Hox10 Patterns Kidney Cortical Stroma an E18.5 Hox10 triple mutant embryo shows a reduced nephrogenic zone in the outer cortex, an expanded inner cortex, a very reduced medullary region as well as hydronephrosis at the ureter-pelvic junction. (E and F). Frontal sections of E16.5 control (E) and Hox10 mutant (F) embryos stained with LTL (green) to label proximal tubules and DBA (red) to label collecting ducts. Mature collecting duct structures do not develop properly and proximal tubules are not localized in a normal pattern in Hox10 mutants (F). Yellow asterisks in (F) indicate fusions to the body wall. Embryos in (E) and (F) were sectioned through the same (frontal) plane. k, kidney; a, adrenal; b, bladder; OC, outer cortex; IC, inner cortex; M, medulla. doi:10.1371/journal.pone.0023410.g001 Hox10 does not effect the early expression of key nephrogenic mesenchyme markers Upon ureteric bud induction, the nascent bud invades the metanephric mesenchyme ventrally. At E11.5, this step occurs indistinguishably in Hox10 mutant animals (Fig. 4C–J). Previous studies have shown that Hox11 genes are specifically required for the differentiation of the metanephric mesenchyme [22]. As depicted in Fig. 4A–B, Hox10 and Hox11 are both expressed in the metanephric mesenchyme surrounding the ureter at E11.5. This overlap of expression domains suggests that Hox10 could also play a role in the early patterning of the nephrogenic mesenchyme [18,22,42]. However, the expression patterns of Hox11, Six2, Wt1 and Eya1, all transcription factors that are important for early nephrogenic mesenchyme events [18,19,22, 23,29,53], are unaffected in Hox10 triple mutants and remain strongly expressed throughout the condensed nephrogenic mesenchyme (Figs 4C–J). In addition, the fact that the expression patterns of these genes are all unaffected indicates that the nephrogenic mesenchyme, a zone of critical importance for the initial events of kidney development, is both properly established and maintained through E11.5 in Hox10 triple mutants. Therefore, despite the clear overlap of expression (Fig 4A–B), Figure 1. Kidney defects in Hox10 paralogous mutants. At E18.5, the kidneys from Hox10 triple