Single-cell transcriptome and accessible chromatin dynamics during endocrine pancreas development.

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate ß-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.

A radial axis defined by semaphorin-to-neuropilin signaling controls pancreatic islet morphogenesis.

The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered. Here, we identify a radial signaling axis that instructs developing islet cells to disperse throughout the pancreas. A screen of pancreatic extracellular signals identified factors that stimulated islet cell development. These included semaphorin 3a, a guidance cue in neural development without known functions in the pancreas. In the fetal pancreas, peripheral mesenchymal cells expressed Sema3a, while central nascent islet cells produced the semaphorin receptor neuropilin 2 (Nrp2). Nrp2 mutant islet cells developed in proper numbers, but had defects in migration and were unresponsive to purified Sema3a. Mutant Nrp2 islets aggregated centrally and failed to disperse radially. Thus, Sema3a-Nrp2 signaling along an unrecognized pancreatic developmental axis constitutes a chemoattractant system essential for generating the hallmark morphogenetic properties of pancreatic islets. Unexpectedly, Sema3a- and Nrp2-mediated control of islet morphogenesis is strikingly homologous to mechanisms that regulate radial neuronal migration and cortical lamination in the developing mammalian brain.

Research Resource: Genetic Labeling of Human Islet Alpha Cells.

The 2 most abundant human pancreatic islet cell types are insulin-producing ß-cells and glucagon-producing α-cells. Defined cis-regulatory elements from rodent Insulin genes have permitted genetic labeling of human islet ß-cells, enabling lineage tracing and generation of human ß-cell lines, but analogous elements for genetically labeling human α-cells with high specificity do not yet exist. To identify genetic elements that specifically direct reporter expression to human α-cells, we investigated noncoding sequences adjacent to the human GLUCAGON and ARX genes, which are expressed in islet α-cells. Elements with high evolutionary conservation were cloned into lentiviral vectors to direct fluorescent reporter expression in primary human islets. Based on the specificity of reporter expression for α- and ß-cells, we found that rat glucagon promoter was not specific for human α-cells but that addition of human GLUCAGON untranslated region sequences substantially enhanced specificity of labeling in both cultured and transplanted islets to a degree not previously reported, to our knowledge. Specific transgene expression from these cis-regulatory sequences in human α-cells should enable targeted genetic modification and lineage tracing.

Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction.

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.

An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

G2 acquisition by transcription-independent mechanism at the zebrafish midblastula transition.

Following fertilization of many animal embryos, rapid synchronous cleavage divisions give way to longer, asynchronous cell cycles at the midblastula transition (MBT). The cell cycle changes at the MBT, including the addition of gap phases and checkpoint controls, are accompanied by activation of the zygotic genome and the onset of cell motility. Whereas the biochemical changes accompanying the MBT in the vertebrate embryo have been extensively documented, the cellular events are not well understood. We show that cell cycle remodeling during the zebrafish MBT includes the transcription-independent acquisition of a G2 phase that is essential for preventing entry into mitosis before S-phase completion in cycles 11-13. We provide evidence from high-resolution imaging that inhibition of Cdc25a and Cdk1 activity, but not Cdk2 activity, is essential for cell cycle lengthening and asynchrony between cycles 9 and 12. We demonstrate that lengthening is not required for initiation of zygotic transcription. Our results are consistent with findings from Drosophila and Xenopus that indicate the central importance of G2 addition in checkpoint establishment, and point to similar mechanisms governing the MBT in diverse species.