Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.     

Snail1 Gene Function During Early Embryo Patterning in Mice

Originally identified as one of two zygotically expressed genes required for gastrulation in Drosophila, the Snail gene and other family members play critical roles in vertebrate development. Functionally, these genes are thought to drive epithelial-mesenchymal transitions at several points during development, and also during the metastatic progression of cancer. Although the Snai2-null mouse is viable and fertile, the early embryonic lethality of Snai1-null mice has precluded the detailed analysis of Snai1 function after gastrulation. We have recently generated a conditional allele of the Snai1 gene and examined its function during the formation of the neural crest and establishment of the left-right axis. We uncovered new details regarding Snai1 function during gastrulation and left-right asymmetry determination, while surprisingly showing that neither the Snai1 nor Snai2 genes are essential for neural crest cell delamination. These results shed new light on the role of Snail family genes in early mouse development, and raise interesting questions concerning the diversity of gene function among vertebrate species.     

Generation and Characterisation of Mice Deficient in the Multi-GTPase Domain Containing Protein,GIMAP8

Background

GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function.

Principal Findings

We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell in vitro in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen.

Conclusions

Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.     

Loss of BH3-only Protein Bim Inhibits Apoptosis of Hemopoietic Cells in the Fetal Liver and Male Germ Cells but Not Neuronal Cells in Bcl-x�Cdeficient Mice

Members of the Bcl-2 family include pro- and antiapoptotic proteins that regulate programmed cell death of developing tissues and death in response to cellular damage. In developing mice, the antiapoptotic Bcl-x_L is necessary for survival of neural and hematopoietic cells, and consequently, bcl-x–deficient mice die around Day 13.5 of embryogenesis. Furthermore, adult bcl-x^+/− heterozygous male mice have reduced fertility because of testicular degeneration. Bax, a multi-BH (Bcl-2 homology) domain proapoptotic member of the Bcl-2 family, is regulated by Bcl-x_L and is required for the neuropathological abnormalities seen in bcl-x–deficient embryos. The BH3 domain only subgroup of the Bcl-2 family includes proapoptotic members that are essential for the initiation of apoptotic signaling. In this study, we investigated the role for Bim, a BH3 domain only protein, in the embryonic lethality and increased developmental cell death in bcl-x–deficient animals and the perturbed testicular function in bcl-x^+/− adults. Our studies show that bim deficiency attenuates hematopoietic cell death in the fetal liver of bcl-x–deficient animals, indicating that Bim contributes to programmed cell death in this cell population. In addition, we found that testicular degeneration of adult bcl-x^+/− males was rescued by concomitant Bim deficiency. However, concomitant Bim deficiency had no effect on the embryonic lethality and widespread nervous system abnormalities caused by bcl-x deficiency. Our work identifies Bim as an important regulator of bcl-x deficiency–induced cell death during hematopoiesis and testicular development. (J Histochem Cytochem 56:921–927, 2008)     

Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function

Background

The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.

Methodology/Principal Findings

Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.

Conclusions/Significance

These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

Unexpected Phenotype of Mice Lacking Shcbp1, a Protein Induced during T Cell Proliferation

T cell development and activation are highly regulated processes, and their proper execution is important for a competent immune system. Shc SH2-domain binding protein-1 (Shcbp1) is an evolutionarily conserved protein that binds to the adaptor protein ShcA. Studies in Drosophila and in cell lines have strongly linked Shcbp1 to cell proliferation, embryonic development, growth factor signaling, and tumorigenesis. Here we show that Shcbp1 expression is strikingly upregulated during the β-selection checkpoint in thymocytes, and that its expression tightly correlates with proliferative stages of T cell development. To evaluate the role for Shcbp1 during thymic selection and T cell function in vivo, we generated mice with global and conditional deletion of Shcbp1. Surprisingly, the loss of Shcbp1 expression did not have an obvious effect during T cell development. However, in a mouse model of experimental autoimmune encephalomyelitis (EAE), which depends on CD4⁺ T cell function and mimics multiple features of the human disease multiple sclerosis, Shcbp1 deficient mice had reduced disease severity and improved survival, and this effect was T cell intrinsic. These data suggest that despite the striking upregulation of Shcbp1 during T cell proliferation, loss of Shcbp1 does not directly affect T cell development, but regulates CD4⁺ T cell effector function in vivo.     

An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis

Homeostasis of the hematopoietic system is tightly regulated by an array of cytokines that control proliferation, differentiation and apoptosis of various cell lineages. To identify genes that are essential for hematopoietic homeostasis, we screened C57BL/6 mice that had been genome-wide mutagenized by N-ethyl-N-nitrosourea (ENU) to produce altered blood cell composition. We identified a mutant mouse line with a drastic reduction in the number of T and B cell lineages in lymphatic tissues and peripheral blood, as well as severe atrophy of the thymus and lymph nodes. Genotyping with a genome-wide single nucleotide polymorphism (SNP) marker set mapped the mutant phenotype to chromosome 3A and subsequent direct DNA sequencing revealed a G-to-A point mutation in the splicing donor site of the third exon of the candidate gene for IL-7, a lymphocyte survival cytokine. Such mutation resulted in skipping of exon 3 and production of an internally truncated IL-7 (ΔE3-IL7). Furthermore, using recombinant proteins produced in a baculoviral system, we demonstrated that ΔE3-IL7 had no detectable anti-apoptotic activity even at a dose that was 30 times more than that required for a wild-type protein to manifest a full activity in a naïve T cell survival assay. Our data suggest that this mutant mouse line?provides an alternative animal model for the study of severe combined immunodeficiency (SCID) syndrome in humans.     

Normal germ line establishment in mice carrying a deletion of the Ifitm/Fragilis gene family cluster

The family of interferon-inducible transmembrane proteins (Ifitm) consists of five highly sequence-related cell surface proteins, which are implicated in diverse cellular processes. Ifitm genes are conserved, widely expressed, and characteristically found in genomic clusters, such as the 67-kb Ifitm family locus on mouse chromosome 7. Recently, Ifitm1 and Ifitm3 have been suggested to mediate migration of early primordial germ cells (PGCs), a process that is little understood. To investigate Ifitm function during germ cell development, we used targeted chromosome engineering to generate mutants which either lack the entire Ifitm locus or carry a disrupted Ifitm3 gene only. Here we show that the mutations have no detectable effects on development of the germ line or on the generation of live young. Hence, contrary to previous reports, Ifitm genes are not essential for PGC migration. The Ifitm family is a striking example of a conserved gene cluster which appears to be functionally redundant during development.     

The Ras GTPase-Activating Protein Rasal3 Supports Survival of Naive T Cells

The Ras-mitogen-activated protein kinase (MAPK) pathway is crucial for T cell receptor (TCR) signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3) is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP) domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.