Single cell transcriptomic profiling of gill cells of zebrafish (Danio rerio) exposed to normoxia and hypoxia
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ABSTRACT: The fish gill is a multifunctional organ containing a variety of specialized cells including respiratory chemoreceptors, neuroepithelial cells (NECs). Although the structure, function and development of the gill have been studied extensively, transcriptomic profiling of individual gill cells is lacking. Using the 10x Genomics Chromium technology, we conducted a single transcriptomic study of cells from distal gill filament of ETvmat2:GFP zebrafish, acclimated to 14 days of normoxia and hypoxia. Overall, approximately 13,000 cells were sequenced with an average depth of 27,000 reads per cell. We identified 16 cell clusters in the gill, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. NECs were identified through expression of vmat2, encoding vesicular monoamine transporter, and showed highly differential expressions of tph1a, sv2, and mitochondrial proteins implicated in O2 sensing. Differential gene expression analysis showed a shift in transcriptome in NECs following 14 days of acclimation to hypoxia. This study presents a comprehensive cell atlas for the zebrafish gill and provides a framework for future investigations of molecular biology and physiology in gills.
Project description:In order to have a comprehensive understanding of endoderm derived mpeg1+ cells in zebrafish gill and intestine, we isolated endoderm-derived GFP+ cells and remaining DsRedx+ cells (hematopoiesis-derived macrophages) from the gill (sox17_gill_GFP, sox17_gill_DsRedx) and intestine (sox17_intestine_GFP, sox17_intestine_DsRedx) of 4-OHT treated Tg(sox17:CreERT2;mpeg1:loxP-DsRedx-loxP-GFP) fish and performed RNA-seq. Both T-SNE analysis and feature gene comparison indicate that the endoderm-derived mpeg1+ cells in the gill and intestine are highly similar to the metaphocytes in the epidermis. We hence also refer to these endoderm-derived mpeg1+ cells as metaphocytes.
Project description:The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. Transcriptome responses to virus were assessed in gills at different stages of disease
Project description:Whereas the gill chambers of extant jawless vertebrates (lampreys and hagfish) open directly into the environment, jawed vertebrates have evolved skeletal appendages that promote the unidirectional flow of oxygenated water over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single operculum covering a large common gill cavity in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here we find that these divergent gill cover patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs, and we identify a deeply conserved Pou3f3 arch enhancer that is present in nearly all jawed vertebrates but undetectable in lampreys. Despite only minor sequence differences, bony fish and cartilaginous fish versions of this enhancer are sufficient to drive the respective single versus multiple gill arch expression. In zebrafish, loss of Pou3f3 gene function or its conserved enhancer disrupts gill cover formation. Conversely, forced expression of Pou3f3b in the gill arches generates ectopic skeletal elements reminiscent of the multiple gill covers of cartilaginous fish. Emergence and modification of this ancient Pou3f3 enhancer may thus have contributed to the acquisition and diversification of gill covers during early gnathostome evolution.
Project description:How novel structures arise during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones1. In contrast, the evolutionary origins of the outer ear, another mammalian innovation, remain a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear.
Project description:How novel structures arise during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones. In contrast, the evolutionary origins of the outer ear, another mammalian innovation, remain a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear.
Project description:Gills of teleost fish represent a vital multifunctional organ; however, they are subjected to environmental stressors, causing gill damage. Gill damage is associated with significant losses in the Atlantic salmon aquaculture industry. Gill disorders due to environmental stressors are exacerbated by global environmental changes, especially with open-net pen aquaculture (as farmed fish lack the ability to escape those events). The local and systemic response to gill damage, concurrent with several environmental insults, are not well investigated. We performed field sampling to collect gill and liver tissue after several environmental insults. Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged gill tissue. The gill damage-associated biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon gill health.
Project description:Dissolved oxygen (DO) is the basis of fish survival, and proper DO level is an important condition to ensure the normal growth of fish. Hypoxic environment is prone to disturb the normal breathing and metabolism of fish, which in turn affects their growth and survival. Gill tissue is the respiratory organs of fish and is in direct contact with the external environment. However, there are few reports on the molecular regulatory mechanism of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) gill tissues in response to hypoxia. Here, we first examine the hypoxia-induced damage of gill tissue by hematoxylin-eosin staining, and then constructed miRNA and mRNA libraries of GIFT gill tissue at 96h of hypoxia stress by a high-throughput sequencing technology, each library has three biological replicates. Gill lamellae of GIFT showed capillary rupture and red blood cell enlargement and overflow under hypoxia stress. Transcription sequencing results showed that the clean reads of miRNA libraries were 9,627,953-13,544,660; the clean reads of mRNA libraries were 43,817,776-53,130,102. Based on the miRNA-mRNA pairs screening principles and mRNA sequencing results, we selected and verified seven differentially expressed miRNAs and their potential target genes. The sequencing results were consistent with the qRT-PCR validation results. These selected miRNA-mRNA pairs are mainly concentrated in the signaling pathways of immune response and metabolic regulation. This study provides new insights into the mechanisms of fish adaptation under hypoxic stress.
Project description:Dissolved oxygen (DO) is the basis of fish survival, and proper DO level is an important condition to ensure the normal growth of fish. Hypoxic environment is prone to disturb the normal breathing and metabolism of fish, which in turn affects their growth and survival. Gill tissue is the respiratory organs of fish and is in direct contact with the external environment. However, there are few reports on the molecular regulatory mechanism of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) gill tissues in response to hypoxia. Here, we first examine the hypoxia-induced damage of gill tissue by hematoxylin-eosin staining, and then constructed miRNA and mRNA libraries of GIFT gill tissue at 96h of hypoxia stress by a high-throughput sequencing technology, each library has three biological replicates. Gill lamellae of GIFT showed capillary rupture and red blood cell enlargement and overflow under hypoxia stress. Transcription sequencing results showed that the clean reads of miRNA libraries were 9,627,953-13,544,660; the clean reads of mRNA libraries were 43,817,776-53,130,102. Based on the miRNA-mRNA pairs screening principles and mRNA sequencing results, we selected and verified seven differentially expressed miRNAs and their potential target genes. The sequencing results were consistent with the qRT-PCR validation results. These selected miRNA-mRNA pairs are mainly concentrated in the signaling pathways of immune response and metabolic regulation. This study provides new insights into the mechanisms of fish adaptation under hypoxic stress.
Project description:This SuperSeries is composed of the following subset Series: GSE22171: Pacific salmon gill samples: fate tracking in river, sampled in ocean GSE22177: Pacific salmon gill samples: fate tracking in river GSE22347: Pacific salmon gill samples: fate tracking at spawning grounds Refer to individual Series
Project description:We compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated susceptible to amoebic gill disease (AGD).