Probes for INS

R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/?-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility

The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but the regulators and significance in the placenta are unknown. We discovered that many murine placental cell types are polyploid. We identified factors that license polyploidy using single-cell RNA seq. c-Myc is a key regulator of polyploidy and placental development and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, c-MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without c-Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal c-Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study combined with the literature suggests c-Myc is an evolutionarily conserved regulator of polyploidy.

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-β-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.