Louis M. Scavo

Insulin and Insulin-like Growth Factor System Components Gene Expression in the Chicken Retina From Early Neurogenesis Until Late Development and Their Effect on Neuroepithelial Cells

To better understand the role of insulin‐related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin‐like growth factor I (IGF‐I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)‐an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF‐I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF‐I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF‐I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9–15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF‐I receptor genes, while IGF‐I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF‐I, already at 10−8 M, increased the proportion of PM1‐positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF‐I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGFs role, local IGFBPs expression can contribute to the fine modulation.

Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors.

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-β in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-γ and TGF-β in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-γ and TGF-β had the most potent and consistent effects in both systems.

MATERNALLY ADMINISTERED DEXAMETHASONE TRANSIENTLY INCREASES APOPTOSIS IN LUNGS OF FETAL RATS

In late gestation, morphological maturation of fetal lung includes septal thinning of potential airspaces, a process accelerated by exogenous glucocorticoids. Apoptosis occurs in normal fetal lung. Glucocorticoids increase apoptosis in several tissues. The authors hypothesized that exogenous glucocorticoids would increase apoptosis in fetal lung, primarily in the interstitium. They administered dexamethasone (DEX), 1 mg/kg, or vehicle (Control) to pregnant rats at 19 days of gestation. Fetuses were delivered at 3, 7, 12, or 24 hours post injection. DEX decreased fetal body weight and lung weight, DNA, and protein 12 hours post injection. Using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) reaction to label apoptotic cells in lung, they calculated an apoptotic index (AI, apoptotic cells/1000 total cells) for each fetus. Average DEX AI (3.6 ± 2.6, mean ± SD) was greater than Control (1.7 ± 0.5) (P <.02). All DEX AIs were greater than Control AIs at 3, 7, and 12 hours, but were similar to Controls at 24 hours post injection. Apoptotic cells appeared to be interstitial, based on colocalization with vimentin staining. Presence of apoptotic cells was confirmed by electron microscopy and detection of the nucleosomal ladder pattern on DNA electrophoresis. The authors conclude that maternal administration of dexamethasone increases apoptosis in fetal lung, primarily in the interstitium. They speculate that apoptosis may contribute to morphological fetal lung maturation induced by endogenous glucocorticoids.