In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

Girbau, Matias; Bassas, Lluís; Alemany, Jorge; Pablo, Flora de

doi:10.1073/pnas.86.15.5868

Cited by 47 publications

(32 citation statements)

References 31 publications

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“…During gastrulation and neurulation, the chick embryo is avascular and prepancreatic. Remarkably, it expresses preproinsulin mRNA, and the insulin receptor is functional at these stages, as indicated by its tyrosine kinase activity and anti-apoptotic effect, features shared in part by other species (8,28,(35)(36)(37)(38). Interference in vivo with insulin receptor synthesis using antisense oligodeoxynucleotides increases apoptosis in the neurulating embryo, demonstrating the physiological relevance of insulin signaling in the regulation of embryonic cell survival/death (this study and Morales et al [8]).…”

Section: Discussionmentioning

confidence: 80%

Unprocessed Proinsulin Promotes Cell Survival During Neurulation in the Chick Embryo

et al. 2002

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We have chosen a vertebrate model accessible during neurulation, the chick, for analysis of endogenous insulin signaling and its contribution to early embryonic cell survival. Unlike rodents, humans and chickens have a single preproinsulin gene, facilitating its prepancreatic expression characterization. We show that in vivo interference with embryonic insulin signaling using antisense oligonucleotides against the insulin receptor increases apoptosis during neurulation. In contrast, high glucose administration does not increase the level of apoptosis in culture or in vivo. Exogenous insulin and, remarkably, proinsulin achieve similar survival protective effects at 10 ؊8 mol/l. The low abundant preproinsulin mRNA from the prepancreatic embryo is translated to a protein that remains as unprocessed proinsulin. This concurs with the absence of prohormone convertase 2 (PC2) in the embryo, whereas PC2 is present later in embryonic pancreas. A C-peptidespecific antibody stains proinsulin-containing neuroepithelial cells of the chick embryo in early neurulation, as well as other cells in mesoderm-and endoderm-derived structures in the 2.5-day embryo. We have determined by 5-RACE (rapid amplification of cDNA ends), and confirmed by RNase protection assay, that prepancreatic and pancreatic proinsulin mRNA differ in their first exon, suggesting differential transcriptional regulation. All these data support the role of endogenous proinsulin in cell survival in the chick embryo during important pathophysiologic periods of early development.

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Section: Discussionmentioning

confidence: 80%

Unprocessed Proinsulin Promotes Cell Survival During Neurulation in the Chick Embryo

et al. 2002

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“…Mice deficient in insulin have shown reduced body size at the time of birth (12), and a similar phenotype was observed in insulin receptor-deficient pups (1, 10). In chickens, insulin and its receptors were shown to be expressed in all stages of early embryonic development, from blastoderm through gastrulation, neurulation, and organ formation (11,22). Clearly, this is before the development of the circulatory system or pancreas formation.…”

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confidence: 99%

Early developmental expression of two insulins in zebrafish (Danio rerio)

