Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2–host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism

Transient development of ovotestes in XX Sox9 transgenic mice

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9(Tg/+) gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3' UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth

A neonatal lethal mutation in FGFR3 uncouples proliferation and differentiation of growth plate chondrocytes in embryos

We have generated the first mouse model of fibro-blast growth factor receptor 3 (Fgfr3) with the K644E mutation, which accurately reflects the embryonic onset of a neonatal lethal dwarfism, thanatophoric dysplasia type II (TDII). Long-bone abnormalities were identified as early as embryonic day 14, during initiation of endochondral ossification. Increased expression of PATCHED: (PTC:) was observed, independent of unaltered expression of parathyroid hormone-related peptide (PTHrP) receptor and Indian Hedgehog (IHH:), suggesting a new regulatory role for Fgfr3 in embryos. We demonstrate that the mutation enhances chondrocyte proliferation during the early embryonic skeletal development, in contrast to previous reports that showed decreased proliferation in postnatal-onset dwarf mice with activating Fgfr3 mutations. This suggests that signaling through Fgfr3 both promotes and inhibits chondrocyte proliferation, depending on the time during development. In contrast, suppressed chondrocyte differentiation was observed throughout the embryonic stages, defining decreased differentiation as the primary cause of retarded longitudinal bone growth in TDII. This model was successfully crossed with a cartilage-specific CRE: transgenic strain, excluding the lung as the primary cause of lethality

Wt1 negatively regulates beta-catenin signaling during testis development

International audiencebeta-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. beta-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of beta-catenin in Sertoli cells during testis formation, we either deleted beta-catenin or expressed a constitutively active form of beta-catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Mullerian duct regression. beta-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized beta-catenin mutants, we then investigated the relationship of Wt1 and beta-catenin in Sertoli cells and found Wt1 inhibits beta-catenin signaling in these cells during testis development. Wt1 deletion resulted in upregulation of beta-catenin expression in Sertoli cells both in vitro and in vivo. Our study indicates that Sertoli cell expression of beta-catenin is dispensable for testis development. However, the suppression of beta-catenin signaling in these cells is essential for proper testis formation and Wt1 is a negative regulator of beta-catenin signaling during this developmental process

β-Catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation

Gene expression profiling of {beta}-catenin, Cripto and Wnt3 mutant mouse embryos has been used to characterise the genetic networks that regulate early embryonic development. We have defined genes whose expression is regulated by {beta}-catenin during formation of the anteroposterior axis and the mesoderm, and have identified Cripto, which encodes a Nodal co-receptor, as a primary target of {beta}-catenin signals both in embryogenesis as well as in colon carcinoma cell lines and tissues. We have also defined groups of genes regulated by Wnt3/{beta}-catenin signalling during primitive streak and mesoderm formation. Our data assign a key role to {beta}-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation

Beta-catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation

Gene expression profiling of {beta}-catenin, Cripto and Wnt3 mutant mouse embryos has been used to characterise the genetic networks that regulate early embryonic development. We have defined genes whose expression is regulated by {beta}-catenin during formation of the anteroposterior axis and the mesoderm, and have identified Cripto, which encodes a Nodal co-receptor, as a primary target of {beta}-catenin signals both in embryogenesis as well as in colon carcinoma cell lines and tissues. We have also defined groups of genes regulated by Wnt3/{beta}-catenin signalling during primitive streak and mesoderm formation. Our data assign a key role to {beta}-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation