CRISPR-TSKO : a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function

The Arabidopsis thaliana checkpoint kinase WEE1 protects against premature vascular differentiation during replication stress

A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress

Myocarditis elicits dendritic cell and monocyte infiltration in the heart and self-antigen presentation by conventional type 2 dendritic cells

Autoimmune myocarditis often leads to dilated cardiomyopathy (DCM). Although T cell reactivity to cardiac self-antigen is common in the disease, it is unknown which antigen presenting cell (ARC) triggers autoimmunity. Experimental autoimmune myocarditis (EAM) was induced by immunizing mice with alpha-myosin loaded bone marrow APCs cultured in GM-CSF. APCs found in such cultures include conventional type 2 CD11b(+) cDCs (GM-cDC2s) and monocyte-derived cells (GM-MCs). However, only alpha-myosin loaded GM-cDC2s could induce EAM. We also studied antigen presenting capacity of endogenous type 1 CD24(+) cDCs (cDC1s), cDC2s, and MCs for alpha-myosin-specific TCR-transgenic TCR-M CD4(+) T cells. After EAM induction, all cardiac APCs significantly increased and cDCs migrated to the heart-draining mediastinal lymph node (LN). Primarily cDC2s presented alpha-myosin to TCR-M cells and induced Th1/Th17 differentiation. Loss of IRF4 in lrf4(fl)(/)(fl).Cd11 cCre mice reduced MHCII expression on GM-cDC2s in vitro and cDC2 migration in vivo. However, partly defective cDC2 functions in lrf4(fl)(/)(fl).Cd11 cCre mice did not suppress EAM. MCs were the largest APC subset in the inflamed heart and produced pro-inflammatory cytokines. Targeting APC populations could be exploited in the design of new therapies for cardiac autoimmunity

The transcription factor Zeb2 regulates development of conventional and plasmacytoid DCs by repressing Id2

Plasmacytoid dendritic cells (DCs [pDCs]) develop from pre-pDCs, whereas two lineages of conventional DCs (cDCs; cDC1s and cDC2s) develop from lineage-committed pre-cDCs. Several transcription factors (TFs) have been implicated in regulating the development of pDCs (E2-2 and Id2) and cDC1s (Irf8, Id2, and Batf3); however, those required for the early commitment of pre-cDCs toward the cDC2 lineage are unknown. Here, we identify the TF zinc finger E box-binding homeobox 2 (Zeb2) to play a crucial role in regulating DC development. Zeb2 was expressed from the pre-pDC and pre-cDC stage onward and highly expressed in mature pDCs and cDC2s. Mice conditionally lacking Zeb2 in CD11c(+) cells had a cell-intrinsic reduction in pDCs and cDC2s, coupled with an increase in cDC1s. Conversely, mice in which CD11c(+) cells overexpressed Zeb2 displayed a reduction in cDC1s. This was accompanied by altered expression of Id2, which was up-regulated in cDC2s and pDCs from conditional knock-out mice. Zeb2 chromatin immunoprecipitation analysis revealed Id2 to be a direct target of Zeb2. Thus, we conclude that Zeb2 regulates commitment to both the cDC2 and pDC lineages through repression of Id2

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest

Background: NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved. Methods: Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-offunction approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool. Results: We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis. Conclusions: We demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins

Overexpression of Gilz protects mice against lethal septic peritonitis

Sepsis in humans and experimental animals is characterized by an acute inflammatory response. Glucocorticoids (GCs) are widely used for the treatment of many inflammatory disorders, yet their effectiveness in sepsis is debatable. One of the major anti-inflammatory proteins induced by GCs is GILZ (Glucocorticoid-Induced Leucine Zipper, coded by the TSC22D3 gene). We found that TSC22D3 mRNA expression is downregulated in white blood cells of human sepsis patients. Interestingly, transgenic Gilz overexpressing mice (Gilz-tg) showed better survival rates in the cecal ligation and puncture (CLP) model of mouse sepsis. To our surprise, Gilz had only mild anti-inflammatory effects in this model, since the systemic pro-inflammatory response was not significantly reduced in Gilz-tg mice compared to control mice. During CLP, we observed reduced bacterial counts in blood of Gilz-tg mice compared to control mice. We found increased expression of Tsc22d3 mRNA specifically in peritoneal exudate cells in the CLP model, as well as increased capacity for bacterial phagocytosis of CD45 Gilz-tg cells compared to CD45 Gilz-wt cells. Hence, we believe that the protective effects of GILZ in the CLP model can be linked to a more efficient phagocytosis

Homelessness and housing problems in admitted psychiatric patients in Flanders, Belgium

Homelessness in psychiatric patients in Flanders, Belgium, has never been investigated. Advocacy groups from patients with lived experience of psychiatric disorders have sounded the alarm on the scarcity of suitable housing options, the strain on psychiatric institutions, and the challenges faced by social service workers. To investigate the extent of the problem a survey on the topic was initiated. A "homelessness-in-mental-health-questionnaire" was designed by experts in the field. The social services of all Flemish psychiatric hospitals and all psychiatric wards in general hospitals were contacted and invited to complete this survey. 24 of 70 contacted services responded. The total number of homeless patients in the inpatient setting on an annual basis are estimated to an average 19.5%. 18% of homeless patients remain longer in admission due to the lack of housing options. 13.7% of homeless psychiatric patients are referred to a community care facility such as an assisted living facility. Social service respondents reported spending an average of 27.4% of their work time on housing issues. The main focus points according to the respondents are the lack of priority measures for homeless psychiatric patients, psychiatric problems as a barrier to housing options and the shortage of adapted housing capacity. The conclusion of this study is the need for comprehensive policy interventions to ensure an adequate supply of suitable social housing for psychiatric patients, accessible mental health care, alternative housing options and crisis accommodation facilities. We propose a 10-point action plan on housing for psychiatric patients for policymakers and politicians

Myocarditis Elicits Dendritic Cell and Monocyte Infiltration in the Heart and Self-Antigen Presentation by Conventional Type 2 Dendritic Cells

Autoimmune myocarditis often leads to dilated cardiomyopathy (DCM). Although T cell reactivity to cardiac self-antigen is common in the disease, it is unknown which antigen presenting cell (APC) triggers autoimmunity. Experimental autoimmune myocarditis (EAM) was induced by immunizing mice with α-myosin loaded bone marrow APCs cultured in GM-CSF. APCs found in such cultures include conventional type 2 CD11b+ cDCs (GM-cDC2s) and monocyte-derived cells (GM-MCs). However, only α-myosin loaded GM-cDC2s could induce EAM. We also studied antigen presenting capacity of endogenous type 1 CD24+ cDCs (cDC1s), cDC2s, and MCs for α-myosin-specific TCR-transgenic TCR-M CD4+ T cells. After EAM induction, all cardiac APCs significantly increased and cDCs migrated to the heart-draining mediastinal lymph node (LN). Primarily cDC2s presented α-myosin to TCR-M cells and induced Th1/Th17 differentiation. Loss of IRF4 in Irf4fl/fl.Cd11cCre mice reduced MHCII expression on GM-cDC2s in vitro and cDC2 migration in vivo. However, partly defective cDC2 functions in Irf4fl/fl.Cd11cCre mice did not suppress EAM. MCs were the largest APC subset in the inflamed heart and produced pro-inflammatory cytokines. Targeting APC populations could be exploited in the design of new therapies for cardiac autoimmunity

Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5 / LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate deprived conditions are TMO5 and cytokinin dependent. In conclusion, cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells