Anders Fjose

A vasa-like gene in zebrafish identifies putative primordial germ cells.

The vasa gene is essential for germline formation in Drosophila. Vasa-related genes have been isolated from several organisms including nematode, frog and mammals. In order to gain insight into the early events in vertebrate germline development, zebrafish was chosen as a model. Two zebrafish vasa-related genes were isolated, pl10a and vlg. The pl10a gene was shown to be widely expressed during embryogenesis. The vlg gene and vasa belong to the same subfamily of RNA helicase encoding genes. Putative maternal vlg transcripts were detected shortly after fertilization and from the blastula stage onwards, expression was restricted to migratory cells most likely to be primordial germ cells.

Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain

During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.

Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube.

Murine and human sequences homologous to the paired box of the Drosophila segmentation gene paired have been reported previously. Here we describe a zebrafish (Brachydanio rerio) paired box‐containing clone, pax[zf‐a], which is clearly distinct from reported vertebrate Pax genes. The putative protein encoded by pax[zf‐a] contains a paired box and a paired‐type homeobox separated by a glycine‐rich, acidic linker and a carboxy‐terminal end which is remarkably rich in serine, threonine and proline residues. By in situ hybridization to embryonic tissue sections and whole mount embryos, pax[zf‐a] transcripts were found within restricted regions of the central nervous system and the eye. In contrast to the murine Pax genes recently characterized, pax[zf‐a] is not expressed in the segmented mesoderm. At the 17 h stage, pax[zf‐a] expression is detected in a defined area of the diencephalon which circumscribes the presumptive thalamus. This suggests an involvement of pax[zf‐a] in pattern formation in the rostral brain. The pax[zf‐a] gene is also expressed throughout the hindbrain and spinal cord. This hybridization signal is restricted to a longitudinal column which includes the basal plate. Later in development, at 36 h post‐fertilization, pax[zf‐a] transcripts are no longer restricted to a specific region of the diencephalon, but are distributed over the entire developing brain.

Isolation of caudal, a Drosophila homeo box-containing gene with maternal expression, whose transcripts form a concentration gradient at the pre-blastoderm stage

We report the isolation and characterization of caudal (cad), a previously unknown Drosophila homeo box‐containing gene from the 38E region on the left arm of the second chromosome. This homeo box has diverged from the prototype sequence in Antennapedia, but contains subregions which are highly homologous. By Northern analysis and in situ hybridization experiments two transcripts of ˜2.4 kb were found to accumulate in nurse cells and in the oocyte during oogenesis. These transcripts generate a transient concentration gradient along the antero‐posterior axis at the syncytial blastoderm stage. At the cellular blastoderm stage transcripts accumulate in a single band from 13–19% egg length at the posterior end. One zygotic transcript of 2.6 kb is detected. At later stages this transcript is localized in ectodermally and endodermally derived tissues such as the proctodeum, the Malpighian tubules and the posterior midgut. The 2.6‐kb transcript is detectable until the onset of metamorphosis.

Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.

The murine homeobox gene Six3 and its Drosophila homologue sine oculis both have regulatory functions in eye development. We report the isolation and characterization of two zebrafish genes, six3 and six6, that are closely related to the murine Six3 gene. Zebrafish six3 may be the structural orthologue, while the six6 gene is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and, subsequently, in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain as reported previously for murine Six3.

Six class homeobox genes in Drosophila belong to three distinct families and are involved in head development

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain.

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.

Molecular cloning and embryonic expression of Xenopus Six homeobox genes.

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.

Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene

The Wnt‐1 (int‐1) gene was originally identified as an oncogene, but its normal function is in embryogenesis. The gene is the vertebrate homologue of the Drosophila segment polarity gene wingless, and encodes a secretory protein. In mouse embryos, Wnt‐1 expression is necessary for proper development of the midbrain and anterior hindbrain. Here we describe the molecular cloning and primary structure of the zebrafish Wnt‐1 gene (denoted wnt‐1). Comparison with its mouse homologue reveals that both the genomic organization of wnt‐1 and the amino acid sequence of the corresponding gene product have been extensively conserved during vertebrate evolution. Moreover, there is probably at least one Wnt‐1‐related sequence in the zebrafish genome. In zebrafish embryos, wnt‐1 is expressed during differentiation of the neural tube. In situ hybridization analysis reveals that the transcripts are confined to the dorsal surfaces of the midbrain, hindbrain and spinal cord, and to lateral cells at the midbrain‐hindbrain junction. Thus, the pattern of wnt‐1 expression in the developing central nervous system of zebrafish is virtually identical to that seen in mouse embryos. Unexpectedly, despite the striking similarities of Wnt‐1 structure and expression in fish and higher vertebrates, we could not identify sequences of obvious homology outside the coding regions, neither in the promoter nor in the introns.

RNA interference: mechanisms and applications.

RNA interference (RNAi) is a phenomenon induced by double-stranded RNA (dsRNA) in which gene expression is inhibited through specific degradation of mRNA. The mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets. This process is related to normal defence against viruses and mobilisation of transposons. Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. RNAi has also been demonstrated in several vertebrate species but with lower efficiency. Development of procedures for in vivo production of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.