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NEUROD1 is critical for the initial cell identity determination and differentiation of the endocrine progenitors in the pancreas [CUT&Tag]

ABSTRACT: While diabetes incidence is gradually rising worldwide, novel therapeutical approaches are required in the search for a replacement of dysfunctional endocrine tissue, especially beta-cells. A promising potential lies in direct cellular reprogramming of similar cell types sharing common multipotent progenitors. With the knowledge of molecular mechanisms determining the endocrine cell fate commitment and endocrine lineage differentiation, other endocrine cells contained within the islets of Langerhans or closely related cells could be trans- or dedifferentiated either in situ or in vitro with their subsequent transplantation into the patient. NEUROD1 is a transcription factor situated on the base of the gene regulatory network of the developing endocrine precursors, directly downstream of endocrine lineage master regulator NEUROG3. While NEUROG3 initiates a rapid cascade of chromatin reorganization in numerous bivalent promoters resulting in spatiotemporal gene expression leading to differentiation of all endocrine cell subtypes from pancreatic multipotent progenitors, the role of NEUROD1 has yet to be clarified. Notably, NEUROD1 can induce neuronal program through pioneering and chromatin remodelling in other cell types. In this study, we performed advanced molecular and phenotypic analyses in early endocrine-specific Neurod1-deficient mouse model, including RNA and CUT&Tag sequencing and lightsheet microscopy, to gain insight into the NEUROD1-regulated transcription network driving endocrine lineage cell fate commitment. Besides a postnatal diabetic phenotype, disrupted endocrine differentiation and associated altered islet architecture, we observed an apparent elevation in the non-endocrine gene expression pattern and uncovered alterations in the epigenetic landscape of characteristic genes, specifically in the H3K4me3 and H3K27me3 distribution. Therefore, we provide evidence that NEUROD1 is critical player responsible for the establishment of the endocrine cell identity, which further affects endocrine development and may serve as a potent proendocrine reprogramming driver.

ORGANISM(S): Mus musculus

PROVIDER: GSE212083 | GEO | 2023/08/17

REPOSITORIES: GEO

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Project description:While diabetes incidence is gradually rising worldwide, novel therapeutical approaches are required in the search for a replacement of dysfunctional endocrine tissue, especially beta-cells. A promising potential lies in direct cellular reprogramming of similar cell types sharing common multipotent progenitors. With the knowledge of molecular mechanisms determining the endocrine cell fate commitment and endocrine lineage differentiation, other endocrine cells contained within the islets of Langerhans or closely related cells could be trans- or dedifferentiated either in situ or in vitro with their subsequent transplantation into the patient. NEUROD1 is a transcription factor situated on the base of the gene regulatory network of the developing endocrine precursors, directly downstream of endocrine lineage master regulator NEUROG3. While NEUROG3 initiates a rapid cascade of chromatin reorganization in numerous bivalent promoters resulting in spatiotemporal gene expression leading to differentiation of all endocrine cell subtypes from pancreatic multipotent progenitors, the role of NEUROD1 has yet to be clarified. Notably, NEUROD1 can induce neuronal program through pioneering and chromatin remodelling in other cell types. In this study, we performed advanced molecular and phenotypic analyses in early endocrine-specific Neurod1-deficient mouse model, including RNA and CUT&Tag sequencing and lightsheet microscopy, to gain insight into the NEUROD1-regulated transcription network driving endocrine lineage cell fate commitment. Besides a postnatal diabetic phenotype, disrupted endocrine differentiation and associated altered islet architecture, we observed an apparent elevation in the non-endocrine gene expression pattern and uncovered alterations in the epigenetic landscape of characteristic genes, specifically in the H3K4me3 and H3K27me3 distribution. Therefore, we provide evidence that NEUROD1 is critical player responsible for the establishment of the endocrine cell identity, which further affects endocrine development and may serve as a potent proendocrine reprogramming driver.

Project description:Atoh8 is a transcription factor of the basic-helix-loop-helix (bHLH) family that is expressed in multiple tissues during embryonic development but whose specific functions remain unknown. The gene encoding Atoh8 is induced by various lineage- determining bHLH transcription factors in cell culture, suggesting a possible common role of this gene in multiple bHLH-driven differentiation programs. In the pancreas, the pro-endocrine bHLH factors Neurogenin3 (Neurog3) and NeuroD1 activate Atoh8 expression. Moreover, Atoh8 is expressed in the embryonic pancreas confirming its participation in the pancreatic transcriptional cascade. This work aims at gaining insight into the molecular function of Atoh8 during the endocrine differentiation program initiated by Neurog3 in the pancreas. To this aim, we have generated a recombinant adenovirus encoding an Atoh8-specific shRNA (Ad-shAtoh8) and used it to down-regulate expression of the Atoh8 gene in Neurog3-expressing pancreatic ductal cells (mPAC), a cellular model of endocrine cell differentiation. Thus, we have compared global changes in gene expression profiles between cells treated with Ad-Neurog3+shControl and cells treated with Ad-Neurog3+shAtoh8 using Affymetrix microarrays. Our results show that Atoh8 silencing significantly affects the expression of 293 genes in Neurog3-expressing mPAC cells. Gene Ontology analysis has revealed cell cycle as the biological function most significantly represented among the modified genes. These results uncover a potential function of Math6 as a regulator of cell cycle progression and provide novel insights into the link between Neurog3 and the regulation of the cell cycle. mPAC cells (mouse pancreatic duct cells) overexpressing Neurogenin3 were divided in 2 groups according to the shRNA encoded by the recombinant adenoviruses used: 1) Scramble shRNA (control for the infection effect), 2) Atonal Homolog 8-specific shRNA. Experiment was performed three independent times (3 independent biological replicates).

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NEUROD1 is critical for the initial cell identity determination and differentiation of the endocrine progenitors in the pancreas [CUT&Tag]

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