1) Directed Differentiation
All pancreatic endocrine cells develop from common progenitor cells that express the bHLH transcription factor Neurogenin 3 (Ngn3). But exactly how Ngn3 promotes the development of one endocrine cell fate over another is not known, and attempts to direct the differentiation of pancreatic endocrine cells by overexpression of Ngn3 alone has mainly resulted in the promotion of only a single endocrine cell fate, the alpha cell. However, to be useful for diabetes it is important to direct the differentiation of beta cell fates over alpha cell fates, and this requires a better knowledge of how beta cells develop from endocrine progenitor cells.
We recently developed a new protocol to maximize production of pancreatic beta cells over alpha cells from naive endoderm (Oropez and Horb 2012). We created a hormone inducible Ngn3 by fusing it with the ligand binding domain of the glucocortocoid receptor (GR), creating Ngn3-GR. This allowed us to temporally control Ngn3 transcriptional activity by simple addition and removal of Dexamethasone (DEX). We found that short-term (4 hours) activation of Ngn3 immediately after gastrulation at stage 12 (14 hpf) resulted in successful expansion of pancreatic beta cells and delta cells, while, importantly, not expanding alpha cells. We next established that insulin expression was first detected as early as 13 hours after Ngn3 activation. This protocol allows us to direct the differentiation of specific cell fates and define in detail the molecular lineage of pancreatic beta cells.
Understanding how to control proliferation of progenitor cells is critical for developing a method to promote regeneration. However, we lack a complete understanding of how cellular proliferation and differentiation are coordinately controlled. Defining how endodermal progenitor cells choose to proliferate or differentiate will help in identifying ways to promote regeneration of pancreatic tissue. We recently identified the RNA binding protein BrunoL1 and showed that it acts to control proliferation and differentiation of endodermal progenitor cells (Horb and Horb 2010). In planarians, the same protein has been found to be essential for neoblast (stem cell) proliferation (Guo et al. 2006). This data suggests a common link between BrunoL1 function and regulation of stem cell proliferation. In our recent manuscript we found that BrunoL1 bound specific target sequences, known as Bruno Response Elements (BRE), located in the 3’UTR of cyclin A2, and promoted increased translation of its mRNA resulting in ectopic proliferation of endodermal progenitor cells. The main goal of this project is to elucidate the mechanism of BrunoL1 translational stimulation.
3) Transdifferentiation of liver to pancreas
During embryogenesis, both the liver and the ventral pancreas arise from the same region within the embryonic endoderm. It appears that the cellular decision to become liver or pancreas is controlled by local signals (e.g., growth factors) in the region. Interestingly it has been argued that these two organs are derived from common progenitor cells, and that their specification may be dictated by a single developmental decision that affects the expression of a relatively small number of genes. If this were the case, this may provide an alternative approach to produce pancreatic cells from hepatic cells.
We have previously shown that expression of a super-active form of the pancreatic homeobox gene Pdx1 in the liver of transgenic Xenopus tadpoles converts the liver into pancreatic tissue containing both exocrine and endocrine cell types. The transdifferentiated b cells were functional and requirement for the transgene was only temporary- once the pancreatic differentiation program is activated the transgene is no longer required. More recently, we have shown that Pdx1-VP16 is able to selectively convert rat liver cells into insulin-producing endocrine cells that are capable of rescuing diabetes in mice. Taken together, these pieces of evidence suggest that self-renewing, pluripotent hepatic stem cells are present in the developing liver and that such cells can be induced to become insulin-producing endocrine cells under appropriate conditions. Similarly, hepatopancreatitc conversion could occur in differentiated adult hepatic cells if conditions are appropriate. This situation provides a unique opportunity to generate large numbers of insulin-producing cells from hepatic cells, perhaps sufficient as a therapy for b-cell replacement for type I diabetes. Current projects in the lab are focused on understanding the mechanism of liver to pancreas transdifferentiation.