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Insulin and its related insulin-like growth factor I and II (IGFs) are multifunctional polypeptidic factors signalling through membrane tyrosine kinase receptors, the insulin receptor and the IGF-I receptor. While in vertebrates the IGFs are locally synthesized in multiple tissues during development, acting in paracrine fashion, insulin is highly expressed in the differentiated pancreas and serves a classic endocrine role as metabolic regulator. We have proposed that in early development insulin and/or its precursor proinsulin acts as well in autocrine/paracrine form to stimulate proliferation and differentiation, either directly or indirectly through the support of cell survival. In the chick embryo (E), the tem~oral expression of preproinsulin mRNA in two model systems, the neurulating embryo and the neuroretina, is complementary to the expression of IGF-I mRNA. In contrast to the expression of the mRNAs for the factors, highly developmentally reguiated, both the insulin receptor and the IGF-1 receptor mRNAs are expressed at all ages in similar levels. There is prepancreatic expression of preproinsulin mRNA in the chick embryo during gastrulation (EO.5) and neurulation (E1-E2) until pancreatic organogenesis (E2.5). IGF-I mRNA expression, however, is not detectable by either RT-PCR (Figure 1) or in situ hybridization until E2-E3. At least the 10 somites embryo (E1.5) in culture is able to translate the preproinsulin mRNA into a secreted product recognized by anti-insulin antibodies (which also crossreact with chicken proinsulin). When insulin is added to this cultured whole embryo, DNA synthesis is stimulated and cell death by apoptosis is reduced. To further characterize the role of autocrine/paracrine acting insulin on cell survival and/or proliferation, we have used an antisense oligonucleotide approach for interference experiments. The addition of preproinsulin antisense oligonucleotides, but not that of sense or random oligonucleotides, to E1.5 in culture, increased the percent of cells that undergo apoptosis, detected by DNAfragmentation revealed by DAPI-staining and TUNEL labelling. This effect is reversed by the co-addition of exogenous chicken insulin. Similarly, antisense oligonucleotides designed for the chicken insulin receptor, applied in ovo, increased the naturally occuring cell death that affects the 10-15 somites embryo. This early embryonic apoptosis is now being characterized in regional and cell subtype specific patterns.

Complementary roles of the insulin family of factors and receptors in early development and neurogenesis.