RHOA GTPase Controls YAP-Mediated EREG Signaling in Small Intestinal Stem Cell Maintenance

RHOA, a founding member of the Rho GTPase family, is critical for actomyosin dynamics, polarity, and morphogenesis in response to developmental cues, mechanical stress, and inflammation. In murine small intestinal epithelium, inducible RHOA deletion causes a loss of epithelial polarity, with disrupted villi and crypt organization. In the intestinal crypts, RHOA deficiency results in reduced cell proliferation, increased apoptosis, and a loss of intestinal stem cells (ISCs) that mimic effects of radiation damage. Mechanistically, RHOA loss reduces YAP signaling of the Hippo pathway and affects YAP effector epiregulin (EREG) expression in the crypts. Expression of an active YAP (S112A) mutant rescues ISC marker expression, ISC regeneration, and ISC-associated Wnt signaling, but not defective epithelial polarity, in RhoA knockout mice, implicating YAP in RHOA-regulated ISC function. EREG treatment or active β-catenin Catnblox(ex3) mutant expression rescues the RhoA KO ISC phenotypes. Thus, RHOA controls YAP-EREG signaling to regulate intestinal homeostasis and ISC regeneration

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Loss of RhoA Exacerbates, Rather Than Dampens, Oncogenic K-Ras Induced Lung Adenoma Formation in Mice

<div><p>Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation, suggesting that RhoA is a useful target in Ras induced neoplasia. However, to date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-Ras^G12D Lox-Stop-Lox murine lung cancer model in combination with a conditional RhoA^flox/flox and RhoC^-/- knockout mouse models. Deletion of the floxed <i>Rhoa</i> gene and expression of K-Ras^G12D was achieved by either CCSP-Cre or adenoviral Cre, resulting in simultaneous expression of K-Ras^G12D and deletion of RhoA from the murine lung. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-Ras^G12D induced lung adenoma initiation. Rather, deletion of RhoA alone exacerbated lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-Ras^G12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. This study suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is likely to lead to more favorable outcomes.</p></div

Breeding Schematic for Transgenic Mice.

<p>Breeding Schematic for Transgenic Mice.</p

Deletion of RhoA and RhoC together does not impair normal lung development.

<p>Rho^WT, RhoC^-/- and DKO mice were mated with CCSP-Cre mice on a K-Ras^WT background. <b>(A & B)</b> Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining. <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 25μm). <b>(E)</b> Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent 100μm). <b>(F)</b> Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent 50μm).</p

RhoA and RhoC are important in combination, but not individually, for K-Ras^G12D induced sporadic lung adenoma formation.

<p>Mice from different Rho backgrounds were administered Adeno-Cre virus endotracheally. Lungs were harvested after 12 weeks. <b>(A)</b> H&E of adenomas (bars represent 100μm). <b>(B)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(C)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Western blots of microdissected tumors (N = normal lung, T = tumor). <b>(E)</b> Quantification of tumor burden. Means represent the quantification of four separate, evenly spaced and similarly sized lung sections, from four mice for each group (n = 4, * = p ≤ 0.001). Data representative of two separate experiments. <b>(F)</b> Quantification of tumor sizes. Data points represent the area of individual tumors expressed in μm². Red bars represent the mean tumor area. <b>(G)</b> Proportion of RhoA-positive and RhoA-null tumors. Displayed as RhoA staining by the level of staining intensity (levels are as follows: no staining; normal = same intensity staining as adjacent normal alveoli; high = greater than adjacent normal alveoli; very high = much greater intensity staining than adjacent normal alveoli).</p

RhoA is not essential for CCSP-promoter driven, K-Ras^G12D-induced, lung adenoma formation in mice.

<p>Rho^WT and RhoA^flox/flox mice were mated with CCSP-Cre mice on a K-Ras^G12D background. <b>(A & B)</b> K-Ras^G12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(E)</b> Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.</p

Neither RhoA nor RhoC is required for K-Ras^G12D-induced adenoma formation in a CCSP-Cre model.

<p>Rho^WT, RhoC^-/- and DKO mice were mated with CCSP-Cre mice on a K-Ras^G12D background. <b>(A & B)</b> K-Ras^G12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(E)</b> Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.</p

CCSP-promoter driven deletion of RhoA does not affect normal lung development.

<p>Rho^WT and RhoA^flox/flox mice were mated with CCSP-Cre mice on a K-Ras^WT background. <b>(A & B)</b> Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining. <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 25μm). <b>(E)</b> Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent 100μm). <b>(F)</b> Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent 50μm).</p