Hox10 Patterns Kidney Cortical Stroma Figure 5. Expression patterns of Hox10, Hox11 and Foxd1 in the early nephrogenic cord. At E11.5, the Hox10 genes (A) exhibit a more anterior boundary of expression in dissected urogentital mesenchyme compared to Hox11 in control mice (Compare arrowheads in A and C). By E13.5, Hox10 (B) and Hoxa11eGFP (D) are both expressed in the nephrogenic cap mesenchyme, but Hox10 genes (B’) are additionally expressed in the cortical stroma cells (black arrow), whereas Hox11 (D’) is expressed in the renal vesicles but not in the cortical stroma (white arrow). (E) At E11.5, Foxd1 expression forms a highly concentrated cap-like pattern localized just anterior to the metanephric kidney. (F–G) As development progresses, these Foxd1-expressing cells are observed in progressively posterior positions (F, G). Panels (B–B’) and (D–D’) show light microscopy and fluorescent images of the same section, respectively. In situ hybridization analyses for Hox10 was performed using probes to all three paralogs (Hoxa10, Hoxc10, Hoxd10). doi:10.1371/journal.pone.0023410.g005 A previous study has shown elevated ectopic levels of Bmp4 can inhibit the proper formation of the kidney capsule [36]. Normally, Bmp4 expression is confined to interior regions of the maturing kidney where it inhibits nephron differentiation [36,60,61]. Consistent with the previous studies and loss of kidney capsule in these mutants, Hox10 mutants are also exposed to elevated levels of Bmp4 expression surrounding the mutant kidney (Compare Figs. 8A, D). Hence, our data suggest that the improper formation of the kidney capsule in Hox10 mutants may be due in part to the exposure of ectopic elevated levels of Bmp4. The loss of proper cortical stromal cell differentiation leads to an improper signaling environment that can secondarily affect ureter branching. Normally, Ret expression is localized in the nephron progenitors at the tips of the UB and provides an inductive signal that stimulates the growth of new ureter branches (Fig. 8B, C; [36]). However, in Hox10 mutants, Ret expression is not solely localized to the UB tips and instead, can be observed in extended regions along the stalks of the nascent ampulla (Fig. 8E, F arrowheads), consistent with the observed branching defects in Hox10 mutant kidneys. Discussion Interestingly, Hox10 and Hox11 mutant mice yield distinct kidney phenotypes despite largely overlapping expression patterns throughout kidney development. Previous studies have shown that the Hox11 genes play a critical role in the formation and invasion of the UB via the upregulation of Gdnf and Six2 in the metanephric mesenchyme [18,22]. As a result of the loss of Gdnf, kidneys do not form in Hox11 mutant mice. In contrast, the initial stages of kidney development are unaffected in Hox10 triple mutants. These mice undergo normal UB induction, and initiate the proper expression of several key transcription factors essential for the formation, proliferation and survival of the nephrogenic mesenchyme. These data indicate that, unlike the situation observed in Hox11 mutants, the nephrogenic mesenchyme is properly formed in Hox10 triple PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23410