Prophylactic Oophorectomy in Carriers of - NEJM In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. Top. b, Dot plot showing variance-scaled, log-transformed expression of marker genes (y-axis) for the identified macrophage subsets (x-axis). performed the imaging experiments. PubMed Central Bian, Z. et al. Sci. d, (left) UMAP projections of the predicted probability of ESGC from our dataset onto Guo et al., 2021 somatic cells manifold using a Support Vector Machine (SVM) classifier. Accordingly, the expression of FOXL2, which is essential for ovarian fate16,17, can already be detected in female ESGCs at this stage (Fig. We used general cell annotations from the main analysis, with the exception of the main gonadal lineages (germ, supporting and mesenchymal) for which we considered the identified subpopulations. Somatic cells are downsampled to account for 150 cells for each cell state in each sex in both species. Estimated abundance for cell types (colour intensity) contributed by each mesenchymal subpopulation to each Visium spot (colour) shown over the H&E images. 10b). To isolate CD45+ and CD45 cells, we used the antibody CD45-BUV395 BD Bioscience 563791 Clone HI30 (RUO) Flow cytometry (dilution 2.5l:100l). Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. Nature 598, 327331 (2021). To study how human pregranulosa cells in the distinct cortical and medullary microenvironments could influence germ cell differentiation, we expanded our CellPhoneDB database to (1) include non-peptide ligands and (2) link receptors with their downstream TFs (CellSign module) (Extended Data Fig. 72. PubMed For female mouse scRNA-seq data, an additional UMAP is coloured by the study of origin. Cancer - Coeliac UK Dots from the same donor or sample share a colour. Knoblaugh, S. E., True, L., Tretiakova, M. & Hukkanen, R. R. in Comparative Anatomy and Histology (eds. 240, 92107 (2001). Histologic examination revealed a poorly differentiated carcinoma with transitional characteristics that are typically associated with Brenner tumors, which are benign ovarian tumors that are thought to be derived from the pluripotent coelomic epithelium . In humans, after 9PCW, sPAX8s remain at the poles of the developing cords in males, where the rete testis develops, but are virtually absent in females. Gigascience 9, giaa151 (2020). 77), HOCOMOCOv10 (ref. PubMed Central Science 273, 12361238 (1996). Am Cancer Soc; Candia Carnevali MD, Lucca E, Bonasoro F. Mechanisms of arm regeneration in the . Finally, we used the function run_palantir (with num_waypoints=500) to estimate the pseudotime of each cell from the root cell. We also excluded extragonadal coelomic epithelium GATA2+. and E.P. Bottom shows UMAP projections of cell lineages (colour) in the human male scRNA-seq (n=133,811), human male scATAC-seq (n=52,285) and mouse male scRNA-seq (n=32,889) datasets. We used this database to link our CellPhoneDB results to the relevant downstream TFs, which were derived from our TF analysis. Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Before meiosis initiation, coinciding with a premeiotic STRA8 surge, we find the activation of ZGLP1, the oogenic TF recently described in mice12 (Extended Data Fig. a, Schematics illustrating the CD45+ enrichment strategy for gonadal and extragonadal samples. Nature 589, 264269 (2021). Donor effects were corrected using Harmony67 (theta=0). Fresh tissue samples of human developing gonads were embedded in cold optimal cutting temperature compound (OCT) medium and flash frozen using a dry ice-isopentane slurry. Mesothelial to mesenchyme transition as a major developmental and We used several single-cell genomics methods: (1) single-cell RNA sequencing (scRNA-seq); (2) single-cell accessible chromatin sequencing (scATAC-seq) and (3) combined single-nucleus RNA and ATAC sequencing (snRNA-seq/scATAC-seq) to profile 347,709, 96,174 and 40,742 cells, respectively (Fig. 4d). Single-cell roadmap of human gonadal development | Nature h, Dot plot showing the variance-scaled, log-transformed expression of microglia markers in the cluster of TREM2+ ftM in both sexes reveals that the few female cells that belong to this cluster do not express the key markers. First, we describe ESGCs, a bipotent transient population whose numbers peak at the time of sex determination and that connects the coelomic epithelium with Sertoli cells and the first wave of pregranulosa cells. In brief, cells were subsampled to balance cell type and sex contribution (n=500 for germ and n=150 for somatic cells). ADS Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a Natl Acad. Scale bars = 100m and 10m in the magnified region. Next, peaks were called separately on each cluster using macs2 (ref. On this treatment, no endogenous fluorescence from the Oct4PE-GFP transgene was observed. et al. Developing gonads were dissected from the mesonephros and both organs were separately dissociated with 0.25% Trypsin/EDTA into single-cell suspensions as described for the human tissue. j, High-resolution imaging of representative gonadal section of a human fetal ovary (19PCW), with intensity proportional to smFISH signal for NTN1 (green, granulosa), FIGLA (yellow, oocytes), DCC (red, oocyte), FOXL2 (magenta, granulosa); n=2. We included six males and three females at E10.5, six males and two females at E11.5, and three males and three females at E12.5. White dashed rectangles highlight the magnified regions from the following organs: testis (top), skin (middle), spinal cord (labelled as CNS = central nervous system) (bottom); n=1 e, High-resolution imaging of representative gonadal sections of two fetal testes (12 and 14PCW), with intensity proportional to smFISH signal to SOX9 (cyan, Sertoli cells), CD68 (red, macrophages), P2RY12 (yellow, TREM2+ ftM); n=5. ftM = fetal testicular macrophages; PCW = post-conceptional week; prob_ = probability. f, High-resolution imaging of a representative transverse section of a human ovary at 21 post-conceptional weeks (PCW), with intensity proportional to smFISH signal for POU5F1 (green, primordial germ cells), DDX4 (red, fetal germ cells), STRA8 (cyan, pre-meiotic germ cells) and FIGLA (yellow, oocytes); n=4. Plots represent estimated abundance for cell types. Guo, J. et al. Pliner et al. Embryos up to 8PCW were staged using the Carnegie staging method47. h, Heatmap showing the expression of TF downstream the receptors (CellSign) upregulated in germ and supporting cells (shown in Fig. Biol. Myeloid cells from fetal liver, skin, kidney, yolk sac, gut, thymus, placenta, bone marrow and brain datasets were selected on the basis of the expression of established myeloid markers (CD14, CD68, CSF1R). Meinhardt, A. For all datasets, we filtered out cells expressing fewer than 300 genes and more than 20% of mitochondrial genes. Dissociated cells were incubated at 4C with 2.5l of antibodies in 1% FBS in Dulbeccos PBS without calcium and magnesium (Thermo Fisher Scientific, 14190136). Kracht, L. et al. As reference, we used scRNA-seq data from individuals of the same sex and gestational stage. DE, ductus epididymidis; DMD, degenerating Mllerian duct; DMN, degenerating mesonephric nephron; ED, efferent ductule; MD, Mullerian duct; RT, rete testis; TC, testis cords; UC, urogenital connection; WD, Wolffian duct; WT, Wolffian tubules; scale bars, 100m unless otherwise specified. & Greenfield, A. Characterizing the bipotential mammalian gonad. PubMed Central A cell atlas of human thymic development defines T cell repertoire formation. Germ, gonadal somatic, endothelial and immune cells were subsequently reanalysed integrating both sexes into the same manifold, using the approach described in the previous paragraph. Nucleic Acids Res. SIGLEC15+ and TREM2+ ftMs are rare populations in comparison to the tissue-repair macrophages characteristic of all developing tissues (2.8% SIGLEC15+ ftMs, 5% TREM2+ ftMs, 92.2% tissue-repair macrophages). 86, 37 (2012). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Zoomed-in UMAPs show SIGLEC15+ and TREM2+ fetal testicular macrophages (ftMs) labelled by sex. Methods 14, 975978 (2017). Science 352, 189196 (2016). Jaurena, M. B., Juraver-Geslin, H., Devotta, A. Syst. 5f,g). CoelEpi, coelomic epithelium; Endo, endothelial; Epi, epithelial; F. Leydig, fetal Leydig; Gi, gonadal interstitial; Mesen, mesenchymal; Oi, ovarian interstitial; OSE, ovarian surface epithelium; preGC, pregranulosa cells; PV, perivascular; sPAX8, supporting PAX8+; Ti, testicular interstitial; SMC, smooth muscle cell. Google Scholar. ADS c, UMAP projections of integrated myeloid cells (colour) from several embryonic/fetal tissues (n=58,948). Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. 48, 838847 (2016). The box extends from the lower to upper quartile values of the data, with a line at the median. Article Wagner, M. et al. Cell 29, 511520 (2014). Scale bars = 100m. Cells were gated on live, singlets and CD45+. PreGC-IIb cells, located in the inner cortex, express ligands involved in meiosis initiation (for example, retinoic acid by ALDH1A1) and oogenesis (for example, BMP2) to support PGC differentiation. Intestinal Epithelial Cells (CECs) were analyzed by flow cytometer. Coelomic Epithelium - an overview | ScienceDirect Topics Comprehensive integration of single-cell data. Molecular Mechanisms Regulating Organ-Specific Metastases in Epithelial and A.M. R.V.-T. supervised the work. volume607,pages 540547 (2022)Cite this article. Extended Data Fig. This single space wall will undergo a mesenchymal epithelial transition. 9 Tissue-resident macrophages in the developing testes. In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. TREM2+ ftMs also have their phagocytosis machinery active (MERTK, AXL, CYBB, BECN1, MTOR) (Extended Data Fig. PubMed 58) analysed by Guo et al.59. Third, we identify a first wave of medullary and a second wave of cortical pregranulosa cells in humans, similar to mice5,21,38. We adopted the cisTopic approach65,66 v.0.3.0 for the core of our downstream analysis. Library preparation was carried out according to the manufacturers protocol. b, UMAP (uniform manifold approximation and projection) of the male and female human (left) and mouse (right) scRNA-seq datasets labelled by donor and sample. To build CellSign, we have manually mined the literature to identify TFs with high specificity for an upstream receptor and recorded the relevant pubmed reference number (Supplementary Table 11c). ISSN 0028-0836 (print). d, High-resolution large-area imaging of representative gonadal section (sagittal) of a human fetal testis (7PCW, Carnegie Stage CS17) with intensity proportional to smFISH signal for GATA4 (green, gonadal), PAX8 (red, sPAX8 population) and GATA2 (cyan, extragonadal); n=5. k, Dot plot showing variance-scaled, log-transformed expression of marker genes expressed in the identified cell populations from the multi-organ integrated fetal myeloid dataset. Single-nuclei suspensions were isolated from dissociated cells when performing scATAC-seq, following the manufacturers instructions, and from frozen tissue sections when performing multiomic snRNA-seq/scATAC-seq. We evaluated the consistency between the main lineages identified in our study with the Smart-seq2 dataset of gonadal cells from Li et al.7 (GSE86146). analysed the data with contributions from T.L., S.D. Similarly, we trained an SVM model on our gonadal macrophages and projected the cell type annotations onto fetal testicular myeloid cells from Chitiashvili et al.58. CAS Nature thanks Humphrey Yao and the other, anonymous, reviewers for their contribution to the peer review of this work. We set the number of terminal states to four, letting CellRank determine the number of initial states. 