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Analysis of immunodeficient zebrafish using InDrops RNA sequencing of the whole kidney marrow. (A–D) 2D projection of tSNE analysis for WT and mutant fish (left) and quantitation of white blood cells within each genotype of fish, demarcated as pie charts (right). (E–H) tSNE visualization showing T, NK, and NKL cell subpopulations within WT and mutant fish and denoted by shaded ovals. Number of cells within each analysis are noted. n = 3 animals for WT and prkdc−/−; n = 2 for il2rga−/− and double-mutant fish.
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Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of defi...
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... scRNA-seq resulted in 19 clusters based on a nearestneighbour algorithm using the R Seurat package 22 ( Figure 2B). Each cluster was classified based on differentially expressed genes ( Figure S4A) and known differentiation markers [37][38][39][40][41][42] (Figure S4B-G). The expression of gata2b was mainly restricted to 'HSPCs' and 'proliferative progenitors' clusters ( Figure S4A). ...
The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2‐related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/−), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/− kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single‐cell (sc) RNA‐seq and single‐nucleus (sn) ATAC‐seq. Sn‐ATAC‐seq showed that the co‐accessibility between the transcription start site (TSS) and a −3.5–4.1 kb putative enhancer was more robust in gata2b+/− zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome‐wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/− chromatin was also more accessible during lineage differentiation. scRNA‐seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down‐regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell‐intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.
... In mammals, haematopoietic stem cells give rise to cells of the immune system [13,14]. In fish, the kidney functions as the primary haematopoietic organ, with the channel catfish kidney (CIK) being susceptible to GCRV [15][16][17][18]. CIK cells are derived from the kidney tissue of grass carp. ...
Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA–mRNA–protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.
... These results suggest that our hmgn2 Δ/Δ is at least a partial loss-offunction mutant and indicate that full-length Hmgn2 is necessary to maintain proper nuclear organization during erythropoiesis. When we performed ATAC-Seq on erythrocytes isolated from hmgn2 ΔΔ is embryos at 72 the double-mutant zebrafish line miR-144 Δ/Δ /hmgn2 Δ/Δ . When we probed erythrocyte development in 72-hpf embryos, we observed that the N:C ratio of erythrocytes of this double mutant is similar to the ratio from wild-type siblings (Fig. 4F), indicating that reduced Hmgn2 activity rescues the effect of the loss miR-144 and suggesting that Hmgn2 is an important player in driving the condensation defects of the miR-144 mutant. ...
... After creating the UMAP and clustering it was observed that one outlier cluster was enriched for genes expressed in sperm and was removed as it was likely contamination on sample collection. Clusters were annotated with the set of marker genes used in ref. 72 and progenitor cell and erythrocyte clusters were further analyzed to compare the expression of hmgn2 in the wild-type and miR-144 mutant genotypes. Expression of marker genes and hmgn2 is shown as a heat map over the UMAP plot, where the color scale represents the natural log of normalized read counts +1, or ln(cpm+1). ...
Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis establish that intact miR-144 target sites in the Hmgn2 3’UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.
... Meanwhile, its small size, high fecundity, rapid external development and transparent embryo enable zebrafish to serve as a valuable tool for high-throughput genetic and chemical screening [22,23]. In an analysis of single-cell RNA-sequencing (scRNA-seq) data of zebrafish hematopoietic cells [24], we found that the zebrafish tdh gene is relatively highly expressed in neutrophils; in contrast, tdh2, a homologous gene that might be generated by the fish-specific whole genome duplication [25] but has much lower homology to mouse Tdh, shows almost no expression in these cells of zebrafish. Therefore, we reasoned that the neutropoiesis of zebrafish may provide a model system to investigate the in vivo function of Tdh. ...
... shinyapps.io/zebrafishblood/) [24]. The results showed that the tdh, but not tdh2, is preferentially expressed in neutrophil and macrophage compared to other cell types (Fig. 1B). ...
... Indeed, these cells have been known to be dependent on glycolysis for energy production and contain fewer mitochondria [26][27][28][29][30], suggesting that the different expression levels of metabolic enzymes may reflect distinct metabolic state of the cells to some extent. We then analyzed the scRNA-seq data of isolated hematopoietic cells from transgenic zebrafish lines containing lineage-specific fluorescent reporters [24] (Fig. 1C,D). The results of our T-distributed stochastic neighborhood embedding (tSNE) analysis showed that tdh was highly expressed in neutrophils that were isolated from the Tg (mpx:EGFP ) zebrafish line, while showing a lower expression in hematopoietic stem/progenitor cells isolated from the Tg (Runx1 +23:EGFP ) and Tg (cd41:EGFP ) lines (Fig. 1E). ...
