Two phases of aging separated by the Smurf transition as a public path to death.

Aging's most obvious characteristic is the time dependent increase of an individual's probability to die. This lifelong process is accompanied by a large number of molecular and physiological changes. Although numerous genes involved in aging have been identified in the past decades its leading factors have yet to be determined. To identify the very processes driving aging we have developed in the past years an assay to identify physiologically old individuals in a synchronized population of Drosophila melanogaster. Those individuals show an age-dependent increase of intestinal permeability followed by a high risk of death. Here we show that this physiological marker of aging is conserved in 3 invertebrate species Drosophila mojavensis, Drosophila virilis, Caenorhabditis elegans as well as in 1 vertebrate species Danio rerio. Our findings suggest that intestinal barrier dysfunction may be an important event in the aging process conserved across a broad range of species, thus raising the possibility that it may also be the case in Homo sapiens.

Expression of Ol-KIP, a cyclin-dependent kinase inhibitor, in embryonic and adult medaka (Oryzias latipes) central nervous system.

From an expression screen in a fish model, the medaka, we have isolated Ol-KIP (Oryzias latipes-kinase inhibitor protein), a new member of the KIP subfamily of cyclin-dependent kinase (Cdk) inhibitors. We have analysed its expression in the developing and adult brain by in situ hybridization and by double labeling with Ol-KIP mRNA and proliferating cell nuclear antigen (PCNA) antibodies. Ol-KIP presents a complex expression pattern in several areas of the embryonic central nervous system, most often in close vicinity to proliferative neuroepithelia. We studied in great detail its expression in the optic tectum: Ol-KIP is expressed in a ring-shaped domain lying exactly between the proliferative and the postmitotic zones of this structure and is, therefore, potentially involved in cell cycle exit. In the adult CNS, Ol-KIP expression persists in numerous nuclei, both close and distant from proliferative ventricular areas. So, Ol-KIP expression is in part compatible with a sustained "stop signal" role for proliferation, but its expression in postmitotic zones suggests that KIP proteins may have late neuronal function(s), in addition to inhibiting Cdks. This first detailed study of the expression profile of a KIP gene in a nonmammalian vertebrate, thus, opens perspectives for analysing the role of these regulators in brain development and function.

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.

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor-derived gametes.

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES-like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma-irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor-derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells.

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.

Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes).

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691-10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54-62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Widespread expression of the eve1 gene in zebrafish embryos affects the anterior-posterior axis pattern.

The zygotic expression of the eve1 gene is restricted to the ventral and lateral cells of the marginal zone. At later stages, the mRNAs are localized in the most posterior part of the extending tail tip. An eve1 clone (pcZf14), containing a poly-A tail, has been isolated. In order to address eve1 gene function, pcZf14 transcript injections into zebrafish embryos have been performed. The injection into uncleaved eggs of a synthetic eve1 mRNA (12 pg), which encodes a protein of approximately 28 kd, produces embryos with anterior-posterior (A-P) axis defects and the formation of additional axial structures. The first category of 24 h phenotypes (87%) mainly displays a gradual decrease in anterior structures. This is comparable to previous phenotypes observed following Xhox3 messenger injection either in Xenopus or in zebrafish that have been classified according to the index of axis deficiency (zf-IAD). These phenotypes result in anomalies of the development of the neural keel, from microphthalmia to acephaly. The second category (13%) corresponds to the phenotypes described above together with truncal or caudal supernumerary structures. Additional truncal structures are the most prominent of these duplicated phenotypes, displaying a "zipper" shape of axial structures including neural keels and notochords. Caudal duplication presents no evident axis supernumerary structures. The observation of these phenotypes suggests an important role for the eve1 gene in mesodermal cell specification and in the development of the posterior region, and more particularly of the most posterior tail tip where endogenous eve1 messengers are found.

The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos.

