Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression
Normal Arx developmental expression patterns were assessed in wild type (WT) mouse pancreata. Immunofluorescence was used to visualize Arx co-expression with islet progenitor markers/hormones with focus on the developmental period E9.5-E15.5. Arx expression was demonstrated on days E9.5-E15.5 in glucagon+ cells and at E13.5 in ghrelin+ cells. Additionally, there were smaller populations of Arx+/Pdx1+, non-hormone expressing cells (E9.5-E15.5), Arx+/Neurog3+ cells (E12.5), and Arx+/glucagon+/ghrelin+ cells (E15.5). To assess the interaction of Nkx2.2 with Arx and subsequent effects on pancreatic cell lineages, immunofluorescence studies were furthered in an Nkx2.2 null mutant model. Arx expression in Nkx2.2Null mutants was localized to ghrelin expressing cells and Nkx2.2 loss led to an increase in the Arx+/ghrelin+ cell population as compared to WT pancreata. From these and other data, the authors hypothesized that Arx and Nkx2.2 interact in such a way as to control ghrelin cell specification in the pancreas.
To elucidate the role of Arx in ghrelin cell specification, immunofluorescence was used to quantify the number of ghrelin expressing cells in WT, single mutant Nkx2.2Null , single mutant ArxΔpanc (Arx removed in Pdx1+ pancreatic progenitor cells), or compound mutant Nkx2.2Null;ArxΔpanc mice. Both Nkx2.2Null and the compound mutant exhibited greater numbers of ghrelin+ cells as compared to WT pancreata however, the production of ghrelin per cell was significantly decreased in the Nkx2.2Null;ArxΔpanc versus Nxk2.2Null mutant. This led the authors to propose that Arx is necessary for the regulation of ghrelin gene expression, rather than for ghrelin cell specification.
Panel 1. Summary of experimental results related to changes in cell populations and gene expression within Nkx2.2 and/or Arx mutant mice. [magnify ]
The Nkx2.2/Arx single and double mutants were analyzed at several developmental time points for expression of specific alpha, beta, PP, or delta cell markers; in order to define how Nkx2.2 and Arx interaction affects the development/specification of endocrine lineages (Panel 1). Previous studies have examined changes in pancreas development in both the Nkx2.2 and the Arx single mutants and these results were recapitulated by the authors to serve as controls for the compound mutant. Immunofluorescence studies showed that all mutant pancreata lost the ability to form alpha cells with a concomitant decrease in POU domain, class 3 (Pou3f4/Brn4 – alpha cell factor) gene expression. Also, Nkx2.2Null;ArxΔpanc mutants displayed an additive decrease in glucagon (Gcg) expression seen at E15.5 but not before E12.5; thereby suggesting an interaction between Nkx2.2 and Arx to allow for a cell differentiation during the secondary transition phase of pancreas development. With regards to beta cell formation, Nkx2.2Null and compound mutants showed an absence of beta cells, a loss of insulin gene expression (Ins1/2), and a decrease in the expression of transcription factors Neurod1, Pdx1, and Nkx6.1. These phenotypes were not observed in the ArxΔpanc mutant. PP cell numbers were decreased in Nkx2.2Null mutants along with pancreatic polypeptide (Ppy) expression but both PP cell number and Ppy expression were restored to wild type levels in the compound mutant, which provides further evidence that Arx and Nkx2.2 interact to mediate PP cell specification/development. ArxΔpanc and compound mutants showed an increase in somatostatin-expressing delta cells however, no change in delta cell numbers were observed with the Nkx2.2Null mutant. Moreover, realtime PCR verified an increase in somatostatin (Sst) levels in the ArxΔpanc mutant and even higher levels of Sst in the compound mutant, confirming that Arx is necessary for delta cell formation but not Nkx2.2. Finally, the authors discovered a population of bihormonal cells in the Nkx2.2Null;ArxΔpanc mutant that co-expressed ghrelin and somatostatin. A smaller number of bihormonal cells were found in the ArxΔpanc mutant but not the Nkx2.2Null mutant, leading the authors to conclude that deletion of both Arx and Nkx2.2 drives a significant increase in the ghrelin+/somatostatin+ cell population.
Endocrine specific Arx deletions in the Nkx2.2 null background (Nkx2.2Null;ArxΔendo) were generated to assess whether Arx functions within the Neurog3+ progenitors to influence the endocrine cell lineage. Immunofluorescence studies with these mutants showed comparable results to the Nkx2.2Null;ArxΔpanc mutants in terms of cell populations and gene expression, demonstrating that Arx functions within the endocrine progenitor population to influence specification/maturation of hormone-expressing cells in the embryonic pancreas.