MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated b-cells remain unclear. Here, we show that miRNA inactivation in b-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient b-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured b-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.

microRNAs (miRNAs) play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas.

The small intestine (SI) of chicks (Gallus gallus) matures rapidly during the initial post-hatch period and acquires digestive, absorptive, and secretive capabilities. The effects of the timing of first feeding on the quantities and distribution of specialized epithelial cells, which generate and maintain SI morphology and functionality, have not yet been examined. In this study, we identified specialized SI epithelial cell sub-types, including stem, progenitor, proliferating, and differentiated cells within crypts and villi of chicks during the first 10 days post-hatch, by in situ hybridization (ISH), immunofluorescence (IF), and histochemical staining. We then examined their quantities and ratios between day of hatch and d10 in chicks that were fed upon hatch [early feeding (EF)], compared to chicks that were fed 24 h post-hatch [delayed feeding (DF)]. Results showed that EF increased total cell quantities in the crypts and villi at days 1, 3, 7, and 10, compared to DF (p < 0.0001). At d3, EF, in comparison to DF, decreased crypt stem cell proportions (p < 0.0001), increased crypt proliferating (p < 0.01) and differentiated (p < 0.05) cell proportions, and increased villus enterocyte proportions (p < 0.01). By d10, EF increased both the quantities and proportions of villus enterocytes and goblet cells, compared to DF. We conclude that feeding upon hatch, compared to 24 h-delayed feeding, enhanced SI maturation and functionality by increasing the quantities and proportions of proliferating and differentiated cells, thus expanding the digestive, absorptive, and secretive cell populations throughout the initial post-hatch period.

Nutritional stimulation of the developing small intestine of chick embryos can be conducted by in-ovo feeding (IOF). We hypothesized that IOF of glutamine and leucine can enhance small intestinal development by promoting proliferation and differentiation of multipotent small intestinal epithelial cells. Broiler embryos ( n = 128) were subject to IOF of glutamine (IOF-Gln), leucine (IOF-Leu), NaCl (IOF-NaCl) or no injection (control) at embryonic d 17 (E 17). Multipotent, progenitor and differentiated cells were located and quantified in the small intestinal epithelium between E 17 and d 7 after hatch (D 7) in all treatment groups by immunofluorescence of SRY-box transcription factor 9 (Sox9) and proliferating cell nuclear antigen (PCNA), in-situ hybridization of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) and peptide transporter 1 (PepT1) and histochemical goblet cell staining. The effects of IOF treatments at E 19 (48 h post-IOF), in comparison to control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and multipotent cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significance, 36% increase in progenitor cell counts. All IOF treatments shifted Lgr5+ stem cell localizations to villus bottoms. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Post–hatch, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to control chicks ( P < 0.05). We conclude IOF of glutamine stimulates small intestinal maturation and functionality during the peri-hatch period by promoting multipotent cell proliferation and differentiation, resulting in enhanced compartmentalization of multipotent and differentiated cell niches and expansions of the absorptive surface area.

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BackgroundNutritional stimulation of the small intestine (SI) of chick embryos can be conducted by enriching the amniotic fluid with nutrients via the in-ovo feeding (IOF) methodology. The impact of IOF of specific nutrients on cellular proliferation and differentiation within the multipotent (MP) and differentiated cell niches of the developing SI have not yet been characterized. In the study, we examined the effects of IOF of 1% glutamine (IOF-Gln), 1% leucine (IOF-Leu) and 0.4% NaCl (IOF-NaCl), compared to non-injected controls, on the proportions and localizations of MP, progenitor and differentiated cells within the SI epithelium of peri-hatch chicks. MP, progenitor and differentiated cells were located and quantified in jejunum sections of all treatment groups at E17 and at E19, and post-hatch at days 0, 1, 3 and 7, by immunofluorescence of Sox9 and PCNA, in-situ hybridization of Lgr5 and PepT1 and histochemical goblet cell staining.ResultsAt E19, 48 h post IOF, the effects of IOF treatments, in comparison to Control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and MP cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significant, 36% increase in progenitor cell counts. Lgr5+ stem cell localizations shifted to villus bottoms in IOF-treated embryos. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Between hatch and D7, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to Control chicks (P<0.05).ConclusionsIOF promotes pre-hatch SI maturation through increased proportions and enhanced compartmentalization of the MP and differentiated cell niches. IOF of glutamine stimulates SI maturation to a greater extent than leucine and NaCl, and elicits further expansions of the crypt and villus epithelium during the first week post-hatch. These findings shed light on the link between primary nutritional stimulation and cellular maturation within the SI epithelium.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling.

Optimal embryonic development and growth of meat-type chickens (broilers) rely on incubation conditions (oxygen, heat, and humidity), on nutrients and on energy resources within the egg. Throughout incubation and according to the embryo’s energy balance, the main energy storage molecules (creatine and glycogen) are continuously utilized and synthesized, mainly in the embryonic liver, breast muscle, and the extraembryonic yolk sac (YS) tissue. During the last phase of incubation, as the embryo nears hatching, dynamic changes in energy metabolism occur. These changes may affect embryonic survival, hatchlings’ uniformity, quality and post hatch performance of broilers, hence, being of great importance to poultry production. Here, we followed the dynamics of creatine and glycogen from embryonic day (E) 11 until hatch and up to chick placement at the farm. We showed that creatine is stored mainly in the breast muscle while glycogen is stored mainly in the YS tissue. Analysis of creatine synthesis genes revealed their expression in the liver, kidney, YS tissue and in the breast muscle, suggesting a full synthesis capacity in these tissues. Expression analysis of genes involved in gluconeogenesis, glycogenesis, and glycogenolysis, revealed that glycogen metabolism is most active in the liver. Nevertheless, due to the relatively large size of the breast muscle and YS tissue, their contribution to glycogen metabolism in embryos is valuable. Towards hatch, post E19, creatine levels in all tissues increased while glycogen levels dramatically decreased and reached low levels at hatch and at chick placement. This proves the utmost importance of creatine in energy supply to late-term embryos and hatchlings.