Role of MNX1 in pancreatic ß-cell formation and function

Currently, much effort is targeted toward development of efficient cell replacement therapy to restore ß-cells in patients with diabetes. Promising sources for this replacement are in vitro differentiated ß-cells generated from stem or precursor populations. However, efficient conversion of such stem cells into mature functional ß-cells is still challenging. In that view, deep understanding of the molecular regulators of ß-cell fate establishment and maintenance is fundamental for successful cell reprogramming strategy. This project aims to elucidate novel regulators of pancreas and ß-cell fate establishment acting downstream of the Mnx1/HB9 transcription factor. While previous studies established Mnx1 as a key-player for ß-cell specification, differentiation and maintenance, its molecular function is poorly understood. Building upon our recent work in zebrafish, we hypothesize that Mnx1 is targeting inhibitors of fate-progression and that repression of these genes is required for proper and efficient ß-cell specification and maturation. To test this, we propose a cross-species approach that provides unique opportunities for clarifying MNX1 activities. We will use the zebrafish as a powerful genetic model for studying mnx1 requirements and to establish functional interactions within the Mnx1-regulated network of pancreas development. Furthermore, recently established genetically modified human induced pluripotent stem cells will be used as the primary model for addressing molecular aspects of MNX1 functions in the context human ß-cell in vitro differentiation. Elucidation of the pancreatic Mnx1 regulated transcriptional network will provide important insights into principle mechanisms of endocrine fate implementation, maintenance and plasticity. These experiments will significantly extend our understanding of ß-cell differentiation, and we expect that knowledge generated during this project will result in improved reprogramming strategies for the generation of functional human ß-cell.

The formation of insulin-producing -cells follows an evolutionarily conserved genetic program. Experiments in model organisms indicated an important role of the transcription factor Mnx1 in determining -cell identity. However, at the beginning of the project, it was not known how the transcriptional repressor Mnx1 translates this activity at the molecular level, and there was evidence of very different cellular consequences upon loss of Mnx1 function in fish, mice and humans. Contrary to these assumptions, we were able to show in this project that both the molecular pancreatic Mnx1 functions and the mutant phenotypes are conserved from zebrafish to humans. Our studies showed that -cell precursors can form in mnx1-mutant zebrafish embryos as well as from MNX1-mutant human stem cells, but that these do not develop into -cells but in the majority into -like cells. In addition, molecular analyses identified several conserved direct Mnx1 target genes with functions both in the general suppression of differentiation and in the establishment of -c However, our experiments also show that insulin-expressing cells can also form independently of Mnx1, and that these cells differ fundamentally from -cells. While the mnx1 mutant zebrafish hardly produce any insulin in the first 2-3 weeks of their development and are correspondingly hyperglycemic, older animals show almost normal blood glucose levels. 'Single-cell RNAseq' and histological analyses confirm that older animals compensate for the missing -cells by massive formation of somatostatin/insulin co-expressing cells, and that this compensation correlates with a general restructuring and massive growth of the pancreas similar to that described in Mnx1 mutant mice. In addition to these important findings on beta-cell formation, the experiments also provided a number of unexpected insights. For example, it was shown that both humans and fish express two Mnx1 proteins of different lengths, and that all knock-down phenotypes previously described in fish are based on the loss of only one of these isoforms. Furthermore, the single-cell analyses of human in vitro differentiation approaches revealed several indications of previously unknown MNX1 functions also outside of -cell differentiation. Taken together, these data establish Mnx1 as the central controller of efficient implementation of -cell differentiation and assign Mnx1 further previously unknown direct and indirect functions in endodermal differentiation