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Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a - and gata3 -dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr' (KA') and V3 interneurons. We show here that the differentiation of the correct number of KA' cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA' cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA' cells of the lateral floor plate and the KA' cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA' cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA' cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA' and KA' cells.

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish sloth(u45) mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution.

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. We report here that the bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together our data demonstrate a role of Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homologue Hairy.

Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. Here, we address the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. First, we find that Fgf signalling is required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. Second, combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with our model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations.

Endoderm originates from a large endomesodermal field requiring Nodal signalling. The mechanisms that ensure segregation of endoderm from mesoderm are not fully understood. We first show that the timing and dose of Nodal activation are crucial for endoderm formation and the endoderm versus mesoderm fate choice, because sustained Nodal signalling is required to ensure endoderm formation but transient signalling is sufficient for mesoderm formation. In zebrafish, downstream of Nodal signals, three genes encoding transcription factors (faust, bonnie and clyde and the recently identified gene casanova) are required for endoderm formation and differentiation. However their positions within the pathway are not completely established. In the present work, we show that casanova is the earliest specification marker for endodermal cells and that its expression requires bonnie and clyde. Furthermore, we have analysed the molecular activities of casanova on endoderm formation and found that it can induce endodermal markers and repress mesodermal markers during gastrulation, as well as change the fate of marginal blastomeres to endoderm. Overexpression of casanova also restores endoderm markers in the absence of Nodal signalling. In addition, casanova efficiently restores later endodermal differentiation in these mutants, but this process requires, in addition, a partial activation of Nodal signalling.

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.

The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos.

Sonic hedgehog (Shh) is a secreted protein that is involved in the organization and patterning of several tissues in vertebrates. We show that the zebrafish sonic-you (syu) gene, a member of a group of five genes required for somite patterning, is encoding Shh. Embryos mutant for a deletion of syu display defects in patterning of the somites, the lateral floor plate cells, the pectoral fins, the axons of motorneurons and the retinal ganglion cells. In contrast to mouse embryos lacking Shh activity, syu mutant embryos do form medial floor plate cells and motorneurons. Since ectopic overexpression of shh in zebrafish embryos does not induce ectopic medial floor plate cells, we conclude that shh is neither required nor sufficient to induce this cell type in the zebrafish.

Zebrafish neurogenin1 encodes a basic helix-loop-helix protein which shares structural and functional characteristics with proneural genes of Drosophila melanogaster. neurogenin1 is expressed in the early neural plate in domains comprising more cells than the primary neurons known to develop from these regions and its expression is modulated by Delta/Notch signalling, suggesting that it is a target of lateral inhibition. Misexpression of neurogenin1 in the embryo results in development of ectopic neurons. Markers for different neuronal subtypes are not ectopically expressed in the same patterns in neurogenin1-injected embryos suggesting that the final identity of the ectopically induced neurons is modulated by local cues. Induction of ectopic motor neurons by neurogeninl requires coexpression of a dominant negative regulatory subunit of protein kinase A, an intracellular transducer of hedgehog signals. Moreover, the pattern of endogenous neurogenin1 expression in the neural plate is expanded in response to elevated levels of Hedgehog (Hh) signalling or abolished as a result of inhibition of Hh signalling. Together these data suggest that Hh signals regulate neurogenin1 expression and subsequently modulate the type of neurons produced by Neurogenin1 activity.

No-isthmus (Noi) is a member of the zebrafish Pax family of transcriptional regulators that is expressed in restricted domains of the developing CNS. In the developing eye and optic nerve, the Noi+ cells are primitive glial cells that line the choroid fissure and optic stalk/nerve to its junction with the optic tract. This pattern of Noi expression is retained in the adult, defining the optic nerve astroglia, which wrap the left and right nerves separately at the midline, thus forming the bodily crossed optic chiasm found in fish. In embryos carrying mutations in the noi gene, the choroid fissure fails to close, glial cells of the optic nerve fail to differentiate and optic axons exhibit abnormal trajectories exiting the eye and at the midline of the diencephalon. Optic axons select inappropriate pathways into the contralateral optic nerve, rostrally towards the anterior commissure and along the ipsilateral optic tract. Noi+ cells also border the pathway of axons in the postoptic commissure, which is located adjacent to the optic chiasm. These postoptic commissural axons are defasciculated and also exhibit pathfinding defects in noi- embryos. These results indicate that Noi is required in cells that line the pathways taken by optic and non-optic commissural axons for guidance across the midline of the diencephalon. We find that expression of two members of the Netrin family of axon guidance molecules and the signalling protein Sonic hedgehog is disturbed in noi- embryos, whereas several members of the Eph family of receptors and ligands show no obvious alterations in expression at the diencephalic midline.

Early morphogenesis of the teleost embryo is characterized by three orchestrated cell movements. Epiboly leads to spreading of the blastoderm over an uncleaved yolk cell while involution around the blastoderm margin and convergence movements towards the dorsal side generate the mes-endodermal inner cell sheet and the axis rudiment, respectively. Irradiation of zebrafish zygotes with ultraviolet light selectively impairs epiboly resulting in embryos with open blastopores but well-formed anterior axes. Gastrulation movements are only marginally affected by ultraviolet irradiation. Involution of marginal cells in epiboly-retarded embryos takes place prior to 50% epiboly and thus appears independent of epiboly. Expression of dorsal and anterior marker genes is unaffected by ultraviolet irradiation. The ultraviolet light effect is not restricted to the zygote stage as irradiation of later embryonic stages also impairs epiboly. The ultraviolet-sensitive targets may thus be maternally encoded components of the machinery driving epiboly. These targets appear to be microtubules: firstly, irradiated embryos show disorganized and less microtubules in the cytoplasmic layer of the yolk sphere; secondly, the ultraviolet light effect can be mimicked by the microtubule-depolymerizing agent nocodazole. We suggest that epiboly is driven, at least partially, by motors that use microtubules radiating from the yolk syncytial layer into the yolk cytoplasmic layer. Together with an observed constrictive behaviour of the blastoderm margin, we propose a two-force model of epiboly: epiboly is initiated and driven by a pulling force dependent on microtubules in the yolk cytoplasmic layer; contraction at the margin operates in addition to aid closure of the blastopore.