Search

Newly differentiated pancreatic β cells lack proper insulin secretion profiles of mature functional β cells. The global gene expression differences between paired immature and mature β cells have been studied, but the dynamics of transcriptional events, correlating with temporal development of glucose-stimulated insulin secretion (GSIS), remain to be fully defined. This aspect is important to identify which genes and pathways are necessary for β-cell development or for maturation, as defective insulin secretion is linked with diseases such as diabetes. In this study, we assayed through RNA sequencing the global gene expression across six β-cell developmental stages in mice, spanning from β-cell progenitor to mature β cells. A computational pipeline then selected genes differentially expressed with respect to progenitors and clustered them into groups with distinct temporal patterns associated with biological functions and pathways. These patterns were finally correlated with experimental GSIS, calcium influx, and insulin granule formation data. Gene expression temporal profiling revealed the timing of important biological processes across β-cell maturation, such as the deregulation of β-cell developmental pathways and the activation of molecular machineries for vesicle biosynthesis and transport, signal transduction of transmembrane receptors, and glucose-induced Ca2+ influx, which were established over a week before β-cell maturation completes. In particular, β cells developed robust insulin secretion at high glucose several days after birth, coincident with the establishment of glucose-induced calcium influx. Yet the neonatal β cells displayed high basal insulin secretion, which decreased to the low levels found in mature β cells only a week later. Different genes associated with calcium-mediated processes, whose alterations are linked with insulin resistance and deregulation of glucose homeostasis, showed increased expression across β-cell stages, in accordance with the temporal acquisition of proper GSIS. Our temporal gene expression pattern analysis provided a comprehensive database of the underlying molecular components and biological mechanisms driving β-cell maturation at different temporal stages, which are fundamental for better control of the in vitro production of functional β cells from human embryonic stem/induced pluripotent cell for transplantation-based type 1 diabetes therapy.

Fluorescent protein (FP) reporter alleles are useful both for identifying and purifying specific cell populations in the mouse. Here, we report the generation of mouse embryonic stem cells that contain a pancreatic and duodenal homeobox 1 (Pdx1) loxed cassette acceptor (Pdx1(LCA)) allele and the use of recombinase-mediated cassette exchange to derive mice that contain a Pdx1(CFP) (Cerulean) reporter allele. Mice with this allele exhibited cyan fluorescence within the previously well-characterized Pdx1 expression domain in posterior foregut endoderm. Immunolabeling showed that endogenous Pdx1 was coexpressed with CFP at all time points examined. Furthermore, fluorescence-activated cell sorting was used to isolate CFP-positive cells from E11.5 and E18.5 embryonic tissues using both 405 and 445 nm lasers, although the latter resulted in a nearly 50-fold increase in emission intensity. The Pdx1(CFP) allele will enable the isolation of specific foregut endoderm and pancreatic cell populations, both alone and in combination with other FP reporter alleles.

SummaryExtracellular signals in development, physiology, homeostasis, and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for being able to systematically compare the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, BMPs, activin A, Wnts, or Notch, and that can be adapted to any pathway that acts via DNA elements.

Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells, it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17(GFPCre) allele and using it to assess the fate of Sox17-expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence-activated cell sorting together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker.