Authors also reported that AFF4 deficiency promotes cancer cell invasion and . and V.L. Google Scholar. Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice. Google Scholar. In brief, cells were subsampled to balance cell type and sex contribution (n=500 for germ and n=150 for somatic cells). . Science 367, eaay3224 (2020). 7d). CAS c, Dot plot showing the variance-scaled, log-transformed expression of genes characteristic of the mesothelial, supporting and epithelial subpopulations in human first trimester (top) and mouse E10.5-E12.5 (bottom) scRNA-seq data. Guo, J. et al. Kleshchevnikov, V. et al. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis. All authors read and approved the manuscript. Furthermore, there is upregulation of HOX factors (for example, HOXA3, HOXD8) and cofactors (for example, PBX3) in distinct oogonia stages. It constitutes the outermost layer of the male and female gonads, thus forming the germinal epithelium of the female or of the male. Channels were as follows: DAPI (excitation 375nm, emission 435480nm), Atto 425 (excitation 425nm, emission 463501nm), Opal 520 (excitation 488nm, emission 500550nm), Opal 570 (excitation 561nm, emission 570630nm) and Opal 650 (excitation 640nm, emission 650760nm). Doublets and low QC cells removed. Development 122, 31513162 (1996). Kracht, L. et al. 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. 2e and Extended Data Fig. Nagaoka, S. I. et al. Niu, W. & Spradling, A. C. Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. Preprint at bioRxiv https://doi.org/10.1101/496521 (2018). Witschi, E. Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal folds. Nat. Mayre, C. et al. 70). Minkina, A. et al. The results of the activity of Venetin-1 against normal lung epithelial cells . Biol. 2d), downregulating RSPO1/WNT4 (Fig. c, Barplot showing the proportions of human (top) and mouse (bottom) cells profiled with scRNA-seq coloured by lineage and classified by sex and developmental stage (indicated in post-conceptional weeks (PCW) or embryonic (E) / postnatal (P) days). Hern, W. M. Correlation of fetal age and measurements between 10 and 26 weeks of gestation. Ovarian carcinoma is the fifth leading cause of death from female cancers [] and comprises several malignancies of epithelial and non-epithelial origins.Epithelial ovarian carcinoma (EOC) is the most predominant type, which, in turn, encompasses several distinct histotypes that are thought to originate in epithelial cells of the female reproductive tract [2,3,4]. Tang, W. W. C. et al. 5d) and express COL1A2, which can potentially interact with the integrins (1/1, 2/1, 10/1 and 11/1) expressed by endothelial and mesenchymal cells (Extended Data Fig. Article After being rinsed with deionized water, slides were dipped in Mayers haematoxylin solution for 90s. Slides were completely rinsed in 45 washes of deionized water, which also served to blue the haematoxylin. Fast, sensitive and accurate integration of single-cell data with Harmony. Colour proportional to scaled log-transformed expression. 2e and Extended Data Fig. 10b). Biotechnol. 1c and Extended Data Fig. Embryos were genotyped to identify the gender. White dashed rectangles highlight enlarged gonadal regions with PAX8high EPCAMlow expression. Latent semantic indexing was clipped at 1.5. For samples at the time of sex specification, we computed RNA velocities61 to model early somatic development with scVelo (v.0.2.4)62 following their tutorial. We uncover a unique composition of extracellular matrix proteins in follicles (Extended Data Fig. Ottolenghi, C. et al. Extended Data Fig. This single space wall will undergo a mesenchymal epithelial transition. SIGLEC15+ ftMs are found in the peritubular spaces surrounding the testis cords, which might aid with mesonephric endothelial cell migration36. An example is netrin-1 (NTN1) and its receptor DCC, which are involved in axon guidance, cell migration and apoptosis (Extended Data Fig. Analysis was done on each sample separately in humans and mice. Next, we refined the annotation accounting for the spatiotemporal dynamics in each sex. In addition, we downloaded a macaque dataset profiling fetal ovaries at stages E84 and E116 (GSE149629)11 and included it in our cross-species comparison of germ and female somatic cells. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. CAS At early 8PCW (Carnegie stage (CS)19CS20), ESGCs (TSPAN8+, LGR5+) reside in the ovarian medulla together with the preGC-I (OSR1+) in females, or the developing testis cords with early Sertoli cells (SOX9+, LGR5) in males. Finally, to identify TF whose activity was upregulated in a specific cell type, we applied the Wilcoxon Rank Sum test from Seurat onto the z-transformed cellTF activity matrix in a one-versus-all fashion. CoelEpi = coelomic epithelium; E = embryonic day; Endo = endothelial; Epi = epithelial; FGC = fetal germ cells; P = postnatal day; PCW = post-conceptional weeks; SMC = smooth muscle cells; Soma = somatic; PV = perivascular; Mese = mesenchymal. Article Cicero predicts cis-regulatory DNA interactions from single-cell chromatin accessibility data. Genome Biol. 8i), as well as new granulosa-to-oocyte interaction candidates for mediating successful follicular assembly (Fig. Folliculogenesis in the adult ovary is characterized by extensive architectural remodeling that culminates in disruption of the coelomic epithelium and extrusion of the ovum at ovulation ( 1, 2 ). Cold Spring Harbor Laboratory https://doi.org/10.1101/2020.11.15.378125 (2020). & Hermoin, L. in Encyclopedia of Reproduction (ed. 91, 403407 (2000). The abundant number of cells profiled in our study allowed us to resolve new somatic cell states, which were not defined in a previous human gonadal scRNA-seq study7 (Extended Data Fig. Mol. Zoomed-in UMAPs show osteoclast and microglia signature macrophages labelled by tissue of origin. coelom [selom] body cavity, especially the cavity in the mammalian embryo between the somatopleure and splanchnopleure, which is both intra- and extraembryonic; it develops into the pleural, peritoneal, and pericardial cavities. Dashed lines outline the cell populations unique to the gonads. 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. m, Dot plot showing variance-scaled, log-transformed expression of immunoregulatory markers in human gonadal macrophages. & Hemberg, M. scmap: projection of single-cell RNA-seq data across data sets. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Our in-depth analysis of human samples covering a broad developmental window allowed us to distinguish two subsets of sPAX8s in humans: early and late sPAX8s. 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. Robustness and applicability of transcription factor and pathway analysis tools on single-cell RNA-seq data. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. 35, D127D131 (2007). Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohns disease. We gratefully acknowledge the Sanger Cellular Generation and Phenotyping Core Facility, Sanger Core Sequencing pipeline and The Flow Cytometry Core Facility from Newcastle University for support with sample processing and sequencing library preparation; F. Ahmed, J. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Specific interacting partners are linked with a matching symbol. protocols.io https://doi.org/10.17504/protocols.io.bwcipaue (2021). TFs whose upregulation is conserved across species are highlighted with bold/coloured labels. cDC, conventional dendritic cells; ftM, fetal testicular macrophages; ILC, innate lymphoid cells; mega, megakaryocytes; MEMP, megakaryocyte-erythroid-mast cell progenitors; mono, monocytes; neutro, neutrophils; NMP, neutrophil-myeloid progenitors; NK, natural killer cells; pDC, plasmacytoid dendritic cell; prec, precursor; Pre-B, pre-B cells; Pre-pro-B, pre-pro-B cells; Pro-B, pro-B cells; prog, progenitor; T, T cells. Nat. Dev. Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. Source data are provided with this paper. Next, we reprocessed the somatic cells (only cells at G1 phase) from each sample independently, performed PCA on the top 2,000 highly variable genes, neighbour identification and UMAP projection to visualize previously annotated cell types. PubMedGoogle Scholar. Methods 16, 12891296 (2019). The whiskers extend from the box to show the range of the data. Dev. We carried out downstream analysis as previously described for UMAP visualization. 5d and Extended Data Fig. Endocrinol. e, Heatmaps showing the expression of human-relevant transcription factors (TF) in human, macaque and mouse germ cells. 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. c, t-SNE (t-distributed stochastic neighbour embedding) projection of somatic cells coloured by Palantir pseudo-time and probability of cells to progress from the gonadal coelomic epithelium GATA4+ in humans between 6-8.5 PCW (left) and mice at E10.5-E11.5 (right). 10d,e), where they are predicted to communicate with Sertoli and germ cells by the interaction between TREM2 and apolipoproteins (CLU, APOA1, APOE) (Extended Data Fig. Dev. To identify marker genes specific to a cluster, we used the TF-IDF approach from the SoupX package v.1.5.0 (ref. b, Dot plot showing variance-scaled, log-transformed expression of marker genes (y-axis) for the identified macrophage subsets (x-axis). For all smFISH panels, unless otherwise specified, white dashed rectangles highlight gonadal regions magnified; scale bars = 100m and 10m in magnified regions; developing testis cords are delineated with dashed lines. In breast cancer, tumor differentiation is related to genetic status, i.e., breast tumors . 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). We computed differential expression using the one-sided Wilcoxon Rank Sum test implemented in the FindAllMarkers function with Seurat v.3.2.2, in a one-versus-all fashion. sPAX8s are located at the site where the rete testis will form in the testis, as shown by Visium (Extended Data Fig. In humans, the undifferentiated bipotent gonads, which emerge on the ventral surface of the mesonephros, commit to ovarian or testicular fate. PreGC-IIa coappear in space (outer cortex) and time (mid-8PCW) with OSE (UPK3B+, LHX2+, IRX3+), and express the retinoic acid inhibitor CYP26B1 (meiosis inhibitor) as well as low amounts of FOXL2. ISSN 1476-4687 (online) Article 5f,g). Using a revised version of CellPhoneDB, we show that the spatial microenvironments defined by the distinct pregranulosa cell subsets in human ovaries regulate germ cell development. a, High-resolution large-area imaging of representative gonadal sections (transverse) of a human ovary (7PCW, CS19; top) and testis (8PCW, CS20; bottom), with intensity proportional to smFISH signal for EPCAM (red, epithelial), NR5A1 (cyan, gonadal somatic) and PAX8 (yellow, sPAX8 and epithelial) (n=2); red blood cells appear as bright autofluorescent cells. Park, J.-E. et al. Mol. Roser Vento-Tormo. 2d). c, UMAP projections of immune cells labelled by sex, PCW and donor. We defined two testis-specific macrophage populations using scRNA-seq and validated them with smFISH: (1) SIGLEC15+ fetal testicular macrophages (ftMs), with an osteoclast-like signature (SIGLEC15, ACP5, ATP6V0D2; refs. e, Barplot showing the proportions of somatic cells in the Guo et al., 2021 dataset coloured by cell state and classified by PCW. & Saint-Jeannet, J.-P. Zic1 controls placode progenitor formation non-cell autonomously by regulating retinoic acid production and transport. LGR5 shows a different expression pattern between humans and mice: in humans, LGR5 is specific to ESGCs; in mice, Lgr5 is upregulated during the second wave of pregranulosa and Sertoli cell formation, with basal expression in ESGCs (Extended Data Fig. Clusters for mesothelial, supporting and gonadal mesenchymal LHX9+ cells were defined in an independent per-lineage reanalysis and projected onto this dataset (Fig. d, Dot plot showing the variance-scaled, log-transformed expression of transcription factors (TFs) with mutually exclusive expression in gonadal and extragonadal mesenchymal and mesothelial cells in human and mouse scRNA-seq data. 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. For the first time, to our knowledge, these markers uniquely expressed by the bipotent supporting progenitor population are defined in humans. 836291) and BBSRC. Sections were cut from fresh frozen or fixed frozen samples embedded in OCT at a thickness of 10m using a cryostat, placed onto SuperFrost Plus slides (VWR) and stored at 80C until stained. Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Reprod. Nature 589, 264269 (2021). OSE activates the primate-specific TF LHX2, which is kept active in preGC-IIa (Fig. To account for the distinct spatial location of cells, we further classified the cells according to their location in the developing ovaries (outer cortex, inner cortex, medulla) as observed by Visium and smFISH. After entering meiosis, oogonia activate DMRTC2 and ZNF711, previously described in mice, together with another DMRT member, DMRTB1, which is analogously upregulated in macaque and mouse oogonia. Nat. 37, 451460 (2019). analysed the data with contributions from T.L., S.D. Gynecol. Li, L. et al. f, Dot plots showing the variance-scaled, log-transformed expression of genes (X-axis) characteristic of ovarian supporting cells (Y-axis) in mouse (left) and macaque (right) scRNA-seq data. Gigascience 9, giaa151 (2020). Gelb, B. D., Shi, G. P., Chapman, H. A. CAS The coelomic hypothesis shown on the left argues that ovarian epithelial tumors arise from coelomic epithelium or its derivatives in cortical inclusion cysts after it has undergone. Signs and symptoms. Google Scholar. Developing granulosa cells in primates upregulate the steroid hormone receptor NR1H4 and the developmental factor PBX3. In humans, we also define a second wave of granulosa cells (preGC-IIa/b) appearing after 8PCW (Fig. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. b, UMAP (uniform manifold approximation and projection) of female (top) and male (bottom) human scATAC-seq datasets labelled by donor and post-conceptional weeks (PCW). Nat. b, Heatmap reporting label transfer scores from human scRNA-seq to scATAC-seq somatic cell data of matched individuals. As reference, we used scRNA-seq data from individuals of the same sex and gestational stage. b, Gating strategy to sort immune cells in gonadal samples for a representative donor (F93). [8-10], the combination of coelomic fluid with 5-fluorouracil has anticancer potential for colorectal cancer. 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. Science 369, 530537 (2020). Sancho, C., Hoo, R. & Vento-Tormo, R. Human embryonic gonad dissociation with Collagenase & Trypsin v3. 48, 838847 (2016). 326, 112120 (2009). Doublets and low-quality control cells were removed. White dashed rectangles highlight gonadal regions magnified; scale bars, 100 and 10m in magnified regions; testicular developing cords are delineated with dashed lines. We then trained a SVM classifier (sklearn.svm.SVC) on the filtered liver dataset and used it to predict cell types on our gonadal immune dataset. d, High-resolution imaging of representative human gonadal sections with intensity proportional to smFISH signal for RNA markers. PubMed Central Figure 1 Diagram of ovarian and tubal anatomy depicting the coelomic and Mllerian models of ovarian cancer development. Mesothelioma is a cancer of the mesothelium, . 18/LO/0822) and Newcastle and North Tyneside 1 Research Ethics Committee (REC reference no. e, High-resolution large-area imaging of a representative section of a mouse fetal ovary (E13.5) with intensity proportional to smFISH signal for Lgr5 (yellow, cortical pre-granulosa), Pax8 (red, sPAX8), Hmgcs2 (green, medullary pre-granulosa) and Gng13 (magenta, cortical pre-granulosa); n=2. f, High-resolution large-area imaging of representative sections of three human fetal testes (sagittal late 8, transverse 11 and transverse 12 PCW; n=3) with intensity proportional to smFISH signal for PAX8 (yellow, sPAX8 population), NR5A1 (cyan, interstitial Fetal Leydig), EPCAM (red, low in supporting cells, high in epithelial cells of the reproductive tubules) and KLK11 (green; coelomic epithelium). 56 implemented in Scanpy score_genes_cell_cycle function. 1b and Supplementary Tables 13). We then trained an SVM classifier (sklearn.svm.SVC) on human data and projected the cell type annotations onto the mouse datasets. Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Flyer points are those past the end of the whiskers. For both germ and early somatic cells, we modelled differentiation trajectories and conducted pseudotime analysis by ordering cells along the reconstructed trajectory with Palantir (v.1.0.0)60 following their tutorial. Labels were transferred using scmap separately for females (left) and males (right), with a cutoff of 0.5. Dots from the same donor share a colour. We uncover previously uncharacterized cellular heterogeneity in the somatic lineage, with relevance for gonadal conditions that have their origin during development, such as differences in sex development. 6, 7476 (2015). The HDBR is regulated by the UK Human Tissue Authority (www.hta.gov.uk) and operates in accordance with the relevant Human Tissue Authority Codes of Practice. Hayman, A. R. et al. The resulting matrix was integrated by sample and origin of the dataset using scVI on the basis of the procedure described above. Before training the model, human cell types were downsampled to the cell type with the lowest number of cells to obtain a balanced dataset. We combined our in-house mouse raw counts matrix with the raw count matrices from the ovarian samples profiled by Niu et al.5, comprising E11.5 to P5 developmental stages (GSE136441)5. In the analysis of human scRNA-seq, we corrected for sample source and donor effect in both the main and the germ and somatic reanalysis. Using Signac (https://satijalab.org/signac/ v.0.2.5), the binary matrix was normalized with TF-IDF followed by a dimensionality reduction step using singular value decomposition. 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. e, Spatial plot showing the variance-scaled, log-transformed expression of TF with mutually exclusive expression in gonadal and extragonadal mesenchymal cells over the H&E images of a late 8 PCW testis, a 12 PCW testis and an 11PCW ovary; n=2. Germ (DAZL+) and supporting (GATA4+, WNT6+) cells are present exclusively within the gonads, whereas other cell types, including coelomic epithelial (UPK3B+) and mesenchymal (PDGFRA+) cells, are present in both the gonads and the mesonephros (extragonadal tissue) (Fig. Google Scholar. We prioritized those TFs that were differentially expressed and active in humans using both the transcriptome and open chromatin data (Extended Data Fig. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. A cell atlas of human thymic development defines T cell repertoire formation. 1c. Subsequently, the automated processing for these sections included heat-induced epitope retrieval at 95C for 5min in buffer ER2 and digestion with Protease III for 15min before probe hybridization. C.I.M., J.P.A.-L., K.R. Knoblaugh, S. E., True, L., Tretiakova, M. & Hukkanen, R. R. in Comparative Anatomy and Histology (eds. Google Scholar. h, (left) Dot plot showing the scaled log-transformed expression of upregulated genes coding for sPAX8 ligands or receptor proteins in the supporting testis cells. 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. c, Heatmap reporting label transfer scores from human scRNA-seq to scATAC-seq female (left) and male (right) data of matched individuals. CAS HGSC is distinct from low-grade serous carcinoma (LGSC) which arises from ovarian tissue, is less aggressive and is present in stage I ovarian . 13, 206218 (2013). The white dashed rectangle highlights the enlarged gonadal region. Supplementary Tables 111 and legends to the tables. Identification, labelling and naming of the unbiased clusters was carried out on each species individually using a manual approach that we validated using a SVM classifier (see Cross-species comparison section below). The ovarian coelomic epithelium covers the ovary as a layer of squamous or cuboidal cells. TF motif activities were computed using chromVar76 v.1.12.2 with positional weight matrices from JASPAR2018 (ref. Hiort, O. et al. 31, 28412855 (2016). The resulting latent representation of each cell in the dataset was used for neighbour identification, Leiden clustering and uniform manifold approximation and projection (UMAP) visualization. Extended Data Fig. ADS Coloured symbols indicate the initial and terminal cell states predicted by CellRank. performed sampling and library prep. 4b and Supplementary Note 6). 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. Tissue-resident macrophages have a role in mouse testicular development and function22,23. (right) UMAP projections of myeloid cells from Guo et al., 2021 showing the expression of SIGLEC15+ ftM marker genes. For the scRNA-seq experiments, cells were loaded according to the manufacturers protocol for the Chromium Single Cell 5 Kit v.1.0, v.1.1 and v.2 (10X Genomics) to attain between 2,000 and 10,000 cells per reaction. During embryonic development, the coelomic epithelium covering the intermediate mesoderm thickens and gives rise to the urogenital ridges, the precursors of the urogenital system (Fig. Mouse fixed frozen sections were subjected to the same manual pretreatment described above. A unique gene regulatory network resets the human germline epigenome for development. d, High-resolution large-area imaging of a representative section of a full male embryo (Carnegie Stage CS19), with intensity proportional to smFISH signal for CD68 (green, macrophages), P2RY12 (red, TREM2+ ftM and microglia), ELAVL3 (cyan, neural cells). Top. 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). Developing ovaries, testes and mesonephros were collected from E10.5, E11.5 and E12.5 mouse embryos carrying the Oct4PE-GFP transgene. We define a supporting-like PAX8+ population (sPAX8s) expressing gonadal (GATA4+, LHX9+ and NR5A1+) and supporting (WNT6+) markers that emerges with the first wave of supporting cells in humans and mice (Fig. The top 2,000 highly variable genes were used for PCA. In brief, we excluded genes expressed by fewer than three cells and excluded cells expressing fewer than 500 genes (or 2,000 genes in mouse), more than 20% mitochondrial content (5% in mouse) or with both more than 10% mitochondrial content and fewer than 1,500 counts correctly mapped to the transcriptome. We set the number of terminal states to four, letting CellRank determine the number of initial states. Each Visium section was analysed separately. 6 Human-mouse comparison and trajectory inference of early gonadal somatic cells.
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