Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction.
Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction.
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作者：
Pauerstein PT;Sugiyama T;Stanley SE;McLean GW;Wang J;Martín MG;Kim SK
在胰腺发育过程中，内分泌前体及其后代分化、迁移和聚集形成新生胰岛。转录因子Neurogenin 3（Neurog 3）是小鼠胰岛发育所必需的，但其在这些动态形态发生步骤中的作用已从固定组织中推断出来。此外，关于NEUROG3在人类胰岛发育中的分子遗传功能知之甚少。我们开发了通过在培养的Neurog3无效突变胎儿胰腺上皮中进行病毒显微注射进行基因转导的方法，从而允许在发育相关的背景下进行遗传互补。此外，我们开发了定量评估单个发育中的胰岛细胞活细胞表型的方法。递送野生型NEUROG3挽救了胰岛分化、形态发生和活细胞变形，而患者来源的NEUROG3R107S等位基因部分恢复了胰岛发育的指标。NEUROG3P39X是一种以前未报道的患者等位基因，未能恢复胰岛分化或形态发生，并且与阴性对照无法区分，这表明它是一种无效突变。我们的系统还允许遗传抑制分析，并揭示了NEUROG3的靶点，包括NEUROD1和RFX6，可以部分恢复Neurog3缺失突变小鼠胰腺中的胰岛发育。因此，这里描述的进展允许前所未有的基因功能的评估，在调节关键的动态方面的胰岛发育的胎儿胰腺。
During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development. We developed methods for gene transduction by viral microinjection in the epithelium of cultured Neurog3-null mutant fetal pancreas, permitting genetic complementation in a developmentally relevant context. In addition, we developed methods for quantitative assessment of live-cell phenotypes in single developing islet cells. Delivery of wild-type NEUROG3 rescued islet differentiation, morphogenesis, and live cell deformation, whereas the patient-derived NEUROG3R107S allele partially restored indicators of islet development. NEUROG3P39X, a previously unreported patient allele, failed to restore islet differentiation or morphogenesis and was indistinguishable from negative controls, suggesting that it is a null mutation. Our systems also permitted genetic suppression analysis and revealed that targets of NEUROG3, including NEUROD1 and RFX6, can partially restore islet development in Neurog3-null mutant mouse pancreata. Thus, advances described here permitted unprecedented assessment of gene functions in regulating crucial dynamic aspects of islet development in the fetal pancreas.