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R Patient

Publications and source records attributed to R Patient.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of the stem cell leukemia (SCL) gene: a tale of two fishes.

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix-loop-helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5' and 3' flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Amino Acid Sequence↗
PubMed2000

Distinct origins of adult and embryonic blood in Xenopus.

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.

Age Factors↗
PubMed1999

The human and mouse GATA-6 genes utilize two promoters and two initiation codons.

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5' noncoding exons are utilized. Both transcript isoforms are expressed in the same tissue-specific and developmental stage-specific pattern, and their ratio appears similar wherever examined. The more upstream noncoding exon showed a substantial degree of homology between the two mammalian species, suggesting a conserved regulatory function. Moreover, in transfection assays we show that elements within this exon act to promote its transcription. Positive regulatory elements that effect transcription from the more downstream exon were not apparent in this assay, revealing a regulatory distinction between the two promoters. We also demonstrate alternative initiator codon usage in both the human and mouse GATA-6 genes. Both isoforms of the protein are synthesized in vitro regardless of which 5' noncoding exon is present in the RNA, although the larger protein has greater transcriptional activation potential in transfection assays. Thus, GATA-6 function in the cell is controlled by a complex interplay of transcriptional and translational regulation.

Alternative Splicing↗
PubMed1999

Gata5 is required for the development of the heart and endoderm in zebrafish.

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.

Amino Acid Sequence↗
PubMed1999

Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF.

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

Activins↗
PubMed1999

The transcription factor GATA6 is essential for early extraembryonic development.

The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue.

Animals↗
PubMed1999

The transcription factor GATA6 is essential for early extraembryonic development.

The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner cell mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue.

Animals↗
PubMed1997

Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors.

Xenopus GATA-6 transcripts are first detected at the beginning of gastrulation in the mesoderm, and subsequent domains of expression include the field of cells shown to have heart-forming potential. In this region, GATA-6 expression continues only in those cells that go on to form the heart; however, a decrease occurs prior to terminal differentiation. Artificial elevation of GATA-6, but not GATA-1, prevents expression of both cardiac actin and heart-specific myosin light chain. This effect is heart-specific because cardiac actin expression is unaffected in somites. Expression of the earlier marker XNkx-2.5 was unaffected and morphological development of the heart was initiated independently of the establishment of the contractile machinery. We conclude that a reduction in the level of GATA-6 is important for the progression of the cardiomyogenic differentiation programme and that GATA-6 may act to maintain heart cells in the precursor state. At later stages, when the elevated GATA-6 levels had decayed, differentiation ensued but the number of cells contributing to the myocardium had increased, suggesting either that the blocked cells had proliferated or that additional cells had been recruited.

Alleles↗
PubMed1997

A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in the zebrafish.

The effects of signal perturbation on expression domains of molecular markers for the mesoderm and ectoderm have been analysed across the dorso-ventral axis in zebrafish embryos. Injection of RNA encoding bone morphogenetic protein-4 (BMP-4) ventralised the embryo, expanding the intermediate mesoderm and non-neural ectoderm at the expense of the dorso-anterior mesoderm and neural plate. A dose-dependent response was observed both morphologically and in expression of gta3, MyoD and pax2. Conversely, increases in dorso-anterior mesoderm and neurectoderm were generated by injection of RNA encoding either a dominant-negative BMP receptor (delta BMPR) or noggin, as demonstrated by goosecoid and pax2 expression. Ventral BMP-4 expression was also inhibited. Thus, patterning of both the mesoderm and the ectoderm during gastrulation appears to depend, directly or indirectly, on the level of BMP activity. Consistent with their locations prior to formation of the neural tube, elevated BMP-4 increased the number of dorsal spinal cord neurons whilst sonic hedgehog and islet1 expression in the ventral spinal cord were reduced. However, the ectopic neurons were not positioned more ventrally, implicating a prepattern in the dorsal neural tube that is independent of the ventral central nervous system.

Animals↗
PubMed1995

Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

Amino Acid Sequence↗
PubMed1995

Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail.

