title: Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction.
abstract: 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.
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
authors.forename: Philip T
authors.initials: PT
authors.lastname: Pauerstein
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
authors.forename: Takuya
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Stanley
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
authors.forename: Graeme W
authors.initials: GW
authors.lastname: McLean
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA seungkim@stanford.edu.
authors.forename: Seung K
authors.initials: SK
authors.lastname: Kim
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; C407737:Neurog3 protein, mouse; C547963:Rfx6 protein, mouse; C093564:Neurogenic differentiation factor 1