Papasani

Robison²,

Hardy

et al. 2006

Physiological Genomics

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We have cloned a second insulin gene in zebrafish and studied temporal and spatial expression of two zebrafish insulin genes. Zebrafish insulin-a (insa) and -b (insb) mRNAs are derived from two different DNA contigs on chromosomes 5 and 14, respectively, representing two different insulin genes. Real-time PCR studies suggest that insa is a maternal and also a postzygotic transcript. insa was observed at 1 h postfertilization (hpf) and was rapidly degraded by 6 and 12 hpf but induced at 24 hpf (i.e., after pancreas formation). Expression levels at 24 hpf were ϳ220-fold higher than at 6 hpf and were significantly different from earlier time points. At 72 hpf (at time of hatching), zebrafish insa mRNA levels tended to be higher than at 24 hpf and were ϳ727-fold higher compared with 6 hpf. This further increase in insa expression may be one of the many rapid physiological changes associated with hatching. insb expression was observed from 1 hpf and was significantly decreased from 12 hpf onward. Its expression levels at 12 and 24 hpf were approximately twofold and sixfold lower, respectively, compared with expression at 6 hpf. insb expression levels at 48 hpf were significantly lower than at 24 hpf but not different from 72 hpf. Expression levels at 72 hpf were ϳ61-fold lower than at 6 hpf. In situ hybridization studies showed insb expression in proliferating blastomeres at 3 and 4 hpf. At later time points, insb expression was restricted to the brain and pancreas (24 and 48 hpf). insa expression was observed in the pancreas at 24 and 48 hpf. Expression of insb in blastomeres and head suggests that insb could be acting as a pro-growth, survival, and neurotrophic factor during development. Pancreatic insa and insb may both be involved in regulation of glucose homeostasis as in mammals.insulin; gene duplication; polymerase chain reaction IT IS BECOMING CLEAR that single gene duplication and whole genome duplication have been typical rather than atypical events in the evolutionary history of many species. Two insulin genes have now been identified in rat, mouse, Xenopus laevis, and channel catfish (5,18,20,32). It was hypothesized that the gene duplication events in rodents were different from those occurring in X. laevis and channel catfish (2, 25, 33). The two X. laevis preproinsulins are more similar to each other than the other species and resulted from an insulin gene duplication that occurred during amphibian radiation (33). Recent evidence suggests that a genome duplication event occurred before teleost radiation in ray-finned fish lineage, after divergence from lobe-finned fishes (2, 25). Phylogenetic analysis using fish insulin genes suggests that insulin gene duplication occurred before the divergence of fugu and zebrafish, suggesting that insulin gene duplication occurred as part of a whole genome duplication event that occurred before teleost radiation (15).Insulin is generally believed to be exclusively expressed by pancreatic ␤-cells. It has multiple regulatory roles including mediation of glucose homeost...

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“…1) from Drosophila (2), chicken (3,4), and mouse (5). In Xenopus laevis large numbers of oocytes and developing embryos are accessible at all stages, making this organism an excellent model for studying the role of growth factors in development (6).…”

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“…Binding Studies. Oocytes, eggs, or embryos (packed volume, 5 ml) were homogenized in 10 ml of 83 mM NaCI/1 mM MgCl2/10 mM Hepes, pH 7.9, and solubilized membranes were prepared (4). Solubilized receptors were then purified on a 2-ml column of wheat germ agglutinin-agarose and radioreceptor assays were done (4) binding oflabeled ligand in the presence of IGF-I at 250 ng/ml or insulin at 1000 ng/ml.…”

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confidence: 99%

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

Scavo

Shuldiner²,

Serrano³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Insulin and insulin-like growth factor I (IGF-I) initiate their metabolic, growth, and differentiation effects through binding to the insulin receptor and the IGF-I receptor, two members of the tyrosine kinase family of receptors. To study the role of these peptides and receptors in early development, we used the polymerase chain reaction and embryo-derived RNA to generate partial cDNA sequences of the insulin receptor and IGF-I receptor from the amphibian Xenopus laevis. Three unique tyrosine kinase-related sequences were obtained. Two of the nucleotide sequences, XTK la and XTK lb, corresponded to peptides that share 92% amino acid identity, and each is 89% identical to the human insulin receptor. The third sequence, XTK 2, corresponds to a peptide that has 92% amino acid identity with the human IGF-I receptor but only 80% identity with XTK la and XTK lb. On the basis of these similarities, the pattern of conserved amino acids, and the tetraploid nature of the Xenopus genome, we suggest that XTK la and XTK lb most likely represent the product of two different nonallelic insulin receptor genes, while XTK2 may be one of the probable two Xenopus IGF-I receptor genes. By reverse transcription-polymerase chain reaction and gene-specific hybridization, expression of the three XTK sequences was detected in the oocyte, unfertilized egg, and embryos through gastrulation, neurulation, and tailbud stages. Competition binding assays with Xenopus membrane preparations demonstrated insulin receptors and IGF-I receptors in older tadpoles. IGF-I receptors were also present in oocytes, eggs, and gastrula embryos. By contrast, insulin binding was present but atypical in oocytes and was barely detected in eggs and gastrula embryos. The expression of receptors for insulin and IGF-I in early Xenopus embryos and their apparent distinct developmental regulation suggest that these molecules and their ligands may be important in early Xenopus development.

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In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

Cited by 47 publications

References 31 publications

Unprocessed Proinsulin Promotes Cell Survival During Neurulation in the Chick Embryo

Unprocessed Proinsulin Promotes Cell Survival During Neurulation in the Chick Embryo

Early developmental expression of two insulins in zebrafish (Danio rerio)

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

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