The work presented in this thesis is a study of human pancreas development. The principle goal of this work is to provide information that can be used in the development of treatments for Type 1 Diabetes and in pancreas regeneration methodologies. The transcription factor (TF) sex determining region Y homeobox gene 9 (SOX9) has been identified as a key factor in human pancreas development but its role has not been well characterized. The expression of SOX9 during early pancreas development was analyzed by immunostaining of fixed embryonic and fetal sections in the context of other developmentally important TFs. Modulators of SOX9 function, downstream targets and upstream regulatory pathways were investigated in human cell lines using coimmunoprecipitation, small interfering RNA (siRNA) knockdown, quantitative polymerase chain reaction (qPCR), luciferase assays and small molecule signaling pathway inhibitors. SOX9 was expressed in epithelial progenitors from initial human pancreas specification, but became excluded from the periphery of the epithelium and developing islet cells as differentiation proceeded. It was co-expressed with important endocrine and exocrine differentiation factors during the early stages of development. Some factors, such as Nirenberg and Kim 2, homeobox family member, drosophila, homolog of, 2 (NKX2.2) showed differing expression profile compared to murine development, while the widespread expression of endocrine factors before expression of the pro-endocrine gene neurogenin 3 (NGN3) suggested that these factors play an important role in initiating endocrine specification. Two transcription factors, GATA-binding protein 4 (GATA4) and neurogenic differentiation 1 (NEUROD1), were found to interact with SOX9 in potentially developmentally relevant complexes. This prompted the search for downstream targets of these transcriptional complexes by in silico analysis, which identified an array of novel potential downstream targets. Luciferase assay analysis of a subset of these genes showed SOX9 to activate a regulatory region of NGN3, and inhibit the regulatory regions of carboxy peptidase A6 (CPA6), v-ets avian erythroblastosis virus E26 oncogene homolog1 (ETS1) and SPONDIN1. An additional target of SOX9, osteopontin (OPN), was identified from a microarray of Sox9 knockout mouse pancreata. Investigation of SOX9 and OPN regulation by the Hedgehog signalling pathway (HH) identified both factors to be regulated by the pathway, suggesting SOX9 may act as a mediator of HH signalling. This is the first study to identify a range of SOX9 targets relevant to human pancreas development. While further characterization is required this work has provided essential clues to the function of SOX9, and provides a detailed framework of SOX9 expression and targets for future pancreatic studies to build upon.

Heterozygous mutations in the transcription factor, hepatocyte nuclear factor 1B (HNF1B), result in multisystem disease including diabetes due to beta-cell dysfunction and pancreatic hypoplasia. However, the mechanisms that underlie development of diabetes in HNF1B mutation carriers are still not fully understood due to lack of an appropriate model system. Human induced pluripotent stem cells (hiPSCs), which are capable of self-renewal and can differentiate into any cell type, provide an advantageous alternative to model human developmental diseases. The aim of this project was to develop a hiPSC based model system to determine the molecular mechanisms by which HNF1B mutations cause pancreatic hypoplasia and diabetes. HNF1B mutant hiPSC lines were produced using CRISPR-Cas9 genome editing. Isogenic HNF1B wild-type, homozygous and heterozygous mutant hiPSC lines were directed to differentiate along the pancreatic lineage and cells were phenotyped at each stage of the differentiation process to check for appropriate expression of lineage markers. The normal expression pattern of HNF1B in human pancreas development was analysed and showed up-regulation of HNF1B at the foregut stage, and during pancreas specification. Homozygous knockout of HNF1B resulted in failure of foregut and pancreatic progenitor development, while heterozygous knockout of HNF1B resulted in impairment of pancreatic progenitor and endocrine cell differentiation as well as impaired insulin secretion upon glucose stimulation. Cell proliferation analyses showed a significant decrease in the proliferation rate in HNF1B heterozygous and homozygous mutant cells compared with wild-type cells at the foregut stage while no change in the apoptosis rate could be detected. RNA-sequencing and ATAC-sequencing, were used to further define the molecular mechanisms controlled by HNF1B and the effect HNF1B on modulation of chromatin accessibility during pancreas development. These results provide further insights into the molecular mechanisms by which HNF1B regulates human pancreas development and function, revealing that HNF1B haploinsufficiency impairs the expansion and maintenance of pancreatic progenitor cells in vitro. In vivo, this would likely result in reduced beta cell numbers at birth and diabetes later in life in patients with HNF1B-associated disease. These mechanisms suggest that the capacity to produce pancreatic progenitor cells during embryonic life could determine individual susceptibility to diabetes.