To analyse the roles of FGF activity and brachyury during gastrulation we have directly compared the consequences of inhibition of FGF-receptor signalling with the phenotype of the zebrafish brachyury mutant, no tail (ntl). We show that expression of ntl is regulated by FGF and that inhibition of FGF receptor-signalling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. We propose a model to explain the FGF-dependent regulation of ntl and ntk that accounts for the above phenotypes. Consistent with this model, over-expression of eFGF led to suppression of anterior fates and development of trunk and tail derivatives only. In addition, widespread activation of convergence and extension movements resulted in the formation of multiple axis-like structures. Expression of eve1 and cad1 was also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorising centre during AP patterning, mediated by FGF activity in this tissue.

Animals↗
PubMed1994

Spatially regulated expression of three receptor tyrosine kinase genes during gastrulation in the zebrafish.

We describe the isolation and early developmental expression of three novel zebrafish genes (rtk1-3) that encode members of the eph family of receptor tyrosine kinases. At the onset of gastrulation, rtk1 is expressed in the shield region corresponding to the future dorsal side of the embryo. As gastrulation proceeds, both rtk1 and rtk2 are expressed within the axial hypoblast along the entire axis of the embryo. After the gastrula stage is complete, expression of both genes is maintained in precursor cells of the notochord in the tail bud but is downregulated in other regions of the axial hypoblast, rtk3 is expressed in anterior axial hypoblast including the 'pillow' at the anterior tip of the hypoblast and in paraxial tissue in posterior regions of the embryo. We show that the precise spatial regulation of expression of rtk genes, ntl and goosecoid along the anteroposterior axis is maintained in embryos that have no dorsoventral axis. This indicates that the mechanisms that regulate gene expression along the anteroposterior and dorsoventral axes of the hypoblast may be independent.

Amino Acid Sequence↗
PubMed1992

Cell lines produce factors that induce fetal hemoglobin in human BFUe-derived colonies.

Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system.

Animals↗
PubMed1989

Activation mechanisms of the Xenopus beta globin gene.

Comparative protein binding studies have been performed on the Xenopus beta globin gene promoter. Erythroblast nuclear extracts 'footprint' over the erythroid-specific consensus sequence, AGGATAAG, which is located immediately upstream of the CCAAT footprint. Nonerythroid cell extracts do not give rise to an AGGATAAG footprint but rather to an extended CCAAT footprint reminiscent of the CCAAT displacement protein (CDP). Erythroblast extracts also protect a sequence similar to the chicken stage selector element (SSE) immediately downstream of the CCAAT box footprint. In contrast to these discrete footprints observed using erythroblast extracts, Xenopus erythrocyte nuclear extracts give rise to more extensive promoter protection. We have previously reported that this promoter is active in transfected HeLa cells when linked to the SV40 enhancer and that transcriptional activation is accompanied by the formation in the chromatin of a nuclease hypersensitive site (HS) in this region. As a first step towards defining the roles of the various promoter-binding proteins in transcriptional activation and HS formation, we transfected deletion mutants of the promoter into HeLa cells. Deletion of the sequences upstream of -116 had no effect on transcription or HS formation. Indeed the upstream boundary of the HS remained unchanged (at around-170) even though plasmid sequences had replaced Xenopus sequences. If the HS boundary reflects resumption of nucleosomal structure, then sequences downstream of -116 must be able to position a nucleosome from at least 50 bp away. beta globin gene activation in a number of transfected cell lines is absolutely dependent on DNA replication. The replication requirement is not a consequence of template copy number or methylation, nor is it dependent on the direction in which the replication fork passes through the gene. We conclude that replication facilitates active transcription complex formation by disrupting a stable association of the template with negative factors, which could include histones. About 200 bp upstream of the Xenopus beta globin gene promoter is a tract of alternating A and T residues which adopts cruciform geometry at low levels of supercoiling. Because of this sensitivity to torsional stress, we have probed the structure of the (AT)n sequence in microinjected Xenopus oocytes, where the Xenopus beta globin gene is transcribed very efficiently. We find that S1 nuclease cleaves specifically in the middle of the (AT)n tract, suggesting that the gene is under torsional stress.

Animals↗