The transcription factors Etv4 and Etv5 mediate formation of the ureteric bud tip domain during kidney development

Abstract

Signaling by the Ret receptor tyrosine kinase promotes cell movements in the Wolffian duct that give rise to the first ureteric bud tip, initiating kidney development. Although the ETS transcription factors Etv4 and Etv5 are known to be required for mouse kidney development and to act downstream of Ret, their specific functions are unclear. Here, we examine their role by analyzing the ability of Etv4 Etv5 compound mutant cells to contribute to chimeric kidneys. Etv4(-/-);Etv5(+/-) cells show a limited distribution in the caudal Wolffian duct and ureteric bud, similar to Ret(-/-) cells, revealing a cell-autonomous role for Etv4 and Etv5 in the cell rearrangements promoted by Ret. By contrast, Etv4(-/-);Etv5(-/-) cells display more severe developmental limitations, suggesting a broad role for Etv4 and Etv5 downstream of multiple signals, which are together important for Wolffian duct and ureteric bud morphogenesis.

Fig. 1.

Etv4^−/−;Etv5^+/− cells, like Ret^−/− cells, are excluded from the ureteric bud tip domain in mutant↔wild-type chimeric mouse kidneys. Wild-type or mutant ES cells, carrying Hoxb7-GFP or Hoxb7-myrVenus, were injected into host blastocysts carrying Hoxb7/Cre and R26R-CFP. In the chimeric embryos, host-derived Wolffian duct (WD) and ureteric bud (UB) cells express CFP (cyan), and ES-derived cells express GFP or myrVenus (green). (A) As previously reported (Chi et al., 2009b), Ret^−/− ES cells contribute poorly to the primary UB tip domain (arrow) and common nephric duct (CND; asterisk) at E10.5. (B,C) At E11.0, the UB tip is composed mainly of wild-type cells. (D-F) Etv4^−/−;Etv5^+/− cells are mostly excluded from the UB tip domain (arrows) (n=11/13), similar to Ret^−/− cells (Chi et al., 2009b). However, they contributed normally to the CND (asterisks) (n=14/16). (G-I) During initial UB branching, Ret^−/− cells contribute mainly to the proximal side of branches (G), and during subsequent branching (H,I) to some trunks, but not tips (arrows) (Chi et al., 2009b; Shakya et al., 2005). (J-O) Two Etv4^−/−;Etv5^+/−↔wild-type chimeras, isolated at E11.5 (J,M) and cultured for 2 days (K,L,N,O). Like Ret^−/− cells, Etv4^−/−;Etv5^+/− mutant cells contribute to UB trunks but not tips (arrows) (n=17/17). (P-R) Control ES cells (Etv4^+/−) contribute throughout the trunks and tips (arrows).

Fig. 2.

Distribution of Etv4^−/−;Etv5^+/− cells in chimeric Wolffian duct and ureteric bud epithelium. (A-F) Sections through Etv4^−/−;Etv5^+/−↔wild-type mouse chimeras at the 36-somite (A-C) and 39-somite (D-F) stages, stained with anti-GFP (green) to reveal mutant cells, with anti-calbindin (red) to label all WD and UB cells, and with Hoechst nuclear stain (blue; A,D). B and C are enlargements of the WDs in A; E and F are enlargements of the emerging UBs in D. The dorsal regions of the WD at the 36-somite stage (i.e. the primary UB tip domains; dashed lines) are devoid of mutant cells, as are the UB tips at the 39-somite stage (arrows). cl, cloaca; nt, neural tube.

Fig. 3.

In Ret-hypomorphic host embryos, Etv4^−/−;Etv5^+/− ES cells contribute strongly to ureteric bud tips. (A-D) Etv4^−/−;Etv5^+/− ES cells were injected into wild-type (A,C) or Ret-hypomorphic (B,D) mouse blastocysts. Kidneys were cultured from E12.5 for 24 (A,B) or 72 (C,D) hours, then stained with anti-pan-cytokeratin (red), which stains all UB cells, and with anti-GFP (green), which stains the Etv4^−/−;Etv5^+/− cells. In wild-type hosts, the Etv4^−/−;Etv5^+/− ES cells contribute to UB trunks, but not (or weakly) to the tips (n=8/8 kidneys). However, in Ret-hypomorphic hosts, the Etv4^−/−;Etv5^+/− cells usually contribute strongly to the entire UB epithelium, including the tips (n=4/5 kidneys).

Fig. 4.

Etv4^−/−;Etv5^−/− cells contribute weakly to the Wolffian duct but not to the ureteric bud in chimeric mouse embryos. (A-F) Etv4^−/−;Etv5^−/−↔wild-type chimeras analyzed at E10.5-12.0. Rostral, left; caudal, right. Mutant cells are green (Venus⁺); wild-type cells are blue (CFP⁺). Six specimens are shown arranged from top to bottom by developmental stage, illustrating the range of mutant cell contributions. B has the most extensive contribution observed in the WD (white arrowheads indicate some of the areas with Venus⁺ mutant cells), but the forming UB tip domain is devoid of mutant cells (open arrowhead). A,D,F show mutant cells at different positions along the WD (white arrowheads) and in a mesonephric tubule (A, yellow arrowhead), but not in the extreme caudal WD (A) or in UBs (D,F) (open arrowheads). In C and E, a longer segment of caudal WD (as well as the UB) is devoid of mutant cells. Asterisks in B and F indicate mutant cells in the CND. The Etv4^−/−;Etv5^−/− cells contribute to the mesonephric tubules, primitive nephrons that form near the rostral end of the WD (A), as do Etv4^−/−;Etv5^+/− cells and Ret^−/− cells (data not shown). (G) The percentage of specimens showing strong, weak or no contribution to different regions. N indicates the number of informative specimens.