5e and Supplementary Table 5). After performing principal component analysis (PCA), neighbour identification and Leiden clustering, the members of the gene cluster including known cycling genes (CDK1, MKI67, CCNB2 and PCNA) were flagged as the data-derived cell cycling genes and discarded in each downstream analysis. e, Barplot showing the proportions of somatic cells in the Guo et al., 2021 dataset coloured by cell state and classified by PCW. g, High-resolution large-area imaging of representative sections of two human fetal ovaries (9 and 11 PCW; n=2) with intensity proportional to smFISH signal for the same panel in f. We then filtered the denoised accessibility matrix from cisTopic to keep only the peaks included in CCANs. Cell Stem Cell 28, 764778.e4 (2021). 3c. k, Schematic illustration of main TFs, receptors, ligands and extracellular molecules regulating germ cell differentiation influenced by the granulosa lineage. Colour legend for the main lineages match those in Extended Data Fig. Albrecht, K. H. & Eicher, E. M. Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor. Popescu, D.-M. et al. Pathology Outlines - Embryology Bozdag, G., Mumusoglu, S., Zengin, D., Karabulut, E. & Yildiz, B. O. Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice. d, Hierarchical clustering of z-scores for each cis-co-accessibility network (CCAN) identified in human ovarian supporting cells in the human scATAC-seq dataset. In humans, we also define a second wave of granulosa cells (preGC-IIa/b) appearing after 8PCW (Fig. Flyer points are those past the end of the whiskers. 60, 533 (1996). Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. 5e). Cell. Dev. Scale bars 1mm (left) and 50m in magnified regions (right). C.S.-S., R.V.-T., J.E., B.C., R.A.B. Niu, W. & Spradling, A. C. Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. To study the unique profile of our gonadal macrophages, we downloaded immune cells from several developing tissues: liver, skin, kidney, yolk sac, gut, thymus, placenta, bone marrow and brain28,31,32,33,34,35. Decoding human fetal liver haematopoiesis. Vu, T. H. et al. The white dashed rectangles (left) highlight the enlarged sample region (right). & Desnick, R. J. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Furthermore, we added a new module to the database called CellSign that links receptors in CellphoneDB to their known downstream TF. For all smFISH panels, unless otherwise specified, white dashed rectangles highlight gonadal regions magnified; scale bars = 100m and 10m in magnified regions. Endocrinol. c, UMAP projections of immune cells labelled by sex, PCW and donor. b, High-resolution large-area imaging of representative gonadal sections of one human testis (12PCW, transverse section), with intensity proportional to smFISH signal for EPCAM (red, epithelial), NR5A1 (cyan, gonadal somatic) and PAX8 (yellow, sPAX8 and epithelial) (n=2). ESGCs appear transiently in the early gonads (roughly 68PCW in humans and E11.5 in mice) and, in males, are the first gonadal somatic cells to express the testis-determining factor SRY, which is required for Sertoli cell commitment2 (Fig. 9ik). PreGC-IIa cells, present in the outer ovarian cortex, express chemoattractants (for example, NRG1) and survival factors (for example, KITLG), with STAT3 downstream of KIT active in PGCs (Fig. The resulting matrix was integrated by sample and origin of the dataset using scVI on the basis of the procedure described above. Science 369, 530537 (2020). SIGLEC15+ ftMs (white arrows) are outside the testis cords in proximity to endothelial cells (n=5). cDC = conventional Dendritic cells; ECM = extracellular matrix; ESGC = early supporting gonadal cells; ftM = fetal testicular macrophages; Gi = gonadal interstitial; ILC prec = innate lymphoid cell precursors; Mac = macrophages; Mast = mast cells; Mega = megakaryocytes; MEMP = megakaryocyte-erythroid-mast cell progenitors; Mono = monocytes; Neutro = neutrophil; NMP = neutrophil-myeloid progenitors; NK = Natural Killer cells; pDC = plasmacytoid Dendritic cell; PV = perivascular; PCW = post-conceptional week; Prec = precursor; Pre_B = pre-B cells; Pre_pro_B = pre-pro-B cells; Pro_B = pro-B cells; Prob_ = probability; sPAX8 = supporting PAX8; Gi = gonadal interstitial; T = T cells; Ti = testicular interstitial. Article & Vento-Tormo, R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Embryol. TF can bind their potential targets); a = TF whose targets are differentially expressed (i.e. All tissue samples used for this study were obtained with written informed consent from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. (right) 8 PCW testis stained for CD68 (red, macrophages), SIGLEC15 (yellow, SIGLEC15+ ftM) and P2RY12 (green, TREM2+ ftM); n=3. b, High-resolution imaging of representative sections of two fetal testes (11 and 12 PCW), with intensity proportional to smFISH signal for EPCAM (cyan, high = epithelial cells; low = Sertoli and germ cells), PDGFRA (green, mesenchymal cells), CD68 (red, macrophages), SIGLEC15 (yellow, SIGLEC15+ ftM); n=7. c, (top left) UMAP (uniform manifold approximation and projection) of myeloid cells from Guo et al., 2021 labelled by PCW. