Fibro-adipogenic progenitors (FAPs) are an interstitial cell population in adult skeletal muscle that support muscle regeneration. During development, interstitial muscle connective tissue (MCT) cells support proper muscle patterning, however the underlying molecular mechanisms are not well understood and it remains unclear whether adult FAPs and embryonic MCT cells share a common lineage. We show here that mouse embryonic limb MCT cells expressing the transcription factor Osr1, differentiate into fibrogenic and adipogenic cells in vivo and in vitro defining an embryonic FAP-like population. Genetic lineage tracing shows that developmental Osr1+ cells give rise to a subset of adult FAPs. Loss of Osr1 function leads to a reduction of myogenic progenitor proliferation and survival resulting in limb muscle patterning defects. Transcriptome and functional analyses reveal that Osr1+ cells provide a critical pro-myogenic niche via the production of MCT specific extracellular matrix components and secreted signaling factors.
The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.
The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a(-/-) mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/β-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.
Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1 FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1 activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.
Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.
Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are currently identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.Summary statementExpression of Osr1 specifically in muscle interstitial fibro-adipogenic progenitors (FAPs) activated by acute injury provides a tool to isolate and trace this population.
BACKGROUND AND AIMS As part of the renal filtration barrier, podocytes are critical to the function of kidney glomeruli. Their loss is one of the hallmarks of chronic kidney disease, including diabetic kidney disease (DKD). Indeed, many drug discovery efforts focus on preventing podocyte apoptosis triggered by diabetic stressors to delay kidney disease progression. In order to discover and characterize new therapeutic agents, phenotypic screens are a powerful tool to directly screen for the desired cellular outcome. These need to be complemented by target-centric assays to support the chemical optimization of compounds. Generally, the deconvolution and confirmation of molecular targets are considered challenging, but necessary in the follow-up of phenotype-based drug discovery programmes to allow progression towards the clinic. We have combined novel technologies like high-throughput next-generation sequencing (NGS) and Cell Painting [1] to develop an unbiased approach for deconvolution of targets from phenotypic screens. As an outcome, we identified a small molecule compound that can protect conditionally immortalized human podocytes (CIHP) from apoptosis in vitro. In a second step, we were able to identify the target responsible for this effect. METHOD Phenotypic screen: CIHP [2] were treated with high glucose and palmitate, a common DKD injury model, as the main driver for apoptosis as evaluated by caspase 3/7 activation [3]. Potentially protective agents were screened against apoptosis from a library of 50 000 small molecules. We selected a hit compound (CpdX) for subsequent target deconvolution to identify the target causing the protective effect. Target deconvolution and compound profiling: We compared the molecular signatures triggered by CpdX treatment to those of a reference panel of known, bio-annotated compounds (#L3500; Selleckchem). Dimensional reduction based on UMAP and PCA applied to transcriptomic data (NGS) as well as Cell Painting fingerprints were used to identify known compounds exhibiting a similar transcriptomic and morphological profile as the CpdX. We hypothesized that the molecular targets of these compounds also constitute likely targets for the new CpdX. RESULTS We developed a screening cascade (Figure 1A) to identify novel targets for kidney disease. A first-pass phenotypic podocyte apoptosis screen delivered a potent compound, able to rescue CIHP from a diabetic challenge in vitro (Figure 1B, C). Integrated analysis and alignment of cellular changes in transcriptomic profile and morphology following CpdX or reference compound treatment enabled us to identify the serine/threonine-protein kinase Ataxia Telangiectasia Mutated (ATM) as one of the putative targets of the orphan compound. Both KU-60019, a known inhibitor of ATM [4] (Figure 1D) and CpdX showed remarkably similar effects in NGS and Cell Painting (Figure 2A, B) and were also equally protecting the cells from apoptosis in vitro (Figure 1B). From this data, we inferred that ATM is a likely effector of CpdX treatment. CONCLUSION The initial lack of specific target candidates following primary phenotypic screens is a widespread problem in drug discovery. In the absence of a target deconvolution strategy, many potential candidates cannot be progressed easily to optimization. Here, we demonstrated that a combination of state-of-the-art technologies used to benchmark hit compounds from phenotypic screens versus known bio-annotated compound libraries represent a viable approach to perform target deconvolution of hits from phenotypic screening approaches. In this work, a phenotypic screen and subsequent deconvolution identified a novel target in combination with a novel compound, making a suitable starting point for progression into a potential kidney therapeutic agent.
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