Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development.

During vertebrate embryonic development, the paraxial mesoderm becomes subdivided into metameric units known as somites. In the zebrafish embryo, genes encoding homologues of the proteins of the Drosophila Notch signalling pathway are expressed in the presomitic mesoderm and expression is maintained in a segmental pattern during somitogenesis. This expression pattern suggests a role for these genes during somite development. We misexpressed various zebrafish genes of this group by injecting mRNA into early embryos. RNA encoding a constitutively active form of notch1a (notch1a-intra) and a truncated variant of deltaD [deltaD(Pst)], as well as transcripts of deltaC and deltaD, the hairy-E(spl) homologues her1 and her4, and groucho2 were tested for their effects on somite formation, myogenesis and on the pattern of transcription of putative downstream genes. In embryos injected with any of these RNAs, with the exception of groucho2 RNA, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment correctly. Activation of notch results in ectopic activation of her1 and her4. This misregulation of the expression of her genes might be causally related to the observed mesodermal defects, as her1 and her4 mRNA injections led to effects similar to those seen with notch1a-intra. deltaC and deltaD seem to function after subdivision of the presomitic mesoderm, since the her gene transcription pattern in the presomitic mesoderm remains essentially normal after misexpression of delta genes. Whereas notch signalling alone apparently does not affect myogenesis, zebrafish groucho2 is involved in differentiation of mesodermal derivatives.

her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a target of NOTCH signalling.

her4 encodes a zebrafish bHLH protein of the hairy-E(spl) family. The gene is transcribed in a complex pattern in the developing nervous system and in the hypoblast. During early neurogenesis, her4 expression domains include the regions of the neural plate from which primary neurons arise, suggesting that the gene is involved in directing their development. Indeed, misexpression of specific her4 variants leads to a reduction in the number of primary neurons formed. The amino-terminal region of her4, including the basic domain, and the region between the putative helix IV and the carboxy-terminal tetrapeptide wrpw are essential for this effect, since her4 variants lacking either of these regions are non-functional. However, the carboxy-terminal wrpw itself is dispensable. We have examined the interrelationships between deltaD, deltaA, notch1, her4 and neurogenin1 by means of RNA injections. her4 is involved in a regulatory feedback loop which modulates the activity of the proneural gene neurogenin, and as a consequence, of deltaA and deltaD. Activation of notch1 leads to strong activation of her4, to suppression of neurogenin transcription and, ultimately, to a reduction in the number of primary neurons. These results suggest that her4 acts as a target of notch-mediated signals that regulate primary neurogenesis.

Overexpression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development.

We describe here the isolation and characterization of a zebrafish Delta homologue (delta D). A PCR fragment was used to obtain overlapping cDNA clones encoding a protein of 717 amino acids with all characteristic features of proteins of this family, a signal peptide, a transmembrane domain, and an extracellular region comprising the DSL domain and eight EGF-like repeats. The gene is transcribed in a complex pattern in the developing nervous system as well as in the hypoblast. Overexpression of this gene following mRNA injections leads to a reduction in the number of islet-I positive cells, which are assumed to be primary neurons, and to various defects in the adaxial mesoderm, as well as in the somites and myotomes. This suggests that delta D, and the Notch signalling pathway are involved in the differentiation of primary neurons within the neural plate, as well as in somite development.