EF-hand domain containing 2 (Efhc2) is crucial for distal segmentation of pronephros in zebrafish

Abstract

BackgroundThe blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function.Resultsefhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development.ConclusionIn this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.Electronic supplementary materialThe online version of this article (10.1186/s13578-018-0253-z) contains supplementary material, which is available to authorized users.

De novo identification of mammalian ciliary motility proteins using cryo-EM

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Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis

“…Indeed, we found that esrrγa deficiency resulted in a decreased MCC cell number [marked by odf3b (also known as cimap1b ), an established marker of terminally differentiated MCCs], and co-injection of esrrγa RNA was sufficient to rescue the splice-blocking morpholino ( Fig. 1 I,J) ( Barrodia et al, 2018 ; Li et al, 2014 ; Liu et al, 2007 ; Xie et al, 2020 ). FISH analysis of the PST domain (marked by the boundaries of trpm7 ) also revealed a shift towards a transporter cell identity ( Fig.…”

Methods to generate and evaluate zebrafish models of human kidney diseases

“…We have shown that the EF-hand domain containing 2 (efhc2) knockdown results in expansion of distal early segments and reduction in CS and distal late segment. The expression of odf3, which marks multiciliated cells of pronephric tubules, was also reduced in efhc2 morphants (Barrodia et al, 2018).…”

“…Defects in cilia present in zebrafish pronephros often lead to body curling, cyst formation and tubule dilation (Sullivan-Brown et al, 2008). Multiciliated cells present in zebrafish pronephros can be visualized by WISH or fluorescence in situ hybridization (FISH) using antisense odf3b or rfx2 riboprobes (Liu et al, 2007;Barrodia et al, 2018). Cilia in zebrafish embryos can be stained using a-acetylated tubulin and g-tubulin can be used to mark the basal bodies (Jaffe et al, 2010;Zaghloul and Katsanis 2011).…”