Biomedical subjects

F Argenton

Publications and source records attributed to F Argenton.

15 recordsLinked to original sources

PubMed2006

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish.

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.

Animals↗

PubMed2001

Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet.

To begin to understand pancreas development and the control of endocrine lineage formation in zebrafish, we have examined the expression pattern of several genes shown to act in vertebrate pancreatic development: pdx-1, insulin (W. M. Milewski et al., 1998, Endocrinology 139, 1440-1449), glucagon, somatostatin (F. Argenton et al., 1999, Mech. Dev. 87, 217-221), islet-1 (Korzh et al., 1993, Development 118, 417-425), nkx2.2 (Barth and Wilson, 1995, Development 121, 1755-1768), and pax6.2 (Nornes et al., 1998, Mech. Dev. 77, 185-196). To determine the spatial relationship between the exocrine and the endocrine compartments, we have cloned the zebrafish trypsin gene, a digestive enzyme expressed in differentiated pancreatic exocrine cells. We found expression of all these genes in the developing pancreas throughout organogenesis. Endocrine cells first appear in a scattered fashion in two bilateral rows close to the midline during mid-somitogenesis and converge during late-somitogenesis to form a single islet dorsal to the nascent duodenum. We have examined development of the endocrine lineage in a number of previously described zebrafish mutations. Deletion of chordamesoderm in floating head (Xnot homolog) mutants reduces islet formation to small remnants, but does not delete the pancreas, indicating that notochord is involved in proper pancreas development, but not required for differentiation of pancreatic cell fates. In the absence of knypek gene function, which is involved in convergence movements, the bilateral endocrine primordia do not merge. Presence of trunk paraxial mesoderm also appears to be instrumental for convergence since the bilateral endocrine primordia do not merge in spadetail mutants. We discuss our findings on zebrafish pancreatogenesis in the light of evolution of the pancreas in chordates.

Amino Acid Sequence↗

PubMed2000

Cloning and expression pattern of a zebrafish homolog of forkhead activin signal transducer (FAST), a transcription factor mediating Nodal-related signals.

Forkhead activin signal transducer (FAST) is a member of the winged-helix family of DNA-binding proteins that has been implicated in mesoderm induction and left-right axis specification during embryonic development in Xenopus and mouse. We have cloned and characterized a zebrafish FAST homolog. Zebrafish fast is expressed maternally and zygotically. Transcripts start regionalizing and decline in level during gastrulation. During somitogenesis, fast is expressed bilaterally in the lateral plate mesoderm, like its mouse homolog. In addition, zebrafish fast is also expressed bilaterally in the dorsal diencephalon, where the nodal-related cyclops gene is only expressed on the left side. It remains to be demonstrated whether FAST expression in the brain can mediate Nodal-induced asymmetric development.

Amino Acid Sequence↗

PubMed1999

Regulatory regions in the promoter and third intron of the growth hormone gene in rainbow trout, Oncorhynchus mykiss walbaum.

The mechanisms involved in the transcriptional regulation of the rainbow trout (Oncorhynchus mykiss) growth hormone (tGH) gene have been investigated. Transient transfection assays, using deletion mutants of the tGH promoter, demonstrated that the -226/+24 5'-flanking region, bearing three binding sites for the pituitary-specific transcription factor GHF1/Pit1 and a cAMP-response element, is necessary and sufficient to confer strong tissue-specific and cAMP-stimulated expression to a luciferase reporter gene. This region is also upregulated by the synthetic glucocorticoid dexamethasone (DEX), the combined effects of cAMP, and DEX being synergistic. Footprinting and gel shift assays revealed that GHF1 binds to a recognition element in the third intron of the tGH gene, suggesting that GHF1 can affect the expression of this gene by interacting with response elements in the transcription unit. These results may be exploited to design tGH gene constructs for the production of autotransgenic fish, in which the expression of the isospecific transgene driven by a constitutive proximal promoter is specifically targeted to the pituitary and physiologically controlled.

Animals↗

PubMed1999

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions.

Animals↗

PubMed1999

Expression patterns of zebrafish sox11A, sox11B and sox21.

