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casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals.

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.

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 HNF3β? 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.

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15‐bp glucocorticoid response element (GRE) or 15‐bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell‐type‐dependent glucocorticoid induction of expression.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10‐fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE‐receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.

Zebrafish embryos represent an attractive system for the study of early vertebrate neurogenesis. The embryos develop outside of the mother and are transparent allowing analysis at the cellular level in the living embryo during all phases of early neurogenesis. The teleostean neural tube is generated by a mechanism different from that of other vertebrates. A massive keel is formed first and the central canal appears by subsequent cavitation. Despite this, however, the organization of the neural plate and the neural keel resembles that of other vertebrates in many aspects. Oriented cell divisions coupled with oriented cell intercalations appear to be involved in the morphogenesis of the neural keel. Embryos mutant in the cyclopsgene show deficiencies in the ventral neural tube. They lack the floor plate and the ventral parts of the diencephalon. Two recently cloned genes, axialand sonic hedgehog, have been implicated in the development of the ventral midline of the neural tube. Expression of axialand sonic hedgehogis impaired by the cyclopsmutation in the midline of the neural plate. This, together with the effects of ectopic expression of the two cloned genes, suggests that axial, sonic hedgehog, and cyclopsare part of the regulatory cascade leading to floor plate formation.— Strähle, U., Blader, P. Early neurogenesis in the zebrafish embryo. FASEB J.8: 692‐698; 1994.

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.

Modulation of gene expression by steroid hormones is mediated by receptor proteins that associate with regulatory elements of responsive genes upon binding the hormone ligand. The finding that two glucocorticoid responsive elements act cooperatively to stimulate transcription of the tyrosine aminotransferase gene prompted us to explore whether synergistic effects also occur when two different steroid hormone receptors are involved. A region of the chicken vitellogenin II gene that displays homologies to glucocorticoid and estradiol responsive elements was tested for its capability to confer estradiol and glucocorticoid inducibility to a heterologous promoter. When positioned immediately upstream of the thymidine kinase gene promoter, this element enhances expression by either steroid. Combination of both hormones results in a synergistic increase of transcription. Mutational analysis shows that sequences that show similarities of glucocorticoid and estradiol responsive elements are absolutely required for hormone induction. Analysis of the dose dependence of induction by both steroids demonstrates that half-maximal activity is observed at lower hormone concentrations when the other steroid is present in saturating amounts, which suggests that the synergistic induction observed with the combination of hormones is based on a functional interaction of the two hormone receptors.

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15‐bp glucocorticoid response element (GRE) or 15‐bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell‐type‐dependent glucocorticoid induction of expression.

Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide variety of developmental processes. Here we describe the cloning of three Wnt family members from the zebrafish, Danio rerio, which encode proteins with homology to murine Wnt-2, -4 and -5A/B. The expression patterns of the latter two zebrafish genes, designated ZfWnt4 and ZfWnt5 show considerable similarity with their homologues in other vertebrates; ZfWnt2, however, is expressed in the developing viscera in a pattern distinct from its closest murine homologue. In the light of the similarities and differences in the patterns of expression of these genes relative to their homologues in other vertebrates, we speculate on their possible functions.

Exposure of vertebrate embryos to ethanol causes cyclopia, but little is known about the underlying mechanisms of this effect. Here we show that cyclopia can be induced in the zebrafish by a short ethanol treatment during early gastrula stages and is accompanied by loss of gene expression characteristic of the ventral aspects of the fore- and midbrain. Interestingly, defects in the expression of ventral brain markers are linked to impaired migration of the prechordal plate mesoderm indicating that the correct position of the prechordal plate mesoderm under the anterior neural plate in the zebrafish embryo is required for specification of the anterior neural midline. Ethanol-induced cyclopia does not, however, impair the induction of anterior neuroectodermal structures in general. Finally, as genes likegoosecoidandislet-1are expressed in prechordal plate cells in a temporal pattern similar to control embryos despite the ectopic position of expressing cells, it appears that regulation of prechordal plate-specific gene expression is largely independent of the final position of the prechordal plate.

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.

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.

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.

To define the recognition sequence of the glucocorticoid receptor and its relationship with that of the progesterone receptor, oligonucleotides derived from the glucocorticoid response element of the tyrosine aminotransferase gene were tested upstream of a heterologous promoter for their capacity to mediate effects of these two steroids. We show that a 15-base-pair sequence with partial symmetry is sufficient to confer glucocorticoid inducibility on the promoter of the herpes simplex virus thymidine kinase gene. The same 15-base-pair sequence mediates induction by progesterone. Point mutations in the recognition sequence affect inducibility by glucocorticoids and progesterone similarly. Together with the strong conservation of the sequence of the DNA-binding domain of the two receptors, these data suggest that both proteins recognize a sequence that is similar, if not the same.

We have characterized the gene for the mouse glucocorticoid receptor. The gene spans approximately 110 kilobases, and glucocorticoid receptor transcripts are assembled from nine exons. Expression of the gene is controlled by at least three promoters, resulting in glucocorticoid receptor transcripts with different 5' nontranslated exons. One promoter is cell-specific, found to be active only in T lymphocytes. The other two promoters are active to various degrees in all cell lines and tissues so far analyzed and are located in a CpG island. The promoter activities are accompanied by DNase I hypersensitivity sites in chromatin. In contrast to a conservation of exon-intron structure, differences in promoter organization suggest a divergence between the evolution of regulatory and coding regions among members of the steroid receptor super-family.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10‐fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE‐receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.