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. Predicted doublets were not excluded from the initial analysis, but used afterwards to flag clusters with high doublet scores. TF motif activities were computed using chromVar76 v.1.12.2 with positional weight matrices from JASPAR2018 (ref. SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. PubMed Coelomic epithelium forms the ovarian surface epithelium Subcoelomic mesoderm forms the ovarian stroma Primordial germ cells migrate from the yolk sac endoderm to the developing ovary Invaginations of coelomic epithelium in the superficial ovarian cortex form the sex cords (pregranulosa cells) 46, D260D266 (2018). LGR5 thus marks different populations in mice and humans, highlighting the need for humanmouse harmonized atlases. La Manno, G. et al. To compare the differentiation of human germ cells with that of other mammals, we integrated our human and mouse gonadal germ cells with more scRNA-seq gonadal germ cells datasets from mouse and macaque5,10,11 (Extended Data Fig. Korsunsky, I. et al. 134, 167194 (2019). The authors declare no competing interests. We then trained an SVM classifier (sklearn.svm.SVC) on human data and projected the cell type annotations onto the mouse datasets. The resulting matrix was further processed to average cells by cell type and peaks by CCAN. Multiplexed smFISH images are available from BioStudies (www.ebi.ac.uk/biostudies), with accession number S-BIAD393. Top-layer groups marker genes by categories. Both sPAX8 subsets show a unique transcriptional pattern of axon guidance factors, suggesting they have a structural and supporting role. Raw counts were normalized and log-transformed, and the 300 most highly variable genes were selected. Methods 15, 359362 (2018). Gut mesodermal tissues originate from the splanchnopleural mesenchyme. Maheshwari, A. 18/NE/0290). We called the peaks using an in-house implementation of the approach described in Cusanovich et al.63 (available at https://github.com/cellgeni/cellatac, revision 21-099). Fresh frozen sections were removed from 80C storage and air dried before being fixed in 10% neutral buffered formalin for 5min. Samples less than 17PCW were also digested using a combination of collagenase and Trypsin/EDTA, a protocol adapted from Wagner et al.50,51. Addressing gaps in care of people with conditions affecting sex development and maturation. https://doi.org/10.1038/s41593-020-0602-1 (2020). Hayman, A. R. et al. Article Colour proportional to scaled log-transformed expression. CoelEpi = coelomic epithelium; E = embryonic day; ESGC = early supporting gonadal cells; FGC = fetal germ cells; Gi = gonadal interstitial; Oi = ovarian interstitial; OSE = ovarian surface epithelium; P = postnatal day; PCW = post-conceptional week; PGC = primordial germ cells; preGC = pre-granulosa cells; TF = transcription factor; Ti = testicular interstitial; sPAX8 = supporting PAX8. For each sequenced multimodal snRNA-seq library, we performed read alignment to the 10X Genomics GRCh38 v.3.1.0 (human) reference genome, quantification and initial quality control using the Cell Ranger ARC Software (v.1.0.1, 10X Genomics) using default parameters. E = embryonic day; Expr = expressed, FGC = fetal germ cells; P = postnatal day; PCW = post-conceptional weeks; PGC = primordial germ cells. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Despite the similar spatiotemporal patterns in humans and mice, we show that certain regulatory programmes differ; for example, LGR5, characteristic of second-wave pregranulosa cells in mice5,38, is restricted to ESGCs in humans. These authors contributed equally: Luz Garcia-Alonso, Valentina Lorenzi, Luz Garcia-Alonso,Valentina Lorenzi,Cecilia Icoresi Mazzeo,Kenny Roberts,Carmen Sancho-Serra,Magda Marekov,Tong Li,Stijn van Dongen,Vladimir Yu Kiselev,Elena Prigmore,Omer Ali Bayraktar,Muzlifah Haniffa&Roser Vento-Tormo, Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK, Joo Pedro Alves-Lopes,Wolfram H. Gruhn&Azim Surani, Physiology, Development and Neuroscience Department, University of Cambridge, Cambridge, UK, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK, Justin Engelbert,Rachel A. Botting,Mary Herbert&Muzlifah Haniffa, Nuffield Department of Womens and Reproductive Health, University of Oxford, Oxford, UK, Great Ormond Street Institute of Child Health, University College London, London, UK, University of Cambridge Centre for Trophoblast Research, Department of Pathology, University of Cambridge, Cambridge, UK, Sorbonne Universit, INSERM, CNRS, Institut de la Vision, Paris, France, Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge, UK, You can also search for this author in Signs and symptoms may include weight loss, fever, malaise, cough, skin and mucosal lesions, diarrhea, and peripheral neuropathy. To speed up computational times, we downsampled our dataset to 50% size. Student Org Marquette University, How Common Is Astigmatism, Nih Bibliography And References Cited, Articles C
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coelomic epithelium cancer

coelomic epithelium cancer

Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We filtered out cells that expressed fewer than 1,000 genes or more than 20% of mitochondrial genes. Colour proportional to scaled log-transformed expression. 70). In short, samples were first digested with collagenase 1A (1mgml1) and liberase TM (50gml1) for 45min at 37C with intermittent shaking. Terminal states were determined automatically by Palantir. 78), SwissRegulon79, HOMER80. To identify marker genes specific to a cluster, we used the TF-IDF approach from the SoupX package v.1.5.0 (ref. 14, 20532062 (2005). Fast, sensitive and accurate integration of single-cell data with Harmony. This sample is also shown in Extended Data Fig. Doublets and low-quality control cells were removed. Dots from the same donor share a colour. Krishnaswami, S. R. et al. Developing liver scRNA-seq raw counts were downloaded from ArrayExpress (E-MTAB-7407), processed with Scanpy v.1.7.0 workflow described above for the main scRNA-seq analysis and filtered on the basis of the expression of CD45 (PTPRC) to exclude non-immune cells. & Hedger, M. P. Immunological, paracrine and endocrine aspects of testicular immune privilege. 