We have identified and characterized zebrafish eve1, a novel member of the Drosophila even-skipped (eve) gene family. eve1 RNAs are expressed initially in late blastulae with a peak during the gastrula stage, at which time expression is confined to ventral and lateral cells of the marginal zone of the zebrafish embryo. Later, eve1 transcripts are located in the most posterior part of the extending tail tip. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish, resulting in embryos with exaggerated dorsoanterior structures. In LiCl-treated embryos, eve1 transcripts are completely absent. eve1 is therefore a marker of ventral and posterior cells. In the light of its ventroposterior expression domain, the localization of eve1 transcripts was analysed in spadetail (spt) and no tail (ntl), two mutants with abnormal caudal development. In sptb140 homozygous mutants, there is an accumulation of cells in the tail region, resulting from inadequate migratory behaviour of precursors to the trunk somites. These cells, in their abnormal environment, express eve1, emphasizing the correlation between ventroposterior position and eve1 expression. In homozygous mutant embryos for the gene ntl (the homologue of mouse Brachyury, originally called Zf-T), posterior structures are missing (M. E. Halpern, C. B. Kimmel, R. K. Ho and C. Walker, 1993; Cell In press). While mutant and wild-type embryos do not differ in their eve1 transcript distribution during gastrulation, eve1 expression is absent in the caudal region of mutant ntl embryos during early somitogenesis, indicating a requirement for ntl in the maintenance of eve1 expression during tail extension. Our findings suggest that eve1 expression is correlated with a ventral and posterior cell fate, and provide first insights into its regulation.

[Ventral and posterior expression of the homeo box gene eve1 in zebrafish (Brachydanio rerio) is repressed in dorsalized embryos]. / L'expression ventrale et postérieure du gène à homéoboîte eve1 de poisson-zèbre (Brachydanio rerio) est réprimée dans les embryons dorsalisés.

We have identified and characterized the zebrafish eve1 homeo box gene, a member of the Drosophila even-skipped (eve) gene family. eve1 is expressed in the most posterior part of the tail bud during somitogenesis. During gastrulation, transcripts are confined to ventral and lateral cells of the marginal zone of the embryo. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish embryos. In LiCl-treated embryos, eve1 transcripts are completely absent, suggesting that eve1 marks the ventral specification of mesoderm in zebrafish gastrulae.

Expression of a zebrafish caudal homeobox gene correlates with the establishment of posterior cell lineages at gastrulation.

This paper deals with the first identification of a caudal cDNA containing a homeobox of the Drosophila caudal family in the zebrafish. A cDNA library from late gastrula stage embryos was constructed and screened with a mouse Cdx 1 homeobox probe. A 1.6 kb cDNA clone containing a homeobox related to other caudal homeoboxes was isolated and called cdx[Zf-cad1]. Analysis of the predicted 301 amino acid translation product reveals additional regions of homology outside the homeodomain with other members of the caudal family. Particularly, the cdx[Zf-cad1] putative protein shares a conserved N-terminal region with its chicken homolog CHox-cad. Transcripts are first detected just before the onset of gastrulation. At the beginning of gastrulation, a single 1.8 kb cdx[Zf-cad1] transcript is located near the blastoderm margin with a high level of expression restricted to the epiblast. At this stage, the hypoblast is clearly negative. At the end of gastrulation, cdx[Zf-cad1] is widely expressed in vegetal (i.e. prospective posterior) epiblast and hypoblast, with a somewhat weaker expression in the dorsal hypoblast. During somitogenesis, cdx[Zf-cad1] exhibits a posterior regionalization in the neurectoderm. In contrast, no expression is detected in the mesoderm of 22 h embryos (late somitogenesis). Posterior endoderm is also positive at this stage. cdx[Zf-cad1] transcripts cease to be detected about 48 h after fertilization. They are undetectable in the adult, particularly in female gonads. The pattern of cdx[Zf-cad1] expression during and after gastrulation is consistent with its possible involvement in the regionalization of the embryo at these stages.(ABSTRACT TRUNCATED AT 250 WORDS)