Abstract
Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17:eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17:eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.
Highlights
Xenopus laevis is an established vertebrate model for studying developmental processes
We find that the cdh17:eGFP transgene is not deleterious to the embryos
79% of genes implicated in human diseases have X. laevis orthologues, including genes that are involved in human kidney disorders [40]
Summary
Xenopus laevis is an established vertebrate model for studying developmental processes. X. laevis produce free-living, relatively transparent embryos, enabling direct visualization of development during chemical and genetic screens. The large size of the embryos and established fate maps allow for targeted microinjection into a selected blastomere to manipulate gene expression in a tissue of interest [4,5,6,7,8,9]. This method of targeted injections directs knockdown or overexpression of genes to a selected subset of tissues in F0 generation embryos. Targeted injections allow for a delivery of constructs to organs of interest, while avoiding tissues that affect early development and prevent assessment of subsequent phenotypes
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