Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements.

Bilaterian animals have a Hox gene cluster essential for patterning the main body axis, and a ParaHox gene cluster. Comparison of Hox and ParaHox genes has led workers to postulate that both clusters originated from the duplication of an ancient cluster named ProtoHox, which contained up to four genes with at least the precursors of anterior and posterior Hox/ParaHox genes. However, the way in which genes diversified within the ProtoHox, Hox and ParaHox clusters remains unclear because no systematic study of non-bilaterian animals exists. Here we characterize the full Hox/ParaHox gene complements and genomic organization in two cnidarian species (Nematostella vectensis and Hydra magnipapillata), and suggest a ProtoHox cluster simpler than originally thought on the basis of three arguments. First, both species possess bilaterian-like anterior Hox genes, but their non-anterior genes do not appear as counterparts of either bilaterian central or posterior genes; second, two clustered ParaHox genes, Gsx and a gene related to Xlox and Cdx, are found in Nematostella vectensis; and third, we do not find clear phylogenetic support for a common origin of bilaterian Cdx and posterior genes, which might therefore have appeared after the ProtoHox cluster duplication. Consequently, the ProtoHox cluster might have consisted of only two anterior genes. Non-anterior genes could have appeared independently in the Hox and ParaHox clusters, possibly after the separation of bilaterians and cnidarians.

Urochordates carry multiple genes for goose-type lysozyme and no genes for chicken- or invertebrate-type lysozymes.

Genome clones and expressed sequence tags (ESTs) from the ascidian Ciona intestinalis and from the larvacean Oikopleura dioica were analysed for the presence of lysozyme-encoding genes. Two genes were found to potentially code for goose-type lysozymes in Oikopleura, while three or possibly more g-type proteins form the lysozyme complement of C. intestinalis, and at least one of these genes from each species is expressed based on EST data. No genes for chicken- or invertebrate-type lysozymes were found in either urochordate species. Consistent with this finding, extracts of Oikopleura animals possessed hydrolysing activity on bacterial cell walls, and this activity was not inhibited in the presence of a known inhibitor of chicken-type lysozyme. A wide range of isoelectric points for the predicted lysozymes from Ciona (pI 4.4, 6.4 and 9.9) and from Oikopleura (pI 5.0 and 8.0) suggests tissue-specific adaptations as well as specific functional roles of the lysozymes. Comparisons of gene structures, encoded sequences, cysteine residue content and their positions in the proteins indicate that the g-type lysozymes of Ciona intestinalis are more closely related to those of vertebrates than are the g-type lysozymes of Oikopleura. Multiple genes from each species may result from separate and lineage-specific duplications followed by functional specialisation.

Stable and full rescue of the pigmentation in a medaka albino mutant by transfer of a 17 kb genomic clone containing the medaka tyrosinase gene.

In the medaka Oryzias latipes, several albino strains have mutations in the tyrosinase gene that have been fully characterized at the molecular level. A genomic clone from wild-type medaka containing the 5 kb tyrosinase gene with its five exons, 10 kb of upstream sequences and 2 kb downstream sequences was introduced into fertilized eggs from a tyrosinase-negative albino strain. We show that the injection of this genomic clone predominantly conferred mosaic expression ending before the hatching stage. A minority of juveniles retained a variable number of pigmented cells, including four individuals keeping one pigmented eye through adulthood. Two of these could be mated, and one of these transmitted the transgene resulting in complete rescue of pigmentation to 16% of its offspring. The resulting transgenic line harbors a single copy of the wild-type tyrosinase gene and all fish are wild-type with respect to pigmentation. These experiments suggest that the tyrosinase genomic clone, or a future shorter version of it, can be used in fish to routinely detect transgenic lines. The apparent faithful and systematic expression of the tyrosinase transgene is most probably due to the presence of a locus control region (LCR) in the injected clone.

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.

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.

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus.

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.

Lymphocyte expression in transgenic trout by mouse immunoglobulin promoter/enhancer.

Two groups of transgenic rainbow trout (Oncorhynchus mykiss, Walbaum) have been produced and compared. One group harbored the reporter gene of chloramphenicol acetyltransferase (CAT) associated with mouse immunoglobulin (Ig) promoter/enhancer (pUCL-CAT-E). The other group carried the same reporter gene under the control of the cytomegalovirus promoter/enhancer (pCMV-CAT). Slot blot analysis of DNA from blood cells and other tissues from pUCL-CAT-E fish showed variation of copy number between the major tissues but not between red and white blood cells. Southern blot analysis indicated that multiple copies organized in concatemers were incorporated into the genome. The pCMV-CAT fish had a pronounced expression of CAT in both white and red blood cells. In contrast, activity of CAT was found in the white blood cells of all pUCL-CAT-E fish but not in their red blood cells. Expression in white blood cells was found preferentially in sIg+ cells, indicating that B cells are the major expressors. High expression was also found in spleen and kidney, but the activity found in thymocytes was equal to the background level. Analysis of some major tissues showed high white blood cell expression associated with low tissue expression, except that liver (known to contain lymphoid tissue in fish) was higher. Thus the regulatory elements of the Ig gene from mouse induce a tissue-specific expression in fish.

