J. Samallo

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos.

Abstract The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

Expression of carp-cdx1, a caudal homolog, in embryos of the carp, Cyprinus carpio.

A carp caudal cDNA of 1.3 kb was cloned after screening an early segmentation stage cDNA library with a probe produced by PCR using conserved homeobox sequences as primers and genomic DNA as template. The homeobox gene was called carp-cdxl. The gene appears highly similar to other vertebrate caudal homologs, especially the zebrafish gene cdx(Zf-cad). The possible relationship to homeobox genes within the Hox-C gene complexes is discussed. A weak expression of the gene, detected by in situ hybridization, was found shortly before gastrulation (at 25% epiboly) in cells likely to have a posterior fate. During gastrulation expression became stronger. At the early segmentation stage, cells of the neural keel in the area of the prospective spinal cord expressed the gene. During the progression of segmentation, expression retracted in a caudal direction. The tailbud expressed the gene throughout, but the somites lost expression shortly after their formation. Only the most lateral mesoderm cells maintained expression in the trunk area. Carp-cdxl was also expressed in the endoderm. At 24 h after fertilization the gene was only expressed in the tailbud. At 48 h, no expression could be detected. The expression pattern suggests a function for carp-cdxl in gastrulation and patterning along the anterior-posterior axis of the embryo.

Isolation of carp cDNA clones, representing developmentally-regulated genes, using a subtractive-hybridization strategy

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.

Teleost yolk cell function on blastoderm differentiation and morphogenesis

Next to a prominent nutritional role during larval growth the teleost yolk cell may function during early developmental processes as a source of inducing signals. In Xenopus mesoderm-inducing signals reside in the vegetal cells of the blastula and exert their function on the more animal located cells in the margin. Because of its position reminiscent of the vegetal cells in Xenopus, the yolk cells of teleost fishes may have a similar inductive capacity. Removal of the blastoderm from the yolk cell (YC) offers an experimental approach to study to what extent blastoderm differentiation and morphogenesis depend on inductive interaction with the YC. Expression of marker genes for dorsal-ventral and anterior/posterior determination such as no tail, goosecoid and caudal was examined in blastoderm explants, which were incubated until sibling controls reached the tail bud stage. Blastoderms removed at 3 h post fertilisation expressed rudimentary dorsal-ventral polarity. Apparently signals leading to this expression had reached the margin of 3-h blastoderms. Blastoderms removed at 4 h showed in addition to dorsal-ventral polarity notochord-like structures and also expression patterns of goosecoid and caudal suggesting that gastrulation movements had occurred.

The carp homeobox gene Ovx1 shows early expression during gastrulation and subsequently in the vagal lobe, the facial lobe and the ventral telencephalon.

Abstract The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections.

TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes.

Abstract. The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.

DNA probes to repetitive sequences for the analysis of porcine genomic DNA with reference to DNA methylation.

Abstract The aim of this study was to isolate probes to repetitive sequences of porcine (Great Yorkshire × Landrace) genomic DNA. The production of transgenic animals involves the isolation of stem cell lines and the understanding of DNA methylation modifications. Probes to repetitive sequences enable the analysis of DNA methylation in the tissues of various embryonic stages of the pig. A primary library of porcine genomic DNA was screened with labeled fragments of porcine DNA, and 6 clones containing repetitive DNA were isolated and analyzed for the presence of potential methylation moieties (CCGG sites). Probes of all 6 clones were tested in a hybridization analysis of HpaII and MspI digests of porcine sperm DNA, and it was found that methylation was not present in the methylation moieties of the repetitive sequences.