1) Define the Ngn3 gene regulatory network
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.
2) Understand molecular action of translational enhancement
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) Genome editing in Xenopus
The genomes of both X. laevis and X. tropicalis have been sequenced providing the necessary information to identify specific sequence targets for the design of single-guide RNAs (sgRNA) and TALEN pairs. We have large colonies of the inbred X. laevis J strain and the X. tropicalis Nigerian strain that were used to sequence their respective genomes. We will use these strains to create various mutants for the community to model human diseases and to enhance gene editing in Xenopus by developing methods to rapidly generate null mutants and standardize protocols for CRISPR/TALEN-mediated insertions Xenopus.
As outlined in the 2014 Xenopus White Paper, a major opportunity identified by the entire Xenopus research community is to accelerate the use of Xenopus as a model for human diseases. In this project we are creating mutations in select genes that are linked to human disease. By creating these mutant animals we will provide individual researchers with new tools to interrogate candidate disease gene function and signaling pathways in a highly amenable model system. Not only does Xenopus provides an effective system for in vivo screening of candidate genetic variants (e.g. from genome-wide association studies), but unbiased studies of organ formation and function can reveal phenotypes similar to human diseases and thus implicate new disease candidates. Since many adult human diseases arise as a result of developmental defects, Xenopus is an excellent model to study the origins of adult disease. Developing these mutants has the potential to change and guide future Xenopus research efforts.
Knock-in strategies using CRISPR/Cas and TALENS has been demonstrated in other organisms, but to date not in Xenopus. The Xenopus oocyte supports homologous recombination and we propose to use CRISPR/Cas and/or TALEN methods to promote homology-directed repair in Xenopus oocytes to engineer specific human disease point mutations and to create new reporter lines.