10a and Supplementary Table 9). To locate cells in the profiled tissues we (1) generated spatial transcriptomics data using Visium and multiplexed single-molecule fluorescent in situ hybridization (smFISH), and (2) isolated the gonad and extragonadal tissue by microdissection to profile each separately. and J.P.A.-L. are supported by Wellcome, CRUK, MSCA (grant no. Kleshchevnikov, V. et al. Administration of CFEF significantly decreased the severity of DSS/AOM-induced CAC in a dose-dependent manner. Specifically, CellRank uses a random walk model to learn directed, probabilistic state-change trajectories and determine initial and terminal states. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Correspondence to 6ac and Supplementary Note 4). We compared the transcriptional signatures of the cell types identified in our human scRNA-seq to their mouse counterparts, considering all developmental stages combined. It is recommended that serological testing should be offered to any children or adults . Prophylactic Oophorectomy in Carriers of - NEJM In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. Top. b, Dot plot showing variance-scaled, log-transformed expression of marker genes (y-axis) for the identified macrophage subsets (x-axis). performed the imaging experiments. PubMed Central Bian, Z. et al. Sci. d, (left) UMAP projections of the predicted probability of ESGC from our dataset onto Guo et al., 2021 somatic cells manifold using a Support Vector Machine (SVM) classifier. Accordingly, the expression of FOXL2, which is essential for ovarian fate16,17, can already be detected in female ESGCs at this stage (Fig. We used general cell annotations from the main analysis, with the exception of the main gonadal lineages (germ, supporting and mesenchymal) for which we considered the identified subpopulations. Somatic cells are downsampled to account for 150 cells for each cell state in each sex in both species. Estimated abundance for cell types (colour intensity) contributed by each mesenchymal subpopulation to each Visium spot (colour) shown over the H&E images. 10b). To isolate CD45+ and CD45 cells, we used the antibody CD45-BUV395 BD Bioscience 563791 Clone HI30 (RUO) Flow cytometry (dilution 2.5l:100l). Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. Nature 598, 327331 (2021). To study how human pregranulosa cells in the distinct cortical and medullary microenvironments could influence germ cell differentiation, we expanded our CellPhoneDB database to (1) include non-peptide ligands and (2) link receptors with their downstream TFs (CellSign module) (Extended Data Fig. 72. PubMed For female mouse scRNA-seq data, an additional UMAP is coloured by the study of origin. Cancer - Coeliac UK Dots from the same donor or sample share a colour. Knoblaugh, S. E., True, L., Tretiakova, M. & Hukkanen, R. R. in Comparative Anatomy and Histology (eds. 240, 92107 (2001). Histologic examination revealed a poorly differentiated carcinoma with transitional characteristics that are typically associated with Brenner tumors, which are benign ovarian tumors that are thought to be derived from the pluripotent coelomic epithelium . In humans, after 9PCW, sPAX8s remain at the poles of the developing cords in males, where the rete testis develops, but are virtually absent in females. Gigascience 9, giaa151 (2020). 77), HOCOMOCOv10 (ref. PubMed Central Science 273, 12361238 (1996). Am Cancer Soc; Candia Carnevali MD, Lucca E, Bonasoro F. Mechanisms of arm regeneration in the . Finally, we used the function run_palantir (with num_waypoints=500) to estimate the pseudotime of each cell from the root cell. We also excluded extragonadal coelomic epithelium GATA2+. and E.P. Bottom shows UMAP projections of cell lineages (colour) in the human male scRNA-seq (n=133,811), human male scATAC-seq (n=52,285) and mouse male scRNA-seq (n=32,889) datasets. We used this database to link our CellPhoneDB results to the relevant downstream TFs, which were derived from our TF analysis. Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Before meiosis initiation, coinciding with a premeiotic STRA8 surge, we find the activation of ZGLP1, the oogenic TF recently described in mice12 (Extended Data Fig. a, Schematics illustrating the CD45+ enrichment strategy for gonadal and extragonadal samples. Nature 589, 264269 (2021). Donor effects were corrected using Harmony67 (theta=0). Fresh tissue samples of human developing gonads were embedded in cold optimal cutting temperature compound (OCT) medium and flash frozen using a dry ice-isopentane slurry. Mesothelial to mesenchyme transition as a major developmental and We used several single-cell genomics methods: (1) single-cell RNA sequencing (scRNA-seq); (2) single-cell accessible chromatin sequencing (scATAC-seq) and (3) combined single-nucleus RNA and ATAC sequencing (snRNA-seq/scATAC-seq) to profile 347,709, 96,174 and 40,742 cells, respectively (Fig. 4d). Single-cell roadmap of human gonadal development | Nature h, Dot plot showing the variance-scaled, log-transformed expression of microglia markers in the cluster of TREM2+ ftM in both sexes reveals that the few female cells that belong to this cluster do not express the key markers. First, we describe ESGCs, a bipotent transient population whose numbers peak at the time of sex determination and that connects the coelomic epithelium with Sertoli cells and the first wave of pregranulosa cells. In brief, cells were subsampled to balance cell type and sex contribution (n=500 for germ and n=150 for somatic cells). ADS Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a Natl Acad. Scale bars = 100m and 10m in the magnified region. Next, peaks were called separately on each cluster using macs2 (ref. On this treatment, no endogenous fluorescence from the Oct4PE-GFP transgene was observed. et al. Developing gonads were dissected from the mesonephros and both organs were separately dissociated with 0.25% Trypsin/EDTA into single-cell suspensions as described for the human tissue. j, High-resolution imaging of representative gonadal section of a human fetal ovary (19PCW), with intensity proportional to smFISH signal for NTN1 (green, granulosa), FIGLA (yellow, oocytes), DCC (red, oocyte), FOXL2 (magenta, granulosa); n=2. We included six males and three females at E10.5, six males and two females at E11.5, and three males and three females at E12.5. White dashed rectangles highlight the magnified regions from the following organs: testis (top), skin (middle), spinal cord (labelled as CNS = central nervous system) (bottom); n=1 e, High-resolution imaging of representative gonadal sections of two fetal testes (12 and 14PCW), with intensity proportional to smFISH signal to SOX9 (cyan, Sertoli cells), CD68 (red, macrophages), P2RY12 (yellow, TREM2+ ftM); n=5. ftM = fetal testicular macrophages; PCW = post-conceptional week; prob_ = probability. f, High-resolution imaging of a representative transverse section of a human ovary at 21 post-conceptional weeks (PCW), with intensity proportional to smFISH signal for POU5F1 (green, primordial germ cells), DDX4 (red, fetal germ cells), STRA8 (cyan, pre-meiotic germ cells) and FIGLA (yellow, oocytes); n=4. Plots represent estimated abundance for cell types. Guo, J. et al. Pliner et al. Embryos up to 8PCW were staged using the Carnegie staging method47. h, Heatmap showing the expression of TF downstream the receptors (CellSign) upregulated in germ and supporting cells (shown in Fig. Biol. Myeloid cells from fetal liver, skin, kidney, yolk sac, gut, thymus, placenta, bone marrow and brain datasets were selected on the basis of the expression of established myeloid markers (CD14, CD68, CSF1R). Meinhardt, A. For all datasets, we filtered out cells expressing fewer than 300 genes and more than 20% of mitochondrial genes. Dissociated cells were incubated at 4C with 2.5l of antibodies in 1% FBS in Dulbeccos PBS without calcium and magnesium (Thermo Fisher Scientific, 14190136). Kracht, L. et al. As reference, we used scRNA-seq data from individuals of the same sex and gestational stage. DE, ductus epididymidis; DMD, degenerating Mllerian duct; DMN, degenerating mesonephric nephron; ED, efferent ductule; MD, Mullerian duct; RT, rete testis; TC, testis cords; UC, urogenital connection; WD, Wolffian duct; WT, Wolffian tubules; scale bars, 100m unless otherwise specified. & Greenfield, A. Characterizing the bipotential mammalian gonad. PubMed Central A cell atlas of human thymic development defines T cell repertoire formation. Germ, gonadal somatic, endothelial and immune cells were subsequently reanalysed integrating both sexes into the same manifold, using the approach described in the previous paragraph. Nucleic Acids Res. SIGLEC15+ and TREM2+ ftMs are rare populations in comparison to the tissue-repair macrophages characteristic of all developing tissues (2.8% SIGLEC15+ ftMs, 5% TREM2+ ftMs, 92.2% tissue-repair macrophages). 86, 37 (2012). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Zoomed-in UMAPs show SIGLEC15+ and TREM2+ fetal testicular macrophages (ftMs) labelled by sex. Methods 14, 975978 (2017). Science 352, 189196 (2016). Jaurena, M. B., Juraver-Geslin, H., Devotta, A. Syst. 5f,g). CoelEpi, coelomic epithelium; Endo, endothelial; Epi, epithelial; F. Leydig, fetal Leydig; Gi, gonadal interstitial; Mesen, mesenchymal; Oi, ovarian interstitial; OSE, ovarian surface epithelium; preGC, pregranulosa cells; PV, perivascular; sPAX8, supporting PAX8+; Ti, testicular interstitial; SMC, smooth muscle cell. Google Scholar. ADS c, UMAP projections of integrated myeloid cells (colour) from several embryonic/fetal tissues (n=58,948). Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. 48, 838847 (2016). The box extends from the lower to upper quartile values of the data, with a line at the median. Article Wagner, M. et al. Cell 29, 511520 (2014). Scale bars = 100m. Cells were gated on live, singlets and CD45+. PreGC-IIb cells, located in the inner cortex, express ligands involved in meiosis initiation (for example, retinoic acid by ALDH1A1) and oogenesis (for example, BMP2) to support PGC differentiation. Intestinal Epithelial Cells (CECs) were analyzed by flow cytometer. Coelomic Epithelium - an overview | ScienceDirect Topics Comprehensive integration of single-cell data. Molecular Mechanisms Regulating Organ-Specific Metastases in Epithelial and A.M. R.V.-T. supervised the work. volume607,pages 540547 (2022)Cite this article. Extended Data Fig. This single space wall will undergo a mesenchymal epithelial transition. 