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Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca influx of -deficient β-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both and in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed "general" coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.
© 2020 by the American Diabetes Association.
The activation of neuronal plasma membrane Ca channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues Arg-Arg-Lys to three alanines disrupts CaMKII binding and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory. Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.
Copyright © 2020 the authors.
Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
Copyright © 2019 Elsevier Inc. All rights reserved.
-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within -infected gastric mucosa. Lineage tracing was induced in (Lrig1/YFP) mice that were uninfected or subsequently infected with or an isogenic mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the mutant. WT -infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic infection stimulates Lrig1-expressing progenitor cells in a -dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.
Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.
BACKGROUND - Plasminogen activator inhibitor-1 (PAI-1) expression increases extracellular matrix deposition and contributes to interstitial fibrosis in the kidney after injury. While PAI-1 is ubiquitously expressed in the kidney, we hypothesized that interstitial fibrosis is strongly dependent on fibroblast-specific PAI-1 (fbPAI-1).
METHODS - Tenascin C Cre (TNC Cre) and fbPAI-1 knockdown (KD) mice with green fluorescent protein (GFP) expressed within the TNC construct underwent unilateral ureteral obstruction and were sacrificed 10 days later.
RESULTS - GFP+ cells in fbPAI-1 KD mice showed significantly reduced PAI-1 expression. Interstitial fibrosis, measured by Sirius red staining and collagen I western blot, was significantly decreased in fbPAI-1 KD compared with TNC Cre mice. There was no significant difference in transforming growth factor β (TGF-β) expression or its activation between the two groups. However, GFP+ cells from fbPAI-1 KD mice had lower TGF β and connective tissue growth factor (CTGF) expression. The number of fibroblasts was decreased in fbPAI-1 KD compared with TNC Cre mice, correlating with decreased alpha smooth muscle actin (α-SMA) expression and less fibroblast cell proliferation. TNC Cre mice had decreased E-cadherin, a marker of differentiated tubular epithelium, in contrast to preserved expression in fbPAI-1 KD. F4/80-expressing cells, mostly CD11c+/F4/80+ cells, were increased while M1 macrophage markers were decreased in fbPAI-1 KD compared with TNC Cre mice.
CONCLUSION - These findings indicate that fbPAI-1 depletion ameliorates interstitial fibrosis by decreasing fibroblast proliferation in the renal interstitium, with resulting decreased collagen I. This is linked to decreased M1 macrophages and preserved tubular epithelium.
© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand ( ) at the genomic locus of each target protein. The resultant knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment () in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the nervous system.
Copyright © 2019 by the Genetics Society of America.
Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
Aberrant activation of the Hedgehog signaling pathway has been linked to the formation of numerous cancer types, including the myogenic soft tissue sarcoma, embryonal rhabdomyosarcoma (eRMS). Here, we report , a novel mouse model in which human GLI2A, a constitutive activator of Hedgehog signaling, induced undifferentiated sarcomas that were phenotypically divergent from eRMS. Rather, sarcomas arising in mice featured some characteristics that were reminiscent of Ewing sarcoma. Even though it is widely understood that Ewing sarcoma formation is driven by gene fusions, a genetically defined mouse model is not well-established. While gene fusions were not present in sarcomas, precluding their designation as Ewing sarcoma, we did find that GLI2A induced expression of known gene targets essential to Ewing pathogenesis, most notably, . Moreover, we found that naïve mesenchymal progenitors originate tumors in mice. Altogether, our work provides a novel genetic mouse model, which directly connects oncogenic Hedgehog activity to the etiology of undifferentiated soft tissue sarcomas for the first time. IMPLICATIONS: The finding that activation of Gli2 transcription factor is sufficient to induce Ewing-like sarcomas provides a direct transformative role of the Hedgehog signaling pathway in undifferentiated soft tissue sarcoma.
©2019 American Association for Cancer Research.