Emx2is Required for Nephron Segment Development in the Zebrafish Pronephros

Abstract

The empty spiracles homeobox gene 2 (emx2)encodes a transcription factor that is expressed during neuronal, auditory, olfactory, and renal development, but its role in the latter has not yet been fully elucidated. Here, our objective is to utilize the zebrafish animal model to investigate the functions of emx2 during the development of nephrons, which are the structural and functional units of the kidney. Using whole mount in situ hybridization (WISH), we detected emx2transcript beginning at the 6 somite‐stage in renal progenitors and this expression continued throughout the process of forming discrete proximal convoluted tubule (PCT) and proximal straight tubule (PST) segment regions at 24 hours post fertilization (hpf). Based on this spatiotemporal pattern of emx2 expression in renal progenitors, we hypothesized that emx2is essential for segmentation of the nephron into discrete proximal and distal regions. To investigate this, we generated emx2‐deficient embryos and compared them to wild‐type (WT) controls at various time points from 24‐96 hpf. Knockdown of emx2resulted in head necrosis and diminished yolk sac extension at 24 hpf, as well as development of smaller head, eye and body, pericardial edema, abnormal fin growth, yolk ball inflammation and vascular hemorrhage in the central nervous system from 2‐5 days post fertilization. We used WISH to stain for each nephron segment in fixed animals and then quantified segment length and cell number. WISH analysis revealed a significantly reduced podocyte area, diminished PCT and reduced multiciliated cell numbers in emx2‐deficient embryos compared to WT embryos. WISH results also revealed secondary phenotypes, such as reduced fin bud area and somite in emx2‐deficient embryos. Additionally, Alcian Blue staining at 96 hpf revealed jaw abnormalities and pharyngeal arch malformation in emx2‐deficient embryos. O‐Dianisidine staining showcased vascular hemorrhage in the eyes and head of emx2deficient embryos. We concluded that emx2is essential for kidney segmentation in zebrafish, especially in podocyte and PCT formation. Additionally, emx2deficiency causes secondary jaw, fin, cartilage blood accumulation and muscle phenotypes. Future studies will delineate the cellular defects in nephron development in emx2 deficient embryos and determine the relationship between Emx2 and pathways which are essential for proximal segment ontogeny, such as retinoic acid signaling.