No transgenic rainbow trout produced with sperm incubated with linear DNA.

We attempted to produce transgenic rainbow trout embryos by fertilizing eggs with sperm incubated with linearized plasmids. One experiment was conducted with the construct pBGH7 in the medium MMSF, with or without DMSO, at 2 concentrations of sperm cells and a relatively low concentration of DNA. The DNA was also in contact with the eggs during insemination and during the first minutes of egg activation. The second experiment was conducted with the construct CMVCAT in the medium MMSF, at 2 concentrations of sperm cells and a much higher concentration of DNA. The DNA was also present during the insemination. DNA analyses and dosages of CAT activity did not permit detection of any transgenic fry. However, one result suggests that sperm cells can capture part of the linear DNA in teh conditions tested.

Transgenesis in fish.

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.

Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.

Gene segregation in induced tetraploid rainbow trout: genetic evidence of preferential pairing of homologous chromosomes.

Gene segregation at six protein loci was analysed in progeny from tetraploid males and females obtained by suppression of first mitosis. The triploid full-sib families from five tetraploid males and the diploid gynogenetic lines from four tetraploid females were examined. The proportions of heterozygous gametes (0.83 on the average) were significantly higher than expected from tetrasomic inheritance (0.667) at all the loci studied. This was explained by preferential pairing of homologous chromosomes. The proportions of heterozygous gametes were significantly different between loci, but the variations were not correlated with the gene--centromere distances. Our results showed that, at least for one locus, the homozygous gametes mainly resulted from pairing of homologous chromosomes rather than from pairing of homologous chromosomes, quadrivalent formation, and chromatin exchanges between homologous chromosomes.

Production of YY rainbow trout males by self-fertilization of induced hermaphrodites.

Dietary administration of various estrogens for three months from swim-up stage resulted in excess of females, the remainder of the treated groups consisting of males and hermaphrodites. Mature hermaphrodites were self-fertilized or mated with standard males and females. These hermaphrodites and some of the estrogen-treated females proved to be genetic males; the frequencies of males obtained from their ova averaged 76.6%, suggesting viability of the YY genotype. Four of nine tested males of those progenies provided all male offspring when mated with standard females.

Chromosome studies of progenies of tetraploid female rainbow trout.

Nine induced tetraploid females were artificially inseminated by UV-irradiated sperm collected from diploid males, in order to induce the gynogenetic development of their ova. Most of the resulting embryos were diploid (or minor aneuploids). Several gynogenetic tetraploids, likely to issue from unreduced ova, were also detected in these progenies. The same females fertilized by normal sperm of diploid males gave a majority of triploids and several pentaploids, while the fertilization by normal sperm of tetraploid males gave rise to a majority of tetraploids and one hexaploid. The same crosses, after the eggs had been heat-shocked to double the maternal genetic contribution, yielded about three-quarters pentaploids and one quarter haploids (normal sperm of diploids), or three-quarters hexaploids and one quarter diploids (normal sperm of tetraploids). These haploids and diploids are likely to result from androgenesis.

Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females - Potential of tetraploid fish.

First generation tetraploids were produced by hydrostatic pressure treatment before the first cleavage and raised until the adult stage. Their survival and growth were severely depressed when compared to the diploid control: after two years, no ovulated females were found although males produced sperm at 1 and 2 years of age and were mated individually with diploid females. The progenies were consistently normal with high survival rates. They were found to be almost all triploids by karyology, which failed to detect a significant rate of aneuploidies. However, the fertilizing ability of tetraploid males was always low (0 to 97% of the control; average 40%). Several arguments presented here support the hypothesis that diploid spermatozoas, which are wider than haploid ones, would be frequently blocked during their penetration through the micropyle canal. Second generation tetraploids were produced after such matings by heat shocks, causing the retention of the second polar body. Their survival and growth were much more satisfactory than in the first generation, although still lower than in diploid and triploid controls issuing from diploid parents. Performances of second generation triploids were comparable to those of diploids, and slightly better than those of conventional triploids issuing from diploid parents. 94.5% of the second generation tetraploids were male.

Techniques of chromosome manipulation in rainbow trout: a new evaluation with karyology.

Experiments, supported by extensive karyology, were carried out to evaluate the different techniques used for chromosome manipulation in rainbow trout. Eggs, when subjected to early heat shocks, changed from haploidy to diploidy and from diploidy to triploidy. In this respect heat shocks differ from pressure shocks which induce gradual transitions between successive ploidy levels. Sperm treatment with dimethylsulphate yields haploid embryos containing residual sperm chromatin fragments, in contrast to treatment with ultraviolet rays.