Pauerstein Philip T, Sugiyama Takuya, Stanley Susan E, McLean Graeme W, Wang Jing, Martín Martín G, Kim Seung K
Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
Diabetes. 2015 Aug;64(8):3037-49. doi: 10.2337/db15-0042. Epub 2015 Apr 21.
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 NEUROG3(R107S) allele partially restored indicators of islet development. NEUROG3(P39X), 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.
在胰腺发育过程中，内分泌前体细胞及其后代分化、迁移并聚集形成新生胰岛。转录因子神经生成素3（Neurog3）是小鼠胰岛发育所必需的，但它在这些动态形态发生步骤中的作用是从固定组织中推断出来的。此外，关于NEUROG3在人类胰岛发育中的分子遗传功能知之甚少。我们开发了通过病毒显微注射在培养的Neurog3基因敲除突变体胎儿胰腺上皮中进行基因转导的方法，从而在与发育相关的背景下实现基因互补。此外，我们还开发了对单个发育中的胰岛细胞活细胞表型进行定量评估的方法。野生型NEUROG3的导入挽救了胰岛分化、形态发生和活细胞变形，而患者来源的NEUROG3（R107S）等位基因部分恢复了胰岛发育指标。NEUROG3（P39X）是一个先前未报道的患者等位基因，未能恢复胰岛分化或形态发生，与阴性对照无差异，表明它是一个无效突变。我们的系统还允许进行基因抑制分析，并揭示了NEUROG3的靶标，包括NEUROD1和RFX6，可以部分恢复Neurog3基因敲除突变小鼠胰腺中的胰岛发育。因此，本文所述的进展使得对调节胎儿胰腺中胰岛发育关键动态方面的基因功能进行了前所未有的评估。
