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Nature Communications May 2024Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of...
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Topics: Animals; Forkhead Transcription Factors; Retinal Neovascularization; Mice; Endothelial Cells; Blood-Retinal Barrier; TOR Serine-Threonine Kinases; Pericytes; Fusion Regulatory Protein 1, Heavy Chain; Retinal Vessels; Humans; Large Neutral Amino Acid-Transporter 1; Mice, Knockout; Mice, Inbred C57BL; Retina; Male; Angiogenesis
PubMed: 38755144
DOI: 10.1038/s41467-024-48134-2 -
Circulation May 2024Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic...
BACKGROUND
Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.
METHODS
We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 ()-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.
RESULTS
Transcriptional regulation and trajectory inference from -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor and upregulation of late anticardiac regulators and in knockout cells reflect a necessary role for in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
CONCLUSIONS
These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.
PubMed: 38752370
DOI: 10.1161/CIRCULATIONAHA.122.061335 -
Frontiers in Cell and Developmental... 2024Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC...
Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor -GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes -GlcNAcase (OGA) and -GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of -depleted (OGAi) and -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34 hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular -GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of and in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of -GlcNAcylation.
PubMed: 38752196
DOI: 10.3389/fcell.2024.1361943 -
BioRxiv : the Preprint Server For... May 2024Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples....
UNLABELLED
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
SUMMARY
We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.
PubMed: 38746120
DOI: 10.1101/2024.04.29.591777 -
Gastro Hep Advances 2024
The IncRNA, Cardiac Mesoderm Enhancer-Associated Noncoding RNA Is Indispensable for Intestinal Smooth Muscle Homeostasis in Female Mice as Revealed by a Novel Endogenous -Encoded Inducible Cre Model.
PubMed: 38737599
DOI: 10.1016/j.gastha.2023.12.012 -
Bio-protocol May 2024Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study...
Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.
PubMed: 38737507
DOI: 10.21769/BioProtoc.4984 -
Stem Cell Research May 2024Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the...
Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the deletion of the Y chromosome Azoospermia factor a (AZFa) has been identified in infertile males with severely impaired spermatogenesis. AZFa, encompassing megabase-scale of the Y chromosome region, poses challenges in modeling AZFa deletion-related male infertility using gene editing tools. Here, we successfully created an AZFa-deleted human embryonic stem cell line utilizing the CRISPR/Cas9 gene editing tool. Our analysis indicates the AZFa-deleted stem cell line holds promise for differentiation into ectoderm, mesoderm, and endoderm, highlighting its potential for further comprehensive study.
PubMed: 38733811
DOI: 10.1016/j.scr.2024.103436 -
International Journal of Molecular... Apr 2024Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The...
Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.
Topics: Melatonin; Mesenchymal Stem Cells; Humans; Cell Differentiation; Cells, Cultured; Adipose Tissue; Neurons; Culture Media, Conditioned; Schwann Cells; Neurogenesis; Adult; Nestin; Glial Fibrillary Acidic Protein; Neuroglia; Synapsins
PubMed: 38732109
DOI: 10.3390/ijms25094891 -
EvoDevo May 2024Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using...
Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.
PubMed: 38730509
DOI: 10.1186/s13227-024-00224-4 -
Cells & Development May 2024The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the...
The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome - a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.
PubMed: 38729574
DOI: 10.1016/j.cdev.2024.203926