The cdx Genes and Retinoic Acid Control the Positioning and Segmentation of the Zebrafish Pronephros

Abstract

Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal ( cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis.

Author Summary

In the kidney, structures known as nephrons are responsible for collecting metabolic waste. Nephrons are composed of a blood filter (glomerulus) followed by a series of specialized tubule regions, or segments, which recover solutes such as salts, and finally terminate with a collecting duct. The genetic mechanisms that establish nephron segmentation in mammals have been a challenge to study because of the kidney's complex organogenesis. The zebrafish embryonic kidney (pronephros) contains two nephrons, previously thought to consist of a glomerulus, short tubule, and long stretch of duct. In this study, we have redefined the anatomy of the zebrafish pronephros and shown that the duct is actually subdivided into distinct tubule segments that are analogous to the proximal and distal segments found in mammalian nephrons. Next, we used the zebrafish pronephros to investigate how nephron segmentation occurs. We found that retinoic acid (RA) induces proximal pronephros segments and represses distal segment fates. Further, we found that the caudal ( cdx) transcription factors direct the anteroposterior location of pronephric progenitors by regulating the site of RA production. Taken together, these results reveal that a cdx-RA pathway plays a key role in both establishing where the pronephros forms along the embryonic axis as well as its segmentation pattern.