The mammal-specific Pdx1 Area II enhancer has multiple essential functions in early endocrine-cell specification and postnatal β-cell maturation

Abstract

The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5′ conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (−2139 to −1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo. Pdx1ΔAREAII/− mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon + α relative to insulin + β cells, associated with the transcriptional and epigenetic derepression of the α-celldetermining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1 ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions.

Defining a Novel Role for the Pdx1 Transcription Factor in Islet β-Cell Maturation and Proliferation During Weaning

“…The reduction in Ngn3 + cell numbers also translated into a lower islet β-cell area in 3-week-old Pdx1 ΔAIV/− mice, which, notably, did not influence expression of key islet β-cell regulators or β-cell function. This observation differs significantly from either the early embryonic deletion of area I, II, and III in vivo ( 11 ) or the Pdx1 ΔAII/− mutant ( 20 ). Thus, severe defects in endocrine progenitor cell formation and insulin + - and glucagon + -coexpressing cells are found in Pdx1 ΔAII/− animals postnatally ( 20 ), phenotypes not observed in Pdx1 ΔAIV/− mutants.…”

“…This observation differs significantly from either the early embryonic deletion of area I, II, and III in vivo ( 11 ) or the Pdx1 ΔAII/− mutant ( 20 ). Thus, severe defects in endocrine progenitor cell formation and insulin + - and glucagon + -coexpressing cells are found in Pdx1 ΔAII/− animals postnatally ( 20 ), phenotypes not observed in Pdx1 ΔAIV/− mutants. Collectively, our embryonic results strongly suggest that area IV has only a very limited impact on developmental regulation imposed by areas I, II, and III.…”

“…2 D and E ). Notably, endogenous Pdx1 ΔAII/− mutant mice produced a much more penetrant developmental phenotype, resulting in a profound loss in α- and β-cell numbers and early postnatal lethality ( 20 ).…”

“…However, what remains to be understood is exactly how each of these enhancer-like domains control Pdx1 expression, appreciating that unique, independent control properties have been found for distal control regions in other cellular contexts (e.g., the globin genes [ 19 ]). Notably, recent analysis of an endogenous Pdx1 area II deletion mutant in a Pdx1 protein null background (i.e., Pdx1 ΔAII/− ) resulted in a profound and specific reduction in pancreatic endocrine progenitor cell formation that caused the loss in hormone-producing cells and early postnatal cell death from hyperglycemia ( 20 ), thus revealing a requirement for area II in controlling Pdx1 transcription during pancreas cell development. In this study, we focused on defining how area IV effected Pdx1 expression.…”

“…Consequently, we generated a new mouse deletion allele, termed Pdx1 ΔAIV , in which endogenous area IV was precisely removed genetically. In contrast to the area II control region mutant mice (i.e., Pdx1 ΔAII/− [ 20 ]), there was only a very modest influence on pancreas cell formation developmentally and no impact on viability in an area IV mutant that also lacked a functional Pdx1 allele (i.e., Pdx1 ΔAIV/− ). However, blood glucose levels were significantly elevated at weaning in male Pdx1 ΔAIV/− mice (i.e., after 3 weeks) and not age-matched female Pdx1 ΔAIV/− or control Pdx1 +/− mice.…”