scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain

Abstract

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

The Pathophysiological Role of Thymosin β4 in the Kidney Glomerulus

“…Tmsb4x transcripts were also detected in both developing and mature zebrafish podocytes by scRNAseq, suggesting that podocyte expression of Tβ4 is conserved in different species. Interestingly, this study identified a switch in aged podocytes from Tmsb4x to Tmsb1, another member of the beta thymosin family found in zebrafish and other bony fish [34]. TMSB4X transcripts are detected in lysates from adult human glomeruli [29]; however, there is little information about the expression of Tβ4 specifically in podocytes in humans.…”

Principles of Zebrafish Nephron Segment Development

“…Continued investigations to elucidate the interrelationships and functions of these identified factors are an exciting future area of investigation. Additionally, data from various single cell sequencing approaches [ 158 , 159 , 160 , 161 , 162 ] are expanding our knowledge of the nephron and will be pivotal to advancing knowledge about nephrogenesis, which can be used in future applications such as regenerative medicine [ 163 ].…”

Characterization of Pipefish Immune Cell Populations Through Single-Cell Transcriptomics

“…A number of teleost studies have utilized cell isolation and single cell transcriptomics to assist with the identification of brain (17,18), kidney (19), intestine (20) and embryonic (21,22) derived cell populations. Moreover, further work identifying teleost immune cell repertoires in a number of model fish species (23)(24)(25)(26)(27)(28), have established a catalogue of putative immune cell markers that extend beyond those used in mammalian cell identification.…”