9 Tissue-resident macrophages in the developing testes. In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. TREM2+ ftMs also have their phagocytosis machinery active (MERTK, AXL, CYBB, BECN1, MTOR) (Extended Data Fig. PubMed 58) analysed by Guo et al.59. Third, we identify a first wave of medullary and a second wave of cortical pregranulosa cells in humans, similar to mice5,21,38. We adopted the cisTopic approach65,66 v.0.3.0 for the core of our downstream analysis. Library preparation was carried out according to the manufacturers protocol. b, UMAP (uniform manifold approximation and projection) of the male and female human (left) and mouse (right) scRNA-seq datasets labelled by donor and sample. To build CellSign, we have manually mined the literature to identify TFs with high specificity for an upstream receptor and recorded the relevant pubmed reference number (Supplementary Table 11c). ISSN 0028-0836 (print). d, High-resolution large-area imaging of representative gonadal section (sagittal) of a human fetal testis (7PCW, Carnegie Stage CS17) with intensity proportional to smFISH signal for GATA4 (green, gonadal), PAX8 (red, sPAX8 population) and GATA2 (cyan, extragonadal); n=5. k, Dot plot showing variance-scaled, log-transformed expression of marker genes expressed in the identified cell populations from the multi-organ integrated fetal myeloid dataset. Single-nuclei suspensions were isolated from dissociated cells when performing scATAC-seq, following the manufacturers instructions, and from frozen tissue sections when performing multiomic snRNA-seq/scATAC-seq. We evaluated the consistency between the main lineages identified in our study with the Smart-seq2 dataset of gonadal cells from Li et al.7 (GSE86146). analysed the data with contributions from T.L., S.D. Similarly, we trained an SVM model on our gonadal macrophages and projected the cell type annotations onto fetal testicular myeloid cells from Chitiashvili et al.58. CAS Nature thanks Humphrey Yao and the other, anonymous, reviewers for their contribution to the peer review of this work. We set the number of terminal states to four, letting CellRank determine the number of initial states. 5e and Supplementary Table 5). After performing principal component analysis (PCA), neighbour identification and Leiden clustering, the members of the gene cluster including known cycling genes (CDK1, MKI67, CCNB2 and PCNA) were flagged as the data-derived cell cycling genes and discarded in each downstream analysis. e, Barplot showing the proportions of somatic cells in the Guo et al., 2021 dataset coloured by cell state and classified by PCW. g, High-resolution large-area imaging of representative sections of two human fetal ovaries (9 and 11 PCW; n=2) with intensity proportional to smFISH signal for the same panel in f. We then filtered the denoised accessibility matrix from cisTopic to keep only the peaks included in CCANs. Cell Stem Cell 28, 764778.e4 (2021). 3c. k, Schematic illustration of main TFs, receptors, ligands and extracellular molecules regulating germ cell differentiation influenced by the granulosa lineage. Colour legend for the main lineages match those in Extended Data Fig. Albrecht, K. H. & Eicher, E. M. Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor. Popescu, D.-M. et al. Pathology Outlines - Embryology Bozdag, G., Mumusoglu, S., Zengin, D., Karabulut, E. & Yildiz, B. O. Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice. d, Hierarchical clustering of z-scores for each cis-co-accessibility network (CCAN) identified in human ovarian supporting cells in the human scATAC-seq dataset. In humans, we also define a second wave of granulosa cells (preGC-IIa/b) appearing after 8PCW (Fig. Flyer points are those past the end of the whiskers. 60, 533 (1996). Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. 5e). Cell. Dev. Scale bars 1mm (left) and 50m in magnified regions (right). C.S.-S., R.V.-T., J.E., B.C., R.A.B. Niu, W. & Spradling, A. C. Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. To study the unique profile of our gonadal macrophages, we downloaded immune cells from several developing tissues: liver, skin, kidney, yolk sac, gut, thymus, placenta, bone marrow and brain28,31,32,33,34,35. Decoding human fetal liver haematopoiesis. Vu, T. H. et al. The white dashed rectangles (left) highlight the enlarged sample region (right). & Desnick, R. J. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Furthermore, we added a new module to the database called CellSign that links receptors in CellphoneDB to their known downstream TF. For all smFISH panels, unless otherwise specified, white dashed rectangles highlight gonadal regions magnified; scale bars = 100m and 10m in magnified regions. Endocrinol. c, UMAP projections of immune cells labelled by sex, PCW and donor. b, High-resolution large-area imaging of representative gonadal sections of one human testis (12PCW, transverse section), with intensity proportional to smFISH signal for EPCAM (red, epithelial), NR5A1 (cyan, gonadal somatic) and PAX8 (yellow, sPAX8 and epithelial) (n=2). ESGCs appear transiently in the early gonads (roughly 68PCW in humans and E11.5 in mice) and, in males, are the first gonadal somatic cells to express the testis-determining factor SRY, which is required for Sertoli cell commitment2 (Fig. 9ik). PreGC-IIa cells, present in the outer ovarian cortex, express chemoattractants (for example, NRG1) and survival factors (for example, KITLG), with STAT3 downstream of KIT active in PGCs (Fig. The resulting matrix was integrated by sample and origin of the dataset using scVI on the basis of the procedure described above. Science 369, 530537 (2020). SIGLEC15+ ftMs (white arrows) are outside the testis cords in proximity to endothelial cells (n=5). cDC = conventional Dendritic cells; ECM = extracellular matrix; ESGC = early supporting gonadal cells; ftM = fetal testicular macrophages; Gi = gonadal interstitial; ILC prec = innate lymphoid cell precursors; Mac = macrophages; Mast = mast cells; Mega = megakaryocytes; MEMP = megakaryocyte-erythroid-mast cell progenitors; Mono = monocytes; Neutro = neutrophil; NMP = neutrophil-myeloid progenitors; NK = Natural Killer cells; pDC = plasmacytoid Dendritic cell; PV = perivascular; PCW = post-conceptional week; Prec = precursor; Pre_B = pre-B cells; Pre_pro_B = pre-pro-B cells; Pro_B = pro-B cells; Prob_ = probability; sPAX8 = supporting PAX8; Gi = gonadal interstitial; T = T cells; Ti = testicular interstitial. Article & Vento-Tormo, R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Embryol. TF can bind their potential targets); a = TF whose targets are differentially expressed (i.e. All tissue samples used for this study were obtained with written informed consent from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. (right) 8 PCW testis stained for CD68 (red, macrophages), SIGLEC15 (yellow, SIGLEC15+ ftM) and P2RY12 (green, TREM2+ ftM); n=3. b, High-resolution imaging of representative sections of two fetal testes (11 and 12 PCW), with intensity proportional to smFISH signal for EPCAM (cyan, high = epithelial cells; low = Sertoli and germ cells), PDGFRA (green, mesenchymal cells), CD68 (red, macrophages), SIGLEC15 (yellow, SIGLEC15+ ftM); n=7. c, (top left) UMAP (uniform manifold approximation and projection) of myeloid cells from Guo et al., 2021 labelled by PCW. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. Predicted doublets were not excluded from the initial analysis, but used afterwards to flag clusters with high doublet scores. TF motif activities were computed using chromVar76 v.1.12.2 with positional weight matrices from JASPAR2018 (ref. SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. PubMed Coelomic epithelium forms the ovarian surface epithelium Subcoelomic mesoderm forms the ovarian stroma Primordial germ cells migrate from the yolk sac endoderm to the developing ovary Invaginations of coelomic epithelium in the superficial ovarian cortex form the sex cords (pregranulosa cells) 46, D260D266 (2018). LGR5 thus marks different populations in mice and humans, highlighting the need for humanmouse harmonized atlases. La Manno, G. et al. To compare the differentiation of human germ cells with that of other mammals, we integrated our human and mouse gonadal germ cells with more scRNA-seq gonadal germ cells datasets from mouse and macaque5,10,11 (Extended Data Fig. Korsunsky, I. et al. 134, 167194 (2019). The authors declare no competing interests. We then trained an SVM classifier (sklearn.svm.SVC) on human data and projected the cell type annotations onto the mouse datasets. The resulting matrix was further processed to average cells by cell type and peaks by CCAN. Multiplexed smFISH images are available from BioStudies (www.ebi.ac.uk/biostudies), with accession number S-BIAD393. Top-layer groups marker genes by categories. Both sPAX8 subsets show a unique transcriptional pattern of axon guidance factors, suggesting they have a structural and supporting role. Raw counts were normalized and log-transformed, and the 300 most highly variable genes were selected. Methods 15, 359362 (2018). Gut mesodermal tissues originate from the splanchnopleural mesenchyme. Maheshwari, A. 18/NE/0290). We called the peaks using an in-house implementation of the approach described in Cusanovich et al.63 (available at https://github.com/cellgeni/cellatac, revision 21-099). Fresh frozen sections were removed from 80C storage and air dried before being fixed in 10% neutral buffered formalin for 5min. Samples less than 17PCW were also digested using a combination of collagenase and Trypsin/EDTA, a protocol adapted from Wagner et al.50,51. Addressing gaps in care of people with conditions affecting sex development and maturation. https://doi.org/10.1038/s41593-020-0602-1 (2020). Hayman, A. R. et al. Article Colour proportional to scaled log-transformed expression. CoelEpi = coelomic epithelium; E = embryonic day; ESGC = early supporting gonadal cells; FGC = fetal germ cells; Gi = gonadal interstitial; Oi = ovarian interstitial; OSE = ovarian surface epithelium; P = postnatal day; PCW = post-conceptional week; PGC = primordial germ cells; preGC = pre-granulosa cells; TF = transcription factor; Ti = testicular interstitial; sPAX8 = supporting PAX8. For each sequenced multimodal snRNA-seq library, we performed read alignment to the 10X Genomics GRCh38 v.3.1.0 (human) reference genome, quantification and initial quality control using the Cell Ranger ARC Software (v.1.0.1, 10X Genomics) using default parameters. E = embryonic day; Expr = expressed, FGC = fetal germ cells; P = postnatal day; PCW = post-conceptional weeks; PGC = primordial germ cells. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Despite the similar spatiotemporal patterns in humans and mice, we show that certain regulatory programmes differ; for example, LGR5, characteristic of second-wave pregranulosa cells in mice5,38, is restricted to ESGCs in humans. These authors contributed equally: Luz Garcia-Alonso, Valentina Lorenzi, Luz Garcia-Alonso,Valentina Lorenzi,Cecilia Icoresi Mazzeo,Kenny Roberts,Carmen Sancho-Serra,Magda Marekov,Tong Li,Stijn van Dongen,Vladimir Yu Kiselev,Elena Prigmore,Omer Ali Bayraktar,Muzlifah Haniffa&Roser Vento-Tormo, Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK, Joo Pedro Alves-Lopes,Wolfram H. Gruhn&Azim Surani, Physiology, Development and Neuroscience Department, University of Cambridge, Cambridge, UK, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK, Justin Engelbert,Rachel A. Botting,Mary Herbert&Muzlifah Haniffa, Nuffield Department of Womens and Reproductive Health, University of Oxford, Oxford, UK, Great Ormond Street Institute of Child Health, University College London, London, UK, University of Cambridge Centre for Trophoblast Research, Department of Pathology, University of Cambridge, Cambridge, UK, Sorbonne Universit, INSERM, CNRS, Institut de la Vision, Paris, France, Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge, UK, You can also search for this author in Signs and symptoms may include weight loss, fever, malaise, cough, skin and mucosal lesions, diarrhea, and peripheral neuropathy. To speed up computational times, we downsampled our dataset to 50% size.

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