Identifying the Origin of Zebrafish (Danio rerio) Renal Progenitor Cells

Abstract

The zebrafish kidney can be distinguished from the human kidney by the presence of adult renal progenitor cells (RPCs) that are capable of neonephrogenesis. These cells can be labeled with eGFP in the Tg(lhx1a:egfp) transgenic line. Using this transgenic line, zebrafish kidney regeneration was shown to utilize tubule repair mechanisms early (from 2 days post-insult) and neonephrogenesis acted later (after 6 days). The fish then totally recovered back to a normal state within 30 days. To better understand the molecular identity of RPCs, an isolation protocol for manual isolation of single and clustered RPCs from Tg(lhx1a:egfp) fish was developed. Gene expression profiling of these cells by RNA-Seq analysis showed early-acting renal genes (such as pax2a and pax8) in both single RPCs and cluster RPCs. Later acting renal genes (such as hnf1ba) were predominantly expressed in the cluster RPCs, supporting the concept that these cells are more mature. Surprisingly, the analysis also revealed a number of somitic genes expressed in RPCs, suggesting a somitic origin for RPCs. Here, genetic lineage labeling of the presomitic mesoderm was performed and showed that mesonephric tubules contain the lineage label, supporting a conclusion whereby RPCs are derived from somitic mesoderm. This finding represented a fundamental shift in our understanding of renal development. The RNA-Seq analysis on RPCs also revealed the expression of a number of genes that are components of major signaling pathways such as the canonical Wnt and PDGF pathways. To investigate the roles of these pathways, kidneys were damaged and exposed to pharmacological agonists and antagonists to these pathways. These results demonstrated that over-activation of the Wnt pathway increases the rate of renal regeneration and inhibition of PDGF signaling precludes neonephrogenesis. This work represented an initial insight into the molecular control of regeneration in the kidney and more work needs to be performed to fully elucidate the specific roles that these pathways manipulate in RPCs. In summary, this thesis uncovered many novel findings regarding the nature of RPCs in zebrafish and it is hoped that it acts as a foundation from which similar cells in humans might be identified and used to treat conditions such as chronic kidney disease.