Morphogenesis of the optic tectum in the medaka (Oryzias latipes): a morphological and molecular study, with special emphasis on cell proliferation.

We analyzed the medaka optic tectum (OT) morphogenesis by using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry (with a new method we developed for pulse-labeling embryos) and in situ hybridization with three probes, two for recently cloned homeobox genes (Ol-Prx3 [Paired-Related-Homeobox3] and Ol-Gsh1 [Genetic-Screen-Homeobox1]) and one for Ol-tailless. The tectal anlage first appears as a sheet of proliferating cells expressing Ol-Gsh1 and Ol-tailless but not Ol-Prx3. Cells subsequently cease to proliferate in a superficial and rostral zone and begin to express Ol-Prx3. When tectal lamination begins, the proliferative zone (mpz) becomes restricted to a crescent at the OT medial, caudal, and lateral margin. This mpz functions throughout the fish's entire life. It produces cells that are added at the OT's edge as radial rows, spanning every layer of the OT. The cells of the mpz continue to express Ol-tailless in the adult, whereas Ol-Gsh1 expression is turned off. When superficial layers form, Ol-Prx3 expression becomes restricted to the underlying deep layer, where it persists in the adult. Ol-Prx3 seems to be a marker for the differentiation of a subset of deep cells and allows analysis of tectal lamination, whereas Ol-tailless and Ol-Gsh1 could be involved in the control of tectal cell proliferation. This study constitutes a first step toward molecular approach to OT development in anamniotes. We compare and discuss the expression patterns of the homologs of the genes studied, and more generally the morphogenetic patterns of the medaka tectum, with those encountered in other cortical structures and in other vertebrate groups.

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2.

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.

Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus.

Here we report the expression pattern of the homeobox Ol-Rx3 gene, a medaka gene homologous to the mouse, Xenopus, zebrafish and Drosophila Rx genes. Ol-Rx3 starts to be expressed, at late gastrula stages, in the presumptive territories of the anterior brain. Subsequently, transcripts are localised in an antero-ventral region of the prosencephalon and in the primordia of the optic vesicles. During organogenesis, distribution of Ol-Rx3 transcripts are gradually restricted to the floor of the diencephalon, the prospective territory of the hypothalamus and the neurohypophysis. During late development and in adult, Ol-Rx3 expression is maintained in hypothalamic nuclei bordering the third ventricle. In the optic vesicles, Ol-Rx3 expression is temporarily switched off when the eye cup morphogenesis is complete, but it is turned on again in the inner nuclear layer of the retina. Thus, the early expression pattern of Ol-Rx3 is in agreement with a conserved role in the specification of the ventral forebrain and eye field. Putative functions linked to late expression domains are discussed in light of the different hypothesis concerning the involvement of vertebrate Rx genes in the maintenance of particular cell fate.

Expression domains of the medaka (Oryzias latipes) Ol-Gsh 1 gene are reminiscent of those of clustered and orphan homeobox genes.

Screening of a medaka (Oryzias latipes) adult brain cDNA library, with a degenerated probe corresponding to the most conserved region of helix III of the homeodomain, led to the isolation of a gene homologous to a murine orphan Hox gene, named Gsh-1. We have called this gene Ol-Gsh 1 (Oryzias latipes-Gsh 1). Molecular analysis of the Ol-Gsh 1 putative protein points to potential functional domains which are highly conserved between fish and mouse genes. Whole-mount in situ hybridization shows that Ol-Gsh 1 is expressed in several waves during embryonic development. Transcripts are found in many regions of the central nervous system: the spinal cord, dorsal rhombencephalon, optic tectum, dorsal diencephalon, hypothalamus anlagen and rostral telencephalon. This multimodal expression pattern, strikingly conserved between fish and mammals, is reminiscent of both clustered and orphan homeobox genes. In addition, each expression wave is initiated in the fish embryo earlier than in the mammalian embryo, relative to the time scale defined by somitogenesis. We propose that Ol-Gsh 1 may be involved in conserved developmental pathways and in particular may be linked to proliferation events. Mouse Gsh-1 was shown to participate in neuro-endocrine functions of the hypothalamus. From late developmental stages onwards, Ol-Gsh 1 expression is also restricted to the hypothalamus. The expression pattern in this structure raises interesting questions concerning a fully or partially conserved function for these genes.