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Amino Acid Sequence↗

PubMed1999

Structure and functional analysis of a tilapia (Oreochromis mossambicus) growth hormone gene: activation and repression by pituitary transcription factor Pit-1.

A gene encoding the Tilapia mossambica (Oreochromis mossambicus) growth hormone (tiGH) was isolated and sequenced. The gene spans 5.6 kb, including 3.7 kb of 5' and 0.2 kb of 3' flanking sequences and a 1.7-kb transcription unit comprised of six exons and five introns. The gene and the 5' flanking region contain several potential binding sites for Pit-1, a key transcription activator of mammalian GH genes. One of these (-57/-42) is highly conserved in fish GH genes. It activates transcription in pituitary cells and binds Pit-1. Transfection of luciferase reporter plasmids containing either the -3602/+19 tiGH sequence or one of its 5' deletion mutants (-2863/, -1292/, and -463/+19) resulted in strong activity in Pit-1-producing rat pituitary GC cells. A dose-dependent activation of the tiGH promoter was achieved in nonpituitary fish EPC and monkey COS cells cotransfected with a rat Pit-1 expression vector, demonstrating the crucial role played by Pit-1 as an activator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosidase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC and EPC cells was strongly repressed by extending its 3' end from +19 to +40, a sequence in which a Pit-1-binding site was identified using gel retardation assays. Point mutations of the site that suppressed Pit-1 binding in vitro restored full tiGH promoter activity. Thus, a Pit-1-binding site located in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene.

3' Untranslated Regions↗

PubMed1998

Cloning of zebrafish neurofilament cDNAs for plasticin and gefiltin: increased mRNA expression in ganglion cells after optic nerve injury.

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.

Animals↗

PubMed1997

The bacteriophage T7 binary system activates transient transgene expression in zebrafish (Danio rerio) embryos.

The bacteriophage T7 binary expression system is widely used in vitro for high level selective expression of cloned genes but its application to in vivo models has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing the T7 RNA polymerase gene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing the Escherichia coli lacZ gene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary system in vivo to a vertebrate model.

Animals↗

PubMed1997

Functional characterization of the trout insulin promoter: implications for fish as a favorable model of pancreas development.

The complex anatomy of the mammalian pancreas, in which the endocrine cells are grouped in islets dispersed among the predominant exocrine component, has hampered study of the molecular events governing the development of pancreatic cell lineages. To investigate whether fish may provide relevant, complementary models of pancreas development, we characterized the trout insulin (tINS) promoter and its molecular interactions with PDX1, a key transcriptional and developmental factor of the mammalian pancreas. Transfection of a luciferase reporter plasmid containing the 280 bp 5'-flanking region of the tINS gene resulted in strong activity in mammalian pancreatic beta cells but not in CHO or pituitary cells. Footprinting assays and cotransfection experiments indicated that mammalian PDX1 binds to and activates the tINS promoter. By microinjecting plasmids to fertilized zebrafish eggs, we showed that the expression of mouse PDX1 is capable of activating the co-injected tINS promoter plasmid in most cell types of the 24-h zebrafish embryo. The conserved role of PDX1 in vertebrate insulin gene regulation opens the possibility to exploit fish models in the study of pancreas development.

Animals↗

PubMed1996

Mechanisms of transcriptional activation of the promoter of the rainbow trout prolactin gene by GHF1/Pit1 and glucocorticoid.

The transcription factor GHF1/Pit1, required for the expression of the prolactin (PRL) and other pituitary-specific genes, is highly conserved from fish to mammals but the mechanisms by which it activates transcription are poorly understood. The activity of the promoter (-627/+15 region) of the rainbow trout PRL (tPRL) gene fused to the luciferase reporter gene was studied using GHF1-expressing rat pituitary GC cells. Nuclear extracts of GC cells produced five GHF1-specific footprints in the tPRL promoter, with the position of the two most proximal ones being highly conserved in trout and mammalian GHF1-regulated genes. Deletional and mutational analyses of the tPRL promoter showed that the most proximal GHF1 site alone is sufficient to confer sub-maximal GHF1-dependent transcriptional activity and that a glucocorticoid response element-like motif mediates dexamethasone stimulation. It is suggested that GHF1 molecules bound to different sites of the tPRL promoter cannot interact simultaneously with the transcriptional apparatus. Moreover, GHF1 and the ligand-bound glucocorticoid receptor tethered to their cognate elements in the promoter could cooperate to enhance transcription by interacting simultaneously with different members of the basal transcriptional complex.

