Abstract
The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of Xenopus laevis consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in Xenopus. dnmbp is expressed in Xenopus embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. Additionally, Dnmbp reduction results in glomus and ciliary defects. dnmbp knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis and also results in edema formation in later stage embryos. Overexpression of dnmbp in the kidney also resulted in disrupted pronephric tubules, suggesting that dnmbp levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.
Highlights
Kidney development is conserved in amphibians and mammals, making Xenopus embryos a good model for studying nephrogenesis
We demonstrate that knockdown, CRISPR knockout and overexpression of dnmbp lead to tubulogenesis and cilia defects in Xenopus pronephric kidneys, indicating that this protein is required for nephrogenesis
Using a commercial antibody against Dnmbp, we found that Dnmbp protein (170 kD) is present in Xenopus embryos from single cell through tadpole stages by Western blot (Figure 1A)
Summary
Kidney development is conserved in amphibians and mammals, making Xenopus embryos a good model for studying nephrogenesis. The Xenopus pronephros consists of a single, large, functional nephron (Brennan et al, 1998; Carroll et al, 1999), making it a simple model for studying vertebrate nephron development. It is possible to modulate gene expression in Xenopus embryos through overexpression, knockdown and knockout experiments via microinjection of RNA constructs, antisense morpholino oligonucleotides (MOs) and CRISPR constructs (Miller et al, 2011; Corkins et al, 2018; DeLay et al, 2018b). The established cell fate maps of the early Xenopus embryo facilitate tissue-targeted modulation of gene expression by microinjection into the appropriate blastomere (Moody, 1987a,b; DeLay et al, 2016, 2018b). Xenopus is a powerful model for studying essential nephrogenesis genes
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