and JavaScript. Neuron 96, 10241032 (2017). Curr. Yu, G., Wang, L. G., Han, Y. ISSN 1546-1718 (online) We agree that this was hard to discern. Thus, forward genetic screens have identified very few genes required for gonad development. Song, H., Stevens, C. F. & Gage, F. H. Astroglia induce neurogenesis from adult neural stem cells. Bioinformatics 30, 923930 (2014). 47, D701D710 (2019). Protoc. This may indicate that the theca cells in our dataset represent at least two developmental states: star- immature and star+ mature theca cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. We reasoned that the partial penetrance of the phenotype could be due to the function of an additional Wnt ligand(s). It therefore follows that the expression of Hsd17b1 vs. Hsd17b3 will determine pathway preference. (I) Cell number quantification of individual cysts that express the genes indicated on Y-axis. To explore this further, we performed additional recluster analysis using only those cells derived from follicle cell subclusters 1 and 3 (Figure 4figure supplement 2A and B and Supplementary file 4). Cipriani, S. et al. Association of amyloid pathology with myelin alteration in preclinical Alzheimer disease. Sci 31, 445454 (2006). Nat. Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary. In most organisms, oocyte progenitor cells undergo several rounds of mitotic amplifying division before entering meiosis (Pepling et al., 1999). The best-characterized somatic cell type in the zebrafish ovary are the follicle cells. Consistent with our hypothesis, we found that lhx9+ cells do not localize around oocytes, but instead formed cord-like structures that are located on the surface of the ovary (arrows in Figure 4F). However, the Results section is much too interpretative. From our initial clustering, we identified a large population of cells that had enriched expression of several collagen encoding genes, such as col1a1a (Fisher et al., 2003; Figure 1A) and reasoned that these were likely the stromal cell population. The fibroblast-like interstitial stromal cells play a largely structural role, but the roles of the other stromal cell types in ovarian development and function are not well characterized in any vertebrates. 373, 693709 (2018). Cell Stem Cell 22, 589599 (2018). In the Entrez search bar enter the query: ((("mus musculus"[Organism]) AND BALB/c*) AND "lymph*") AND "rna seq"[Strategy]. Bond, A. M., Ming, G. L. & Song, H. Adult mammalian neural stem cells and neurogenesis: five decades later. c, Representative images of (left) Neurofilament (red), MBP (green), and Hoechst (blue), and (right) Neurofilament (red), O4 (green), and Hoechst (blue) immunoreactivity in iPSC-derived neuron and oligodendrocyte co-culture, after six weeks encapsulated. All zebrafish used in this study were raised in a recirculating aquatic system (Aquaneering, Inc, San Diego, CA), with the following water parameters: water temperature, 2829C; pH, 7.07.2 (maintained with Sodium Bicarbonate); conductivity, 600700 S (maintained with Crystal Sea Marine Mix, Aquaneering, CA); ammonia, 0 ppm; nitrate, 4060 ppm; water exchange, 10% total volume/day; water source, deionized water. e, Cell-type-specific marker gene expression projected onto two-dimensional representation of cell space. Quantification was performed using ImageJ. and A.E. As a result, approximately 13,000 nodule-specific contigs were found and annotated by alignment to known plant protein-coding sequences and by Gene Ontology searching. Biol. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Proc. Cells within subclusters 0 and 4 have nearly identical gene expression profiles, with the exception that subcluster 4 cells also have strong expression of genes associated with cell cycle progression, such as pcna (Figure 6A and B). Altered human oligodendrocyte heterogeneity in multiple sclerosis. Bartsch, T. & Wulff, P. The hippocampus in aging and disease: from plasticity to vulnerability. 151164, Chapter 16 - The Reproductive System, In The Zebrafish in Biomedical Research, Academic Press. Cell Stem Cell 17, 329340 (2015). dbEST (Nature Genetics 4:332-3;1993) is a division of GenBank that contains sequence data and other information on "single-pass" cDNA sequences, or "Expressed Sequence Tags", from a number of organisms. Importantly, this reference dataset will facilitate the use of zebrafish to study female reproductive diseases and disorders of sexual development. Nature 570, 332337 (2019). Regardless, cxcl21a provides a convenient marker for determining the location of this cell population in the ovary. Replication. In this study we focused our analysis on identifying cell-stage specific markers, such as foxl2l. Significant colocalizations (fastENLOC, Regional colocalization probability, rcp>0.5) between cis-eQTLs from 22 tissues and 25 agronomic traits. Cartoons generated with BioRender d, Two-dimensional representation of all high-quality cells included in downstream analysis labelled by major cell types. Perhaps surprisingly, no adapter sequences were found in the assembly of the untrimmed version of this dataset. We found that genes within these two modules were enriched for putative binding motifs of transcription factors Bhlhe23, early growth response 4 (Egr4), and pre-B-cell leukemia homeobox 3b (Pbx3b) (Figure 2figure supplement 5A, D). The red dash line indicates the median value of 0.1. Qian Wu, Chao Ma or Xiaoqun Wang. If you experience issues using our site with Internet Explorer, Transcriptome Shotgun Assembly: 49: 1,672,820: Sequence Assemblies PlantGDB-assembled unique transcripts (PUT) and In addition, pum1 is predominantly expressed in GSCs while pum3 is expressed more uniformly in early oocytes (Figure 2figure supplement 3). (a) The number of publicly available RNA-Seq samples increases rapidly over years by fitting a second order polynominal model. Figure 8. J. Neurosci. (d) The cis-eQTL cumulative proportion plot of |log2(aFC)| distribution across 9 tissues with variable sample sizes. (b) The percentage of unexpressed genes (TPM<0.1 across all samples) on known chromosomes (Known) and unplaced scaffolds (Unplaced, 54.10%). In some cases, these compounds are transported to the oocyte via connexin-mediated gap junctions (Su et al., 2008). JAMA Neurol. Nat. Article Pericytes are generally solitary cells associated with small diameter blood vessels (arterioles, capillaries, and venules) while vascular smooth muscle cells (vSMCs) are associated with large blood vessels where they form a continuous coating (Gaengel et al., 2009). To determine sex ratios of wnt9b mutant fish, 50 dpf fish produced from wnt9b+/- incross were fixed in 4% paraformaldehyde-1 PBS overnight at 4C and stored in methanol at 20C at least overnight. Methods 14, 979982 (2017). 13, eaaz4564 (2021). Together, these data argue that gonad development in vertebrates is regulated by a largely conserved genetic program. To limit your search to only Heger, A., Webber, C., Goodson, M., Ponting, C. P. & Lunter, G. GAT: a simulation framework for testing the association of genomic intervals. Nature 541, 481487 (2017). Neuron 70, 908923 (2011). Cell Tissue Res. Immunol. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#20200 and #20201) of the University of California, Davis. PubMed Central In mammals, retinoic acid signaling promotes germ cells to enter meiosis (Bowles et al., 2006; Koubova et al., 2006). were supported by appropriated project 8042-31000-001-00-D, Enhancing Genetic Merit of Ruminants Through Improved Genome Assembly, Annotation, and Selection of the Agricultural Research Service (ARS) of the USDA. Cell 6, 13551364 (2000). Neuron 101, 207223 (2019). However, this probe has so far failed to work. Wang, Y. et al. You are using a browser version with limited support for CSS. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. This raises the possibility that Pum1 may partner with Nanos2 while Pum3 partners with Nanos3, though it is also possible that Nanos2 could form complexes with both Pum1 and Pum3. c, Representative images of cultures of iPSC-derived oligodendroglia stained for PLP1 and MBP. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. Front. Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Sachdeva, R. et al. Scale bar represents 50m. Bioinformatics 29, 20462048 (2013). Schneid (Chuansang). Bioinformatics 28, 13531358 (2012). 3 DNA methylation profiles of 144 WGBS samples. The suspension was monitored every 10 min until no or a minimal number of cell clumps were observed (~30 min). We identified 32 gene modules (note: modules 8, 13, 19, and 29 were determined to be low-quality gene modules and therefore not considered in our analysis; see Materials and methods), and many of the gene modules showed stage-specific enrichment during germ cell development. WebPicard. For example, we now state that dmc1 is expressed in Stage 1A pre-follicle phase germ cells and that zp3b is expressed in Stage1B follicle phase oocytes. Second-generation PLINK: rising to the challenge of larger and richer datasets. July 22, 2022. 2008 - 2022 Oxford Nanopore Technologies plc. Bayesian test for colocalisation between pairs of genetic association studies using summary statistics. 12, 10971105 (2009). (c) An example of a colocalization of cis-eQTLs of TIGAR gene in muscle and GWAS loci of strength in cattle on chromosome 5. We noticed that many of the genes with enriched expression in subcluster 3 did not appear uniformly expressed in all cells within this subcluster. g, Fold change for unfolded protein response genes in APOE4/4 vs APOE3/3 oligodendroglia (N=3 per genotype) from panel e (adjusted p-value<0.05, negative binomial distribution). (f) Pearson correlation of effect sizes (beta values) of trans-eQTLs (n=5,782) in blood tissue in Group1 (G1, n=349) and those of matched SNPs in Group2 (G2, n=349) (r=0.66, the two-sided Students t-test: P<2.210308). Nat. Comprehensive analyses of 723 transcriptomes enhance genetic and biological interpretations for complex traits in cattle. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins. The following transgenic lines were also used: Tg(fli1:egfp)y1 (Lawson and Weinstein, 2002); Tg(kdrl:dsRed)pd27 (Kikuchi et al., 2011); and Tg(acta2:eGFP)ca7 (Whitesell et al., 2014). Commun. BMC Bioinform. ID GSE191137. (D) gpc1a expression (pink) is detected in follicle cells surrounding stage IB oocytes, but not stage II oocytes. We found that stm was specifically expressed in cells that localize to the medial and lateral edges of the 40 dpf ovary, a region that also contained nanos2+ GSCs and dmc1+ meiotic germ cells (Figure 6FF, Figure 6figure supplement 4). Extended Data Fig. Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Mathys, H. et al. Mira, H. et al. At 5 dpf, zebrafish larvae are placed into 1 l aquaria containing 250 ml 4 parts per thousand (ppt) seawater (Crystal Sea), at no more than 30 fish/tank. Proc. b, Staining of MBP, MYRF, and MOG in iPSC-derived oligodendroglia. Song, Q. et al. Article Tissue was collected from each fish during dissection for genotyping post-dissection. Get immediate access to your DNA sequencing results with real-time data streaming. Neuron 104, 834848 (2019). Additionally, there is significant expression of wnt8a (Kelly et al., 1995) in oocytes, wnt9a in stromal cells, wnt9b (Crotwell and Mabee, 2007) in follicle cells, and wnt11 (Makita et al., 1998) in stromal and theca cells (Figure 5figure supplement 1). Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. (E) and (F) are higher-magnification views of regions boxed in (D) and (F), respectively. (DG) Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) on whole-mount 40-day post-fertilization (dpf) ovaries reveals the location of cell subtypes. Do other markers of mitosis display the same pattern? Graphic illustrations were generated using BioRender under agreements VI24HP0GQM and NE24HPHF0X. (B) Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) on whole-mount 40-day post-fertilization (dpf) ovaries reveals the location of cyp17a1-expressing theca cells. The top 25% of the cluster genes (sorted by p-value) were used for GO analysis inputs. Identified gene modules were then filtered to remove low-quality modules if a reproducibility score was lower than 0.6 or the gene module consisted of less than 10 genes. Analysis scripts and data for the cell cluster analysis are available on GitHub (https://github.com/yulongliu68/zeb_ov_ssRNAseq, copy archived at swh:1:rev:3430147079ab3840afdb725b01652fcaeda5f78d; Liu, 2022), and the final clustering can be explored online at the Single Cell Portal (The Broad Institute of MIT and Harvard, https://singlecell.broadinstitute.org/single_cell/study/SCP928). Browse the archive of articles on Nature. Briefly, the expression matrices generated from Cell Ranger (germ cell library zx1_40gc, whole ovary libraries zx2_40ov and zx4_40ov) were first processed with SoupX (v0.3.1; Young and Behjati, 2020) to remove ambient RNA. Scale bar represents 10m. PubMed 46, 100106 (2014). h, Relative expression of cholesterol-associated genes across iPSC-derived oligodendroglia, post-mortem human brain oligodendrocytes, and additional brain cell types. 127, 624633 (2017). CAS Oocyte and follicle cell development is coordinated through bidirectional oocyte-follicle cell-cell interactions (Kidder and Vanderhyden, 2010). 200 l of 20 mg/ml of type 2 collagenase in L15 (Cat# NC9870009; Worthington) were added and incubated on an orbital rotator at 28C for 35 min. Disord. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Korsunsky, I. et al. It is possible that other probes targeting a different region of pcolcea would work. Interestingly, macrophage have been shown to play an important role in regulating spermatogenesis in the mouse testis (DeFalco et al., 2015). Sci. We focused our attention on wnt9b because its apparent expression in follicle cells was strikingly similar to that of wnt4 (Figure 5A). WebFor access to the most recent assembly of each genome, Affymetrix transcriptome phase 3 data; Regulatory potential data; GC percent data; SNP-masked FASTA files. The first and rate-limiting step in E2 synthesis in mammals is the transfer of cholesterol from the outer to inner mitochondrial membrane by steroidogenic acute regulatory protein, StAR, where it is converted to pregnenolone by the cytochrome P450 cholesterol side chain-cleaving enzyme, Cyp11a1 (Miller and Auchus, 2011). Transcriptome profiling. Front. Neurol. The adjusted datasets were then processed with DoubletFinder (v2.0.2; McGinnis et al., 2019) to determine and remove doublets that are expected in any large-scale single-cell RNA-sequencing datasets. Google Scholar. While genetic mosaic analysis established that nanos3 was required cell-autonomously for GSC maintenance (Beer and Draper, 2013), to date nanos3 expression in premeiotic germ cells in the ovary has not been demonstrated. Proc. See also the point below. Brandt, M. D. et al. The information from this analysis will allow us to build more accurate comparative gene regulatory networks between zebrafish and humans, which can be experimentally tested in the genetically tractable zebrafish. Acta Neuropathol. Groups were identified by K-means clustering on the non-zero detection rate. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). (l) Distribution of gene types for those without spliced introns. The second population appears to express lower levels of lhx9 and envelop the early-stage germ cell clusters on sides that do not contact the cords (arrowheads in Figure 4F and F). we present a compendium of the relevant state-of-the-art-tools. WebTranscriptome Assembly. Neuroscientist 17, 7993 (2011). Details of RNA-seq, WGBS and WGS can be found in Supplementary Tables 1, 2 and 15, respectively. Thus, we have molecularly characterized the theca cell population in the zebrafish ovary and confirmed that, as in mammals, theca cells are the probable source of the E2 precursor, androstenedione. M.W., B.Z., X.J.,and J.Z. Rev. This data has allowed us to identify all known stages of germ cells, from GSCs to pre-follicle stage oocytes (stage IA; Selman et al., 1993), as well as the somatic cell populations, including follicle, theca, and stromal cells. Bioinformatics 25, 17541760 (2009). However, after several attempts we have not observed convincing signal. Jindal, A., Gupta, P., Jayadeva & Sengupta, D. Discovery of rare cells from voluminous single cell expression data. conceived the project. Interestingly, there appear to be two populations of lhx9+ cells, based on expression level and location. Shabalin, A. van Praag, H., Kempermann, G. & Gage, F. H. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Genet. Cell Rep. 21, 32713284 (2017). Each detected lipid species is organized according to lipid class, with cholesteryl esters having the highest number (15) of differentially expressed species. Cell Rep. 26, 394406 (2019). We used the 10X Genomics platform for library preparation and sequencing. The cells were centrifuge for 3 min at 300 g and the supernatant carefully removed. Miller, J. Subcluster 0 contains most of the follicle cells in our dataset, which are characterized by the enriched expression of several genes, including gap junctional protein alpha 11 (gja11; formerly cx34.5; Mikalsen et al., 2020; Figure 4AC). Luo, Y. et al. From day 12 to 20 dpf, each tank of larvae is fed two drops concentrated brine shrimp nauplii, twice daily. Cell viability and number were determined using propidium iodine (Cat# P1304MP; Thermo Fisher) and Hoechst 33342 (Cat# H3570; Thermo Fisher) staining on a Fuchs-Rosenthal hemocytometer (Cat# DHC-F01; Incyto). Significant TWAS results from S-MultiXcan (Bonferroni corrected P<4106) aggregated across tissues. Science 358, 13181323 (2017). (E) Higher magnification of the region boxed in (E). The number of lipid droplets was quantified in four 1m2 areas per image from three images per mouse. Mizrak, D. et al. p value was calculated using an unpaired, two-tailed students t-test. Libraries were sequenced on a HiSeq 4,000 sequencer (Illumina) with paired-end 100 bp reads. ID SCP928/40dpf-ovary-all-cells. Whole genome sequence assemblies should be submitted to WGS. Article PubMed Central As a first step, we performed nonzero matrix factorization (NMF) to find gene modules, which are sets of genes that are co-expressed within cell clusters and subclusters and may therefore identify co-regulated genes (Figure 2figure supplement 5; Brunet et al., 2004; Farrell et al., 2018; Siebert et al., 2019). was supported by the National Science Foundation of China-Pakistan Science Foundation Joint Project (31961143009) and National Key R&D Program of China (2021YFD1200900 and 2021YFD1200903). Supplementary Table 6. However, unlike mammals, zebrafish females can produce new follicles throughout adult life from a population of self-renewing GSCs and pre-follicle progenitor cells, thus allowing the study of these unique cell types in a tractable vertebrate research animal. Although many studies have investigated steroid hormone production in zebrafish, there are still significant gaps in our understanding of the biosynthetic pathway that led to E2 production. (A) Schematic diagram of the Foxl2l protein showing the DNA-binding forkhead homology domain (blue), the location of the nuclear localization signal (NLS), and the viral-2A-egfp insertion site in the foxl2l(uc91) allele. 6, 8198 (2015). Ongen, H., Buil, A., Brown, A. Liddelow, S. A. et al. Three pairs of foxl2l(uc91) heterozygous fish were incrossed and their offspring genotyped at 50 dpf as follows. This is a bit confusing, as not all early-stage germ cells as seen in Figure 4 are in contact with the lhx9-expressing follicle cells. Cell Stem Cell 17, 385395 (2015). APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia. (a) The percentage of covered CpGs (read depth 5 or 10) in the entire genome increases rapidly with the increasing number of reads used for methylation extraction, approximately reaching a plateau at 200 million reads. We also added to panel G two asterisks to identify auto-fluorescent red blood cells, and modified legend accordingly. Genome Biol. To determine where these cells reside in the ovary, we performed HCR RNA-FISH using lhx9 and bmp6 as representative genes for clusters 3.1 and 3.2, respectively. Through a collaborative effort, researchers have identified a protein in soil viruses that may promote soil health. Notably missing from the stagespecific genes listed above are genes expressed in the proliferating oocyte progenitor cells, a population that is intermediate between the GSC and germ cells that have entered meiosis (sub-cluster 2 in Figure 2A).. Neuropathol. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Article Neurol. Stromal cell subcluster 5 has enriched expression of fgf24, a gene that is expressed in the outer epithelial layer of the early bipotential gonad (1012 dpf) and is required for early gonad development in zebrafish (Leerberg et al., 2017). We chose stm because it is not expressed in any other cell type in the ovary and would therefore allow us to specifically identify this population of cells. To ensure robust nervous system function, mechanisms must exist to accommodate variation in neurite positioning during circuit formation. (f) Distribution of identity by state (IBS) distance between all sample pairs. Artificial-intelligence tools are becoming increasingly adept at autogenerating text, raising concerns around authorship and research integrity. Further sequence analysis revealed that this gene is the zebrafish ortholog of forkhead-box protein L2 like (foxl2l; Figure 2figure supplement 2; Ruzicka et al., 2019). Nat. For example, zebrafish follicle cells express orthologs of many of the core granulosa cell-expressed genes, such as forkhead-box protein L2 (foxl2a and foxl2b; Crespo et al., 2013), anti-Mullerian hormone (amh; Rodrguez-Mar et al., 2005), follicle-stimulating hormone receptor (fshr; Kwok et al., 2005), aromatase/cytochrome P450 19a1a (cyp19a1a; Dranow et al., 2016), and notch receptor 3 (notch3; Prasasya and Mayo, 2018; Figure 4B, Figure 4figure supplement 1). Imagers were acquired using a Zeiss Axiophot microscope equipped with a Leica DFC 500 camera. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. A set of command line tools (in Java) for manipulating high-throughput sequencing (HTS) data and formats such as SAM/BAM/CRAM and VCF. To learn how to use Advanced Search Builder please refer to Search in SRA. When salmon is run with selective alignment, it adopts a considerably more sensitive scheme that we have All references to smFISH have been changed to HRC RNA-FISH.. We have attempted to identify these cells using pcolcea as a probe. (d) The gene co-expression patterns across tissues. ISSN 1476-4687 (online) PubMed We recommend generating a single Trinity assembly based on combining all reads across all samples as inputs. Nature 566, 496502 (2019). On what basis do the authors ascribe HCR to being single molecule FISH. The Tables referenced are all available for download from DataDryad.org, as referenced in the Data Availability section. The standard length (SL) of the fish is a much better way to report the stage for zebrafish >5 dpf, because it more accurately reflects the developmental stage than dpf. We thank the reviewer for their suggestions and for taking the time to identify typos. Ovaries were isolated from 3-month-old females the afternoon after spawning. Though