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Evidence that the insulin-like growth factors play a role in embryonic as well as postnatal growth and central nervous system development has accumulated recently from studies using knock-out mice models. However, no effects of IGF-I and II have been demonstrated prior to organogenesis in these studies. We summarize here results supporting the role of insulin (or its precursor proinsulin) in vertebrate development prior to the expression of IGFs. (Pro)insulin mRNA is expressed in the chick embryo during neurulation and early organogenesis and its inhibition by antisense oligodeoxynucleotides increase apoptosis. In another system, proliferative neuroretina, (pro)insulin expression predominates over IGF-I expression. Modulation of apoptosis by (pro)insulin in retina may be largely responsible for the observed stimulation of DNA synthesis and neuronal differentiation. These effects are elicited as well by IGF-I, expressed later in neuroretina. Thus, these polypeptides have complementary expression in early embryos which suggests coordinated actions during development.

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.

The characterization of (pro)insulin as an early embryonic growth factor requires demonstration of its expression and cellular effects in vivo. By in situ hybridization, we found widespread preproinsulin transcripts in the chick embryo throughout gastrulation and neurulation, before the beginning of preproinsulin-like growth factor I expression and pancreatic organogenesis. To analyze the prepancreatic (pro)insulin effect on apoptotic cell death, we treated embryos with antisense oligodeoxynucleotides in ovo and in vitro. The specific effect of two preproinsulin messenger RNA (mRNA) antisense oligodeoxynucleotides was confirmed by the decrease in a biosynthetically labeled protein immunoprecipitated with antiinsulin Igs. Insulin receptor mRNA antisense oligodeoxynucleotide applied in ovo increased by 2.7-fold the level of apoptosis in the 1.5-day embryo (neurulation) compared with that in its random sequence control. In a whole embryo culture, apoptosis increased by 25-35% with the addition of preproinsulin or insulin receptor mRNAs antisense oligodeoxynucleotides, respectively, whereas it decreased by 64% after 10 h in the presence of 10(-8) M chicken insulin. Exogenous insulin also rescued the death induced by preproinsulin antisense oligonucleotides. These findings provide evidence for an autocrine/paracrine role ofpreproinsulin gene products acting through the insulin receptor in the control of cell survival/death during early embryonic development.

Despite the absence of a pancreas, which develops between embryonic day 3 (E3) to E4, previous studies showed that insulin receptors are widely expressed in chicken embryos from the blastoderm stage (unincubated embryo, E0) through gastrulation (E0.5-E1), neurulation (E1.5-E2), and organogenesis. We now characterize prepancreatic preproinsulin gene expression and its regulation, using a highly sensitive modification of the polymerase chain reaction. We found preproinsulin messenger RNA (mRNA) expression at all stages, from the unincubated chicken blastoderm through early organogenesis, with the highest expression in embryos undergoing gastrulation. In situ hybridization analysis of E1-E1.5 embryos in toto showed widespread distribution of preproinsulin mRNA in a pattern similar to that of insulin receptor mRNA. In contrast, insulin-like growth factor-I mRNA expression appeared later than preproinsulin mRNA in the embryo; it was first demonstrable in the head portion of E3 and was found in head, trunk, and caudal regions by E4. With a novel culture system for chicken embryos during neurulation, we examined whether glucose regulated prepancreatic preproinsulin mRNA expression. Embryos cultured in glucose-free medium had increased preproinsulin mRNA with respect to the value in ovo, but the addition of 17 mM glucose had no stimulatory effect. In marked contrast, in organ cultures of E13 pancreas, insulin mRNA expression decreased in glucose-free medium by 50% relative to that in ovo. The addition of glucose restored the levels to a concentration similar to that found in ovo. Exogenous insulin added to cultured E1.5 embryos increased protein and DNA synthesis. We conclude that the preproinsulin gene is widely expressed in chicken embryo structures throughout gastrulation and neurulation. This prepancreatic preproinsulin mRNA is differentially regulated compared to the pancreatic mRNA. Preproinsulin gene products may have a role in cell proliferation, differentiation, or survival in very early avian embryos at a time when insulin-like growth factor-I expression is absent or undetectable.