Diese Dissertation analysiert zum ersten Mal STARD13, ein Protein mit einer RhoGAP-Domäne, und dessen Rolle als essentiellen Regulator der Pankreasarchitektur im Mausembryo. Es wird gezeigt, dass Stard13 anfangs im pankreatischen Endoderm exprimiert wird und später in den “Epithelspitzen” angereichert ist. Konditionelle Ablation von Stard13 im Mauspankreas beeinflusst die normale Epithelmorphogenese und die Organisation der “Epithelspitzen”. Das beeinträchtigt die Proliferation der Pankreasvorläuferzellen und führt zu Organhypoplasie. Dabei reguliert STARD13 örtlich und zeitlich Rho-Signale, die für die Morphogenese essentiell sind. Desweiteren werden die Mechanismen, die für die Entwicklung des Pankreasepithels in ein funktionierendes Organ notwendig sind, neu beleuchtet. Es wird zum Beispiel eine funktionelle Verbindung zwischen Rho-vermittelter Kontrolle der Epithelumgestaltung und der Determinierung der Organgröße hergestellt. Dabei spielt die reziproke Interaktion von actin-MAL-SRF and MAPK Signalen eine wichtige Rolle. / The development of functional organ architecture relies on coordinated morphogenesis and growth. In the developing pancreas, the branching epithelium is organized in discrete domains that delineate one specific domain of progenitor cells at the tip of the branches. Very little is known about branching morphogenesis in the pancreas and how it is coordinated with proliferation. This thesis presents the first analysis of the RhoGAP-domain-containing protein STARD13 and its role as an essential regulator of pancreas tissue architecture in the mammalian embryo. It is shown that Stard13 is expressed in the pancreatic endoderm and enriched at the distal tip of the branching epithelium. Conditional ablation of Stard13 expression in the mouse pancreas disrupts epithelial morphogenesis and tip domain organization, resulting in hampered proliferation of pancreatic progenitors and subsequent organ hypoplasia. Stard13 acts by regulating Rho signaling spatially and temporally during pancreas development. This thesis provides new insights into the mechanisms that shape pancreatic epithelium to create a mature organ and establishes a functional link between Rho-mediated control of epithelial remodeling and organ size determination, involving reciprocal interaction of actin-MAL-SRF and MAPK signaling.

This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.

The mouse pancreas is a mixed exocrine and endocrine glandconsisting of three lobular compartments: the splenic, duodenal and gastric lobes. During embryogenesis, the pancreas forms from two progenitor populations located on the dorsal and ventral side of the primitive gut tube. These anlagen are brought in close proximity as the gut elongates and rotates, and fuse to form a single organ. The splenic and duodenal lobes develop from the dorsal and ventral anlagen, respectively. In the adult pancreas, exocrine tissue secretes digestive enzymes intothe gut lumen to support nutrient uptake. The endocrine Islets of Langerhans are scattered throughout the exocrine tissue and aid in regulation of energy homeostasis through the secretion of hormones. One of the key players in energy homeostasis is the pancreatic ß-cell, which is the most abundant cell type of the islets. The β-cells regulates blood glucose levels through the action of insulin. Conditions where this regulation does not function properly are gathered under the common name of Diabetes mellitus. Type 1 diabetes (T1D) is characterized by insulin deficiency due to autoimmune destruction of the ß-cells. Using recently developed protocols for optical projection tomography (OPT) whole-organ imaging, we have revealed new spatial and quantitative aspects on ß-cell mass dynamics and immune infiltration during the course of T1D development in the non-obese diabetic (NOD) mouse model. We show that although immune infiltration appears to occur asynchronously throughout the organ, smaller islets, mainly located in the periphery of the organ, preferentially loose their ß-cells during early stages of disease progression. Larger islets appear more resistant to the autoimmune attack and our data indicate the existence of a compensatory proliferative capacity within these islets. We also report the appearance of structures resembling tertiary lymphoid organs (TLOs) in association with the remaining islets during later phases of T1D progression. OPT has already proven to be a useful tool for assessments of ß-cellmass in the adult mouse pancreas. However, as with other techniques, previous protocols have relied on a tedious degree of manual postivacquisition editing. To further refine OPT-based assessment of pancreatic ß-cell mass distribution in the murine pancreas, we implemented a computational statistical approach, Contrast-Limited Adaptive Histogram Normalisation (CLAHE), to the OPT projection data of pancreata from C57Bl/6 mice. This methodology provided increased islet detection sensitivity, improved islet morphology and diminished subjectivity in thresholding for reconstruction and quantification. Using this approach, we could report a substantially higher number of islets than previously described for this strain and provide evidence of significant differences in islet mass distribution between the pancreatic lobes. The gastric lobe stood out in particular and contained a 75% higher islet density as compared to the splenic lobe. Although the development of the early pancreatic buds has been relatively well studied, later morphogenetic events are less clear and information regarding the formation of the gastric lobe has largely been missing. Using OPT we have generated a quantitative three-dimensional road map of pancreatic morphogenesis in the mouse. We show that the gastric lobe forms as a perpendicular outgrowth fromthe stem of the dorsal pancreas at around embryonic day (e) 13.5, which grows into a mesenchymal domain overlaying the pyloric sphincter and proximal part of the glandular stomach. By analyzing mutant mice with aberrant spleen development, we further demonstrate that proper formation of the gastric lobe is dependent on the initial formation of the closely positioned spleen, indicating a close interplay between pancreatic and splenic mesenchyme during development. Additionally, we show that the expression profile of markers for pancreatic multipotent progenitors within the pancreas is heterogenous with regards to lobular origin. Altogether, our studies regarding the morphogenesis and adult constitution of the mouse pancreas recognize lobular heterogeneities that add important information for future interpretations of this organ.

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