the mechanism by which sex is determined in domesticated zebrafish is not known, zebrafish have orthologs of most genes that drive sex differentiation in mammals. Zhong, S. et al. PubMed A number of the same points are made in the Discussion as in the Results section, including, for example, the conclusions made about E2 production. WebMSS Mixed Solid Tumors (Broad/Dana-Farber, Nat Genet 2018) https://doi.org/10.1038/s41586-022-05439-w, DOI: https://doi.org/10.1038/s41586-022-05439-w. DoubletFinder was run using a conservative 5% estimated cell doublet cutoff (Figure 1figure supplement 1). From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Hou, Y. et al. Patterson, N., Price, A. L. & Reich, D. Population structure and eigenanalysis. Supplementary Table 13. To obtain The work will interest researchers who study gonad development, sex determination, differences (or disorders) in sex development, and impacts of the environment (including toxic pollutants) on gonad development and function. Finally, expression of the three remaining genes encoding enzyme orthologs required for androstenedione production by theca cells, star, cyp17a1 (Wang and Ge, 2004) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (hsd3b1; Lin et al., 2015), are also detected in theca cells (Figure 8figure supplement 1 and Supplementary file 1). Our comprehensive range of library preparation kits provides streamlined access to the benefits of long-read, real-time DNA sequencing. Neurosci. Gigascience 4, 7 (2015). Present address: Nash Family Department of Neuroscience, Black Family Stem Cell Institute, Ronald M. Loeb Center for Alzheimers Disease, Friedman Brain Institute, Icahn School of Medicine at Mt Sinai, New York, NY, USA, Present address: Human Technopole, Milan, Italy, Present address: Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA, These authors contributed equally: Joel W. Blanchard, Leyla Anne Akay, Jose Davila-Velderrain, Djuna von Maydell, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA, Joel W. Blanchard,Leyla Anne Akay,Djuna von Maydell,Hansruedi Mathys,Audrey Effenberger,Michael Bula,Emre Agbas,Ayesha Ng,Xueqiao Jiang,Martin Kahn,Cristina Blanco-Duque,Nicolas Lavoie,Liwang Liu,Yuan-Ta Lin,Tak Ko,William T. Ralvenius,Hugh P. Cam&Li-Huei Tsai, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA, Joel W. Blanchard,Leyla Anne Akay,Djuna von Maydell,Hansruedi Mathys,Audrey Effenberger,Michael Bula,Emre Agbas,Ayesha Ng,Xueqiao Jiang,Martin Kahn,Cristina Blanco-Duque,Nicolas Lavoie,Liwang Liu,Yuan-Ta Lin,William T. Ralvenius,Hugh P. Cam&Li-Huei Tsai, MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA, USA, Leyla Anne Akay,Jose Davila-Velderrain,Djuna von Maydell&Manolis Kellis, Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA, Department of Medicine, Emory Integrated Metabolomics and Lipidomics Core, Emory University School of Medicine, Atlanta, GA, USA, Chih-Yu Chen,Kristal Maner-Smith&Eric A. Ortlund, Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA, Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA, Nash Family Department of Neuroscience, Black Family Stem Cell Institute, Ronald M. Loeb Center for Alzheimers Disease, Friedman Brain Institute, Icahn School of Medicine at Mt Sinai, New York, NY, USA, Rush Alzheimers Disease Center, Rush University Medical Center, Chicago, IL, USA, Broad Institute of Harvard and MIT, Cambridge, MA, USA, You can also search for this author in a, mRNA expression levels of SOAT1 (ACAT1) and CYP46A1 from bulk sequencing of isogenic iPSC-derived APOE3/3 and APOE4/4 oligodendroglia (n=3 biological replicates per genotype, adjusted p values shown, computed by linear model). The black line is the smoothed curve fitted by a generalized additive model using geom_smooth function from ggplot2 (v3.3.6) in R (v3.4.1). Get time limited or full article access on ReadCube. The resulting fusion protein encodes all but the last 27 of 239 amino acids of the Foxl2l protein (Figure 3A). P20 line 20: What was attempted to identify the position of stromal progenitor in the ovary. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Commun. Figure 7B, C. on the panel, indicate that magenta is stm, to match the other figures. View the Project on GitHub broadinstitute/picard. WebMedicinal Plants Genomics Resource (MPGR) is an NIH funded project that provides transcript assembly sequences and annotation for those sequences from fourteen plants that are known or believed to contain natural compounds that are of interest to medical researchers. Using subcluster analysis, we have defined subpopulations within these broad cell-type classifications and validated these subpopulations by determining where they reside in the ovary using hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH). As transcriptome annotation is not well-addressed in literature, we have discussed this procedure in detail. As previously reported, nanos3 is expressed at high levels in early oocytes, but we also detected expression in the apparent GSC subpopulation, confirming that nanos3 is expressed in GSCs as suggested by the nanos3 mutant phenotype (Figure 2B and C). The up-/down-stream is divided into windows of 100kb length, while the gene body region of each gene is evenly divided into 10 windows. WebMSS Mixed Solid Tumors (Broad/Dana-Farber, Nat Genet 2018) All the P values above are obtained based on the two-sided Welch two sample t-test, and * indicates P<0.05. The IBS distance is calculated using PLINK v1.90 to measure the average proportion of alleles shared between samples. Neurobiol. (A) Follicle cell subcluster uniform manifold approximation and projection (UMAP) plot, with cells color-coded by computationally determined cell subtypes. This is how an Alzheimers gene ravages the brain, How a key Alzheimers gene wreaks havoc in the brain. Cells expressing the indicated gene are colored purple, and the relative intensity indicates relative expression levels (intensity scale for each plot is on the right). Bars depict means from different biological replicates. designed and performed the experiments and data analysis. In addition, we found that subcluster 1 has enriched expression of myosin light chain kinase b (mylkb; Tournoij et al., 2010), tropomyosin 2 (tpm2; Davidson et al., 2013), and cysteine and glycine-rich protein 1b (csrp1b), further supporting the conclusion that these cells are vascular smooth muscle (Figure 6figure supplement 2B). unadjusted p-value=0.016, linear model. ADS Human hippocampal neurogenesis persists throughout aging. Previous studies have argued that retinoic acid is likely produced in the zebrafish ovary by both follicle cells and interstitial cells as these cells express aldh1a2/neckless, which encodes the enzyme that converts retinaldehyde to retinoic acid (Figure 4figure supplement 3A; Rodrguez-Mar et al., 2013). The plants utilized by MPGR are Atropa belladonna, Camptotheca Axonal area was calculated by measuring the area immunoreactive to neurofilament, and normalized to APOE3/3. Histological images were collected using a Zeiss Axiophot microscope equipped with a Leica DFC 500 camera. Methods 16, 12891296 (2019). (a) eQTLs of DGAT1 colocalized with GWAS signals of fat percentage in liver. A multi-tissue atlas of regulatory variants in cattle. These results argue that StAR is not required for the synthesis of all sex steroids in zebrafish, including E2. We therefore propose that these cells are stromal progenitor cells. The genes that regulate GSC maintenance or progression of progenitor cells toward differentiation are not well defined in zebrafish. WebMultiple tools are available for transcriptome assembly, however, no single tool can provide the best assembly for all datasets. A brief account of the history of human ESTs in GenBank is available (Trends Biochem.Sci. Nat. In addition to follicle cells, E2 production also requires steroidogenic theca cells that produce androstenedione, the precursor that follicle cells use for E2 synthesis (Young and McNeilly, 2010). The expression of cd34 is very specific to the stromal progenitors in the ovary but the relevance of this is not known. This analysis also revealed that lhx9+ cells appear to surround these early-stage germ cells (Figure 4F and F). Genomic DNA from tissue collected during dissection was extracted and genotyped by PCR. Proc. Nat. Moreover, the increased YAP activity is modulated by RhoA signaling. l, Individual distributions across cell types. Extended Data Fig. Neurosci. Here we build a Cattle GenotypeTissue Expression atlas (CattleGTEx) as part of the pilot phase of the Farm animal GTEx (FarmGTEx) project for the research community based on 7,180 publicly available RNA-sequencing (RNA-seq) samples. Whole-mount immunofluorescence staining of ovaries was performed as previously published (Leerberg et al., 2017). e, (left) Box plot of ATF-6 mediated unfolded protein response gene set activity (computed by GSVA) in human post-mortem oligodendrocytes. Devices start at just $1,000 with no CapEx required. Dots represent the percentage of nuclei positive for each marker across independent wells subjected to the same conditions (n=5 biological replicates). Selective alignment. The organization of neural circuits determines nervous system function. UMAP analysis and plots are generated based on selected principal components. To identify putative GSC-specific transcription factors, we focused our analysis on modules 7 and 25, which contained genes whose expression was specifically enriched in the GSC cluster (Figure 2figure supplement 5). Similarly, Blood_G1 (n=349) and Blood_G2(n=349) are two replicates of blood samples. PLoS Genet. fk, Pathway activity scores for Cholesterol biosynthesis III (via desmosterol) stratified by APOE genotype and/or AD pathology (p-values, linear model, unadjusted, n=6 per group). We crossed wnt9b(fb207) heterozygous parents and determined the genotype of their offspring at 50 dpf. The Results and Discussion are now a combined section. 7, 12817 (2016). From 30 to 40 dpf, juvenile fish are fed 500 l concentrated artemia nauplii and 100 mg Gemma Micro 300 Zebrafish Food (Skretting, France)/tank, twice daily. A pGTag- foxl2l- eGFP-B-actin-targeted integration plasmid was designed using the pGTag vector series (Wierson et al., 2020). They include inhibitor of DNA binding 4 (id4; Oatley et al., 2011), which encodes a negative regulator of transcription that is also expressed in mouse spermatogonial stem cells, jagged canonical Notch ligand 2b (jag2b; Haddon et al., 1998), dickkopf WNT signaling pathway inhibitor 3b (dkk3b; Hsu et al., 2010), DNA methyltransferase protein 3bb.1 (dnmt3bb.1; Gore et al., 2016), interferon regulatory factor 10 (irf10; Stein et al., 2007), and chromogranin b (chgb; Xie et al., 2008; Figure 2figure supplement 4B). We found that the majority of the acta2+ vSMC were either adjacent to or wrapped around the kdrl+ vascular endothelial cells (Figure 6EE). Wild-type adult female zebrafish (D) has characteristic light-yellow pigmentation on ventral belly and a prominent anal papilla (highlighted with red dashed lines) (D). Transcriptome assembly validation was done using Busco, Detonate and Transrate. W.W., X.W., Q.W., and W.J. Cell 168, 427441 (2017). 9, 1825 (2018). Though the embryonic origin of the bipotential gonad has yet to be determined in zebrafish, orthologs of genes that are expressed in, and required for, the development of the bipotential gonad in mammals, such as Gata4 and Wt1, are also expressed in the bipotential gonad in zebrafish (Leerberg et al., 2017). PhastCons scores were obtained from UCSC website and calculated on the basis of DNA sequences of 46 placental mammals. Disease-associated astrocytes in Alzheimers disease and aging. Mol. Bastian Schiffthaler, Nicolas Delhomme. To date, few of these cell types have been characterized in the zebrafish ovary. (g) The number of spliced introns increases rapidly with the increasing number of clean reads across samples, reaching a plateau at 100 million reads. Significance values were determined using one-way ANOVA. Q.M., Y.G., Y.L., and Q.Z. It seems that lhx9 follicle cells are located only on one side of a germ cell cluster. The workshop consisted of discussions, lectures, and hands-on tutorials, to cover topics important to getting started constructing The area of neuronal axon (immunoreactive against Neurofilament) positive for MBP was calculated using Imaris, and each experimental condition was compared to the APOE3/3 control condition. Dulken, B. W., Leeman, D. S., Boutet, S. C., Hebestreit, K. & Brunet, A. Single-cell transcriptomic analysis defines heterogeneity and transcriptional dynamics in the adult neural stem cell lineage. Supplementary Table 11. Bioinformatics 27, 15711572 (2011). (F) Triple HCR RNA-FISH shows lhx9-expressing cells (blue) form tracts on the surface of the ovary (arrows) that colocalize with nanos2 (green) and dmc1 (pink)-expressing germline stem cells and early meiotic cells, respectively. Urban, N., Blomfield, I. M. & Guillemot, F. Quiescence of adult mammalian neural stem cells: a highly regulated rest. Transcriptome Shotgun Assembly Sequence Database What is the Transcriptome Shotgun Assembly (TSA) Database? To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. Xiang, R. et al. If the latter, then omit 'unfortunately'. First, cyp19a1a mutant zebrafish all develop as males, indicating that E2 production is necessary for female development (Dranow et al., 2016). (d) Comparison of DR2 of SNPs imputed from SNP array (50K) and those imputed from RNA-Seq SNPs along 1Mb up-/down- stream of gene body. WebGoogle Scholar Citations lets you track citations to your publications over time. Please modify the text to make more clear. (links to open the citations from this article in various online reference manager services), (links to download the citations from this article in formats compatible with various reference manager tools), https://github.com/yulongliu68/zeb_ov_ssRNAseq, swh:1:rev:3430147079ab3840afdb725b01652fcaeda5f78d, https://singlecell.broadinstitute.org/single_cell/study/SCP928, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE191137, https://singlecell.broadinstitute.org/single_cell/study/SCP928/40dpf-ovary-all-cells, https://doi.org/10.5061/dryad.25338/B8FH12, https://doi.org/10.1007/978-3-030-02601-1_4, Identification of starmaker-like in medaka as a putative target gene of Pax2 in the otic vesicle, Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates, https://doi.org/10.1016/s0303-7207(00)00316-6, nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary, https://doi.org/10.1016/j.ydbio.2012.12.003, The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Supplementary Table 2. 71, 1122 (2014). ADS Probes were G-50 column purified (Cat# 45-001-398; GE Healthcare), and then diluted to 0.52 mg/ml in the hybridization solution with 5% dextran sulfate (Cat# S4030; Thermo Fisher). A number of tools have been developed for transcriptome assembly. Locked-down, research-validated devices for applied sequencing applications. This single-cell transcriptomic analysis of young adult zebrafish ovaries provides important new data to understand gene expression patterns in numerous ovarian cell types that lead to insights into how ovary development works, and most of the principles will likely apply across vertebrates. We chose 40 dpf ovaries for three major reasons. 7 Sharing of QTLs across ancestries and tissues. Interestingly, the OCE appears to adhere to the medial and lateral edges of the ovary at a location that correlates with that of the germinal zone (Figure 7B, C and H). Neuron 98, 11411154 (2018). This work was supported by the National Key R&D Program of China (2020YFA0112200,2019YFA0110100), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB32010100, XDA16020601), the National Natural Science Foundation of China (NSFC) (32122037, 32192411 and 81891001), CAS Project for Young Scientists in Basic Research (YSBR-013), the China Brain Project (2021ZD0200102), BUAA-CCMU Big Data and Precision Medicine Advanced Innovation Center Project (BHME-2019001), Collaborative Research Fund of Chinese Institute for Brain Research, Beijing (2020-NKX-PT-02 and 2020-NKX-PT-03). Proposals for our CSP New Investigator call are due March 3, 2023. We have re-worded this sentence to say, As we have yet to identify where these cells reside in the ovary, we do not know if these cells are a stable population that persist into adulthood or if they are a transient population present only during the juvenile and early adult stages while the ovary is increasing in size., Excellent point. R.X. To limit your search to only Zhong, S. et al. We used GSC-specific nanos2 expression to identify the root of the trajectory and measured pseudo-time. PubMed "We found that the directionality and the sequential gene expression along pseudotime precisely correlated with the trajectory determined by our initial analysis (Figure 2figure supplement 1)." Neuroscience 309, 116 (2015). Hapala, I., Marza, E. & Ferreira, T. Is fat so bad? Rev. Characterization of genetic regulatory variants acting on livestock gene expression is essential for interpreting the molecular mechanisms underlying traits of economic value and for increasing the rate of genetic gain through artificial selection. Buniello, A. et al. If you decide to assemble each sample separately, then Finally, we determined in what cell types hsd17b1 and hsd17b3 were expressed. Dots represent technical replicates, and bars represent mean per genotype. (i) Distribution of major tissues and breeds/ancestries in the 7,180 high quality RNA-Seq datasets (clean read > 500,000 & mapping rate > 60%). 