐repeat domain‐containing protein recently shown to be required for endothelin‐1 (edn1) expression and subsequent lower jaw development. Previous reports detected the Wdr68 protein in multiprotein complexes containing mammalian members of the dual‐specificity tyrosine‐regulated kinase (dyrk) family. Here we describe the characterization of the zebrafish dyrk1b homolog. We report the detection of a physical interaction between Dyrk1b and Wdr68. We also found perturbations of nodal signaling in dyrk1b antisense morpholino knockdown (dyrk1b‐MO) animals. Specifically, we found reduced expression of lft1 and lft2 (lft1/2) during gastrulation and a near complete loss of the later asymmetric lft1/2 expression domains. Although wdr68‐MO animals did not display lft1/2 expression defects during gastrulation, they displayed a near complete loss of the later asymmetric lft1/2 expression domains. While expression of ndr1 was not substantially effected during gastrulation, ndr2 expression was moderately reduced in dyrk1b‐MO animals. Analysis of additional downstream components of the nodal signaling pathway in dyrk1b‐MO animals revealed modestly expanded expression of the dorsal axial mesoderm marker gsc while the pan‐mesodermal marker bik was largely unaffected. The endodermal markers cas and sox17 were also moderately reduced in dyrk1b‐MO animals. Notably, and similar to defects previously reported for wdr68 mutant animals, we also found reduced expression of the pharyngeal pouch marker edn1 in dyrk1b‐MO animals. Taken together, these data reveal a role for dyrk1b in endoderm formation and craniofacial patterning in the zebrafish. genesis 48:20–30, 2010. © 2009 Wiley‐Liss, Inc.     

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

Background  

The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.     

Hot +TIPS: guidance cues signal directly to microtubules

Plus end tracking proteins (+TIPS) bind to microtubules. Results reported by Lee et al. and Zhou et al. in this issue of Neuron demonstrate that, via intracellular pathways signaling to +TIPs, microtubules can be a direct target of guidance cues in steering growth cones and regulating axon elongation.     

casanova plays an early and essential role in endoderm formation in zebrafish.

The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to be explored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutants lack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression of genes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective from the onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formation in Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicating that casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunner cells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies of casanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as has been previously proposed. In addition, although different populations of mesodermal precursors are generated normally in casanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possible role for the endoderm in morphogenesis of these organs.