Animals↗

PubMed1996

A TGACG motif mediates growth-hormone factor-1/pituitary-transcriptional-activator-1-dependent cAMP regulation of the rainbow trout growth-hormone promoter.

The mechanisms involved in the regulation of the rainbow trout growth hormone (tGH) gene promoter by the pituitary-specific transcription factor GHF1 (growth hormone factor 1), also called Pit1 (pituitary transcriptional activator 1), and cAMP have been investigated in mammalian and fish cells. The -340 to +24 5'-flanking Fegion of the tGH gene focused to the luciferase gene was activated in rat pituitary GC cells and in HeLa cells cotransfected with an effector plasmid encoding rat GHFI. GC cell nuclear extracts produced four GHFI-specific footprints (sites Fl to F4) on the tGH promoter, each containing multiple W4NCAT (W, A or T) or closely related motifs. Mutational analysis performed in GC cells indicated that the proximal Fl site alone can direct transcription, but that the region encompassing the F2 and F3 sites is necessary for optimal activation and contains a TGACG motif (cAMP-response element, CRE) conferring cAMP responsiveness. The role of the TGACG motif in mediating cAMP regulation of the tGH promoter was confirmed in primary cultures of trout pituitary cells. Cotransfection studies in carp EPC cells using an effector plasmid encoding trout GHF1 demonstrated the GHF1 dependence of cAMP stimulation. Gel shift and southwestern experiments revealed nuclear proteins of 43 kDa and 30 kDa in GC and fish cells, respectively, that bind specifically to the tGH CRE, suggesting the involvement of CRE-binding-protein/activating-transcription-factor-l-related peptides in cAMP response. Incidentally, and in contrast with previous reports, we found the rat GH promoter, that lacks TGACG motifs, unresponsive to cAMP. Thus, the CAMP stimulation of the tGH gene is more similar to its human counterpart. that is also GHF1 dependent and mediated by TGACG motifs in the promoter. It is suggested that control of GH gene expression has evolved modularly, through various assortments of the same regulatory units, rather than molecularly, through innovative units.

Animals↗

PubMed1996

An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos.

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

Animals↗

PubMed1993

The transcriptional regulation of the growth hormone gene is conserved in vertebrate evolution.

Growth hormone (GH) gene expression in mammals is regulated by the interaction of the transcription factor Pit-1 with two binding sites within the proximal promoter. Four sequences, homologous to the mammalian Pit-1 motif occur in the rainbow trout (Oncorhynchus mykiss) GHII (rtGH) gene promoter, two of which partly overlap. The three regions containing these putative Pit-1 binding sequences were protected from deoxyribonuclease I digestion by nuclear extracts of GC cells, a rat pituitary tumor cell line producing Pit-1. In gel shift assays, nuclear proteins from GC cells and from trout pituitaries were found to interact specifically with one of these protected sites. Transfection experiments showed that the rtGH promoter is transcriptionally active in GC cells, the response being strongly enhanced in the presence of a cAMP analogue. The results demonstrate that rat Pit-1 binds to and activates the rtGH promoter, indicating that the basic mechanisms regulating GH gene transcription have been conserved between fish and mammals.

Animals↗

PubMed1990

Centrosome competition: a possibility?

A recent theory on the evolution of sexuality, has hypothesized heritable variation in the functional properties of centrosomes, leading to competition for the organization of the mitotic spindle when different centrosomes enter a common cytoplasm. We present here data on polyethylene glycol-induced polykaryocytes of cultured Chinese hamster fibroblasts indirectly supporting centrosome competition. On the assumption that sib centrosomes are similar and variation increases with cell generations, the frequencies of multipolar mitoses were compared in cultures fused under conditions favoring sib cell fusion or fusion of distantly related cells. Multipolar mitoses were considerably more frequent in the former, when the average difference between pairs of centrosomes was assumed to be too small for one centrosome to "outcompete" the other.

Animals↗