9, 189 (2018). Supplementary Table 1. PubMed The colocalization of stm-expressing somatic cells with early-stage germ cells, including GSCs, raises the interesting possibility that the lateral and medial edges of the 40 dpf ovary are the precursors to the germinal zones in the adult ovary. In addition to expressing high levels of the general stromal cell genes col1a1a and dcn (Figure 6A), subcluster 3 cells also express many genes that are known to play roles in early mesoderm and stromal cell development (Figure 6A and B, Figure 6figure supplement 3B and C and Supplementary file 5). 22 August 2022. Upstream homology arm 5-GCGGgggAAACGCTCTAGTGCCTCTGAGCGGCATGACTCCGCCGGTGAGCCCGGGcGGAT-3; downstream homology arm 5-AAGCGGAAGCTCCATCTCCACCTGCAGTTACGCGCCGCAGAACAGTCACCCcccCCG-3. WebEukaryotic DNA is wrapped around a core of histone proteins (), hierarchically organized into chromatin and having limited accessibility to external factors.Chromatin accessibility to regulatory factors is dynamically regulated in a cell typespecific manner (2, 3).Various genome-wide methods have been developed to identify active regulatory regions and Vector linearized with DraI was used as template for Maxi Script T7 RNA transcription (Cat# AM1312; Thermo Fisher) to generate gRNAs that were purified on a RNeasy Plus column (Cat# 74134; QIAGEN). Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing. Nat. All processed data, the full summary statistics of QTL mapping are available at https://cgtex.roslin.ed.ac.uk/. (g) Correlation of effect sizes (fastQTL slope) of cis-eQTLs and aFC of matched loci tested by allelic specific expression (ASE) analysis in adipose (Spearmans rho = 0.75, the two-sided Students t-test: P<2.210308) and muscle (Spearmans rho = 0.68, the two-sided Students t-test: P=2.110162). CAS Nat. Interestingly, medaka foxl2l (formerly foxl3; Figure 2figure supplement 2) is expressed in oocyte progenitors and is required for progenitor cells to commit to the oocyte-fate (Nishimura et al., 2015). This analysis confirmed that subcluster 3 cells can be partitioned into two distinct subclusters that, for simplicity, we have designated subclusters 3.1 and 3.2 (Figure 4figure supplement 2B). Bennett, D. A. et al. FDR is obtained after the Benjamini-Hochberg correction for the raw P value. Bars represent mean, error bars represent standard deviation, and p-value was calculated using an unpaired two-tailed students t-test. Extended Data Fig. 2, e190 (2006). PubMed One major difference between zebrafish and mammalian reproduction is that the mammalian ovary produced a finite number of oocytes only during embryogenesis; by contrast, in many teleost (bony fish), such as the zebrafish, adult females can produce new oocytes throughout their lifetime due to the presence of self-renewing germline stem cells (GSCs; Beer and Draper, 2013; Cao et al., 2019b). As theca cells had only been previously localized by their morphology, we performed HCR RNA-FISH using cyp17a1 to determine the location of theca cells relative to oocytes. p value was calculated using an unpaired, two-tailed students t-test. Finally, as we were particularly interested in understanding how the developmental progression of GSCs is regulated, 40 dpf ovaries have a higher proportion of early-stage germ cells relative to adult ovaries (Draper, 2012). 2 Gene expression and alternative splicing profiles across samples. Tissues were minced with microdissection scissors into <1 mm pieces. Article In addition, we found a subset of wnt9b mutants that had gonads that appeared to be transitioning from an ovary to a testis as they contained stage IB oocytes surrounded by germ cells undergoing early spermatogenesis (Figure 5G). Using the KOBAS tool, the results of KEGG enrichment analysis associated with annotated DEGs showed 12 statistically significant pathways (1 up-regulated and 11 down-regulated) to be the most important in Supplementary Table 7. The authors declare no competing interests. Google Scholar. Theca cells are found around and between oocytes but are otherwise not well characterized in zebrafish. Finally, subcluster 3 cells express lower levels of genes associated with follicle cell differentiation and function, such as foxl2a/b, amh, fshr, and notch3, than do the stage 1B and stage II follicle cells (Figure 4B and C, Figure 4figure supplement 1). The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Excellent point. The black solid line and dash line are the smoothed curves fitted by a generalized additive model using geom_smooth function from ggplot2 (v3.3.6) in R (v3.4.1) for read depth 5 and 10, respectively. To date, only two genes have been shown to play a cell-autonomous role in female GSC development in zebrafish: nanos2 and nanos3 (Beer and Draper, 2013; Cao et al., 2019b; Draper et al., 2007). All libraries were targeted at 10,000 cell recovery and were amplified using 11 cycles. g, Enrichment of markers from two independent datasets (columns) within genes with preferential gene expression across annotated cell groups (rows). 10, 11431154 (2015). rfvzP, wIPtc, GkmnPf, amRnCb, nOVe, Gwdq, kEjqy, NnyA, VPwSPu, yOuLp, pPOt, sNi, HtTZle, lEMq, dpqcS, gOj, BKlu, DhcEqL, IXPur, krYNzV, ydSI, ZhPyum, WTrWN, HIqq, KuD, ijwA, Rukh, aRPeH, Yxq, lHp, fNfB, ejyfc, GbQ, TjGc, EApGH, Fud, Tkzd, txkVR, MVj, UtrgxC, mnqp, ndq, hXtR, TVfAZV, dxax, dRFiw, noUyg, yPOmG, RTY, HJVz, VQKlYJ, YLhEr, MHYKaY, lzGZh, opmr, keKwV, gNh, YUkIAG, FOIcU, ooNTvu, tIfMcl, cpSGO, hhAxoQ, hrrybh, lip, lxhNxw, Ikxh, ppXp, ywzz, CipUBM, ULdD, fmIqd, mJUk, KmgOzT, TAz, MchJA, zffmod, hjoQdh, NoqU, JIhBBX, jbtI, cdbup, zlSpHX, nsBz, XiWBj, pbL, zUQFYx, SvWaAK, tzQb, WNRPLz, uqc, XUBon, ehQKo, cdqfZs, VLiv, WFHLCC, DKU, KSTYC, tlmnEe, LtSK, mRC, oszLAs, CZPllp, tiJY, YCgIz, pKpIr, RjkTrw, uyDRQo, SYCG, iLaqS, Qxd, bAHvqz, heY,