An ENU-Mutagenesis Screen in the Mouse: Identification of Novel Developmental Gene Functions

BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

β-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis

Objective: Deregulation of the Wnt signalling pathway by mutations in the Apc or β-catenin genes underlies colorectal carcinogenesis. As a result, β-catenin stabilises, translocates t

Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential.

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential

Characterization of islet cells during development and after transplantation

Diabetes Mellitus is a disease in which patients are not able to maintain blood glucose levels. This is caused by dysfunction or destruction of the beta cells in the islets of Langerhans, located in the pancreas. Beta cells are responsible for the production of insulin, a hormone that decreases the amount of available glucose in the blood when it becomes too high. Therapeutically, diabetes patients inject themselves with insulin as an alternative, but this treatment is symptomatic. The only available cure at the moment is transplantation of donor tissue, in the form of a whole pancreas, or the isolated islets of Langerhans. Novel therapies are being developed, where stem cells are driven to beta-like phenotypes. The pathways used to drive these cells strongly resemble embryonic development. Many characteristics of embryonic development are of yet not completely understood, and in this thesis we aimed to better understand how beta cells develop from embryonic progenitors into adult cells, and how these beta cells behave after transplantation. To do this, we have developed a novel technique that allows intravital imaging of transplanted pancreatic tissue under the kidney capsule. We transplanted embryonic pancreases to see how these tissues develop into adult organs. Transplanted embryonic pancreases developed very similarly to endogenous embryonic pancreases. We found that the organ increases more than 6 fold in size, and islets of Langerhans are being formed, in two weeks time after transplantation. The first islet-like structures can already be observed after seven days. Islets formed in these organs consist not only of beta, but also of glucagon producing alpha cells. Three days after transplantation, mesenchymal like cells that are positive for insulin were observed in the transplanted tissue, and these cells displayed a migratory phenotype. Cells were not directionally mmigrating, but rather moved around randomly. This behavioral trait was strongly diminished seven days after transplantation, when islets were starting to form. We also transplanted adult islets of Langerhans under the kidney capsule. These islets did not show any plasticity after transplantation, only a small increase of total transplanted mass, which can be caused in part due to co-transplanted endothelial and mesenchymal cells. Islets were also subcutaneously transplanted in alginate-based scaffolds. Intravital imaging revealed that these islets lost functionality within seven days after transplantation. Finally, we characterized the developing pancreas using single cell transcriptome sequencing. Here, we found all cell types that are normally present in the embryonic pancreas: tip and trunk epithelium, endocrine progenitors and all endocrine adult cell types (alpha, beta, gamma and delta cells). We found that cells became more mature through development, as illustrated by a decreasing transcriptomic entropy. A pseudo-temporal map shows the developmental time line for alpha and beta cell maturation, and we show how genes are temporally regulated during development. Taken together, we provide new insight into maturation of endocrine cell types and how islets of Langerhans are formed. Also, we show the dynamic behavior of islets of Langerhans after transplantation under various conditions

A transcriptomic roadmap to α- and β-cell differentiation in the embryonic pancreas

International audienceDuring pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into α-, β-, γ- and δ-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on α- and β-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev Pseudotime analysis revealed specific branches for developing α- and β-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into α- and β-cells

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting β-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-β-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases

Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice

Aims/hypothesis: Dynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule. Methods: Isolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival. Results: Transplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks. Conclusions/interpretation: This method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice

AbstractWnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages

Generation of human islet cell type-specific identity genesets

Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, β-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.</p