l ‐threonine dehydrogenase (Tdh) is an enzyme that links threonine metabolism to epigenetic modifications and mitochondria biogenesis. In vitro studies show that it is critical for the regulation of trimethylation of histone H3 lysine 4 (H3K4me3) levels and cell fate determination of mouse embryonic stem cells (mESCs). However, whether Tdh regulates a developmental process in vivo and, if it does, whether it also primarily regulates H3K4me3 levels in this process as it does in mESCs, remains elusive. Here, we revealed that, in zebrafish hematopoiesis, tdh is preferentially expressed in neutrophils. Knockout of tdh causes a decrease in neutrophil number and slightly suppresses their acute injury‐induced migration, but, unlike the mESCs, the level of H3K4me3 is not evidently reduced in neutrophils sorted from the kidney marrow of adult tdh ‐null zebrafish. These phenotypes are dependent on the enzymatic activity of Tdh. Importantly, a soluble supplement of nutrients that are able to fuel the acetyl‐CoA pool, such as pyruvate, glucose and branched‐chain amino acids, is sufficient to rescue the reduction in neutrophils caused by tdh deletion. In summary, our study presents evidence for the functional requirement of Tdh‐mediated threonine metabolism in a developmental process in vivo . It also provides an animal model for investigating the nutritional regulation of myelopoiesis and immune response, as well as a useful tool for high‐throughput drug/nutrition screening.
... Clusters 7, 10, 11, and 12, characterized by lineage-specific genes like tuba7l, dnai1.1, kif17; clcnk, hoxb8a, cldnh; muc2.2, agr2, itln1; and slc26a1, slc5a12, slc13a3 respectively, represented kidney multiciliated cells, kidney distal tubule cells, kidney mucin cells, and kidney proximal tubule cells ( Figure 1C and L-O; Tang et al., 2017). These renal cells are remnants in the preparation of leukocytes. ...
The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of the kidney in fish immune systems remains to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cells types from zebrafish kidneys via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in the kidney, suggesting the kidney acts as a secondary lymphoid organ. These results indicated that the fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune systems, and highlighted the multifaced biology of kidneys in ancient vertebrates.
... To analyze the expression pattern of osr2 within zebrafish kidneys, we employed a previously published single-cell RNA-sequencing dataset (GSE100910) [26]. For the purpose of data preprocessing, normalization, dimensionality reduction, and subsequent clustering analysis (resolution = 0.4), we applied the Seurat package (version 4.2.0), a widely utilized computational tool in the field of single-cell genomics. ...
... B: PCR analysis of the 3′ and 5′ junctions of F1 progenies from the 3# founder. The RNA sequencing (scRNA-seq) data (GSE100910) [26]. Analyzing scRNA-seq data revealed that osr2 is expressed in proximal tubule epithelial cells (PTECs) (Fig. 1A and B). ...
The proximal convoluted tubule (PCT) of the kidney is a crucial functional segment responsible for reabsorption, secretion, and the maintenance of electrolyte and water balance within the renal tubule. However, there is a lack of a well-defined endogenous transgenic line for studying PCT morphogenesis. By analyzing single-cell transcriptome data from the adult zebrafish kidney, we have identified the expression of odd-skipped-related 2 (osr2, which encodes an odd-skipped zinc-finger transcription factor) in the PCT. To gain insight into the role of osr2 in PCT morphogenesis, we have generated a transgenic zebrafish line Tg(osr2:EGFP), expressing enhanced green fluorescent protein (EGFP). The EGFP expression pattern closely mirrors that of endogenous Osr2, faithfully recapitulating its native expression profile. During kidney development, we can use EGFP to track PCT development, which is also preserved in adult zebrafish. Additionally, osr2:EGFP-labeled zebrafish PCT fragments displayed short lengths with infrequent overlap, rendering them conducive for nephrons counting. The generation of Tg(osr2:EGFP) transgenic line is accompanied by simultaneous disruption of osr2 activity. Importantly, our findings demonstrate that osr2 inactivation had no discernible impact on the development and regeneration of Tg(osr2:EGFP) zebrafish nephrons. Overall, the establishment of this transgenic zebrafish line offers a valuable tool for both genetic and chemical analysis of PCT.
... While the nephron is organized into these specialized segments, recent RNA sequencing studies have provided a new appreciation for the complexity of these cell types (Tang et al., 2017;Liao et al., 2020;McEvoy et al., 2022;Ransick et al., 2019). This complexity is further amplified in the context of various disease states and cancers (Hinze et al., 2022;Zhang et al., 2021). ...
While traditionally recognized as a sex hormone, estrogen has a potent effect on the development of tissues beyond those of the reproductive system. Estrogen synthesis enzymes and estrogen receptors are broadly expressed in vertebrate tissues, further indicating their importance in various processes. These include the tissues of the zebrafish, which is a particularly suitable model for studying early development due to its rapid ex utero ontogeny and conserved genetic and cellular composition with other vertebrates. In this review, we provide readers with an overview of estrogen signaling, discuss important attributes of the zebrafish animal model with a special focus on the kidney, and explore recent insights from zebrafish studies about the roles of estrogen signaling in organogenesis across germ layer derivatives that range from the kidney to the brain and liver.
... Eslec specifically labels zebrafish eosinophilic cells Eosinophils are characterized in adult zebrafish 21 , but their developmental origins remain unclear due to a lack of lineage-restricted markers. To identify genes that specifically mark the eosinophil lineage from early life to adulthood, we reanalyzed a single-cell RNA-Seq (scRNA-Seq) dataset containing zebrafish kidney cells 27 . The zebrafish kidney marrow (KM) is analogous to the mammalian BM 20 . ...
Eosinophils are a group of granulocytes well known for their capacity to protect the host from parasites and regulate immune function. Diverse biological roles for eosinophils have been increasingly identified, but the developmental pattern and regulation of the eosinophil lineage remain largely unknown. Herein, we utilize the zebrafish model to analyze eosinophilic cell differentiation, distribution, and regulation. By identifying eslec as an eosinophil lineage-specific marker, we establish a Tg(eslec:eGFP) reporter line, which specifically labeled cells of the eosinophil lineage from early life through adulthood. Spatial-temporal analysis of eslec⁺ cells demonstrates their organ distribution from larval stage to adulthood. By single-cell RNA-Seq analysis, we decipher the eosinophil lineage cells from lineage-committed progenitors to mature eosinophils. Through further genetic analysis, we demonstrate the role of Cebp1 in balancing neutrophil and eosinophil lineages, and a Cebp1-Cebpβ transcriptional axis that regulates the commitment and differentiation of the eosinophil lineage. Cross-species functional comparisons reveals that zebrafish Cebp1 is the functional orthologue of human C/EBPεP27 in suppressing eosinophilopoiesis. Our study characterizes eosinophil development in multiple dimensions including spatial-temporal patterns, expression profiles, and genetic regulators, providing for a better understanding of eosinophilopoiesis.
... A comprehensive understanding of cellular heterogeneity is essential for unravelling the developmental processes and disease pathogenesis of organs. The identification and functional characterization of stem cells and their diverse lineages pose significant challenges in the field of developmental biology [122]. Conventional 2D in vitro models lack the capacity to replicate organ-level cell interactions, underscoring the necessity of 3D organoids for exploring cellular heterogeneity [123]. ...
While cartilage tissue engineering has significantly improved the speed and quality of cartilage regeneration, the underlying metabolic mechanisms are complex, making research in this area lengthy and challenging. In the past decade, organoids have evolved rapidly as valuable research tools. Methods to create these advanced human cell models range from simple tissue culture techniques to complex bioengineering approaches. Cartilaginous organoids in part mimic the microphysiology of human cartilage and fill a gap in high-fidelity cartilage disease models to a certain extent. They hold great promise to elucidate the pathogenic mechanism of a diversity of cartilage diseases and prove crucial in the development of new drugs. This review will focus on the research progress of cartilaginous organoids and propose strategies for cartilaginous organoid construction, study directions, and future perspectives.
... The current research underscores the complexity of fish kidney cellular composition. For instance, in zebrafish kidneys, at least seven unique clusters have been discerned through single-cell RNA sequencing, which includes three epithelial cell types from the proximal, intermediate, and distal tubules, as well as mucinous cells, kidney stem/progenitor cells, multiciliated cells, and vascular endothelium cells [22]. Interestingly, even during fatal infections, MRV remains restricted to certain cells within target organs, notably the renal tubules [7]. ...
Mandarin fish ranavirus (MRV) infection poses a substantial challenge to the mandarin fish culture industry as no effective preventive or therapeutic measures currently exist. The creation of a highly permissive cell line from a natural host is crucial for developing a vaccine for MRV and understanding its pathogenic mechanisms. In this research, the mandarin fish (Siniperca chuatsi) kidney cell line (SCK) was isolated from mandarin fish kidneys. Subsequently, SCK-a to SCK-g monoclonal cell lines were derived from the SCK cell population, distinguished by morphological variations. Notably, MRV infection induced an advanced cytopathic effect (CPE) in almost all cells of the SCK-f clone. Further tests showed that MRV achieved a peak viral titer of 1010.7 50% tissue culture infectious dose (TCID50)/mL and consistently exceeded 1010 TCID50/mL across nine passages in SCK-f cells. Electron microscopy verified the MRV virion integrity within SCK-f. In vivo experiments revealed that MRV infections led to cumulative mortality rates of 86.9% in mandarin fish and 88.9% in largemouth bass (Micropterus salmoides). Such results suggest that SCK-f is highly permissive to MRV. This study underscores the importance of cellular diversity in developing viral permissive cell lines. The SCK monoclonal cell line pool may offer potential for generating highly permissive cell lines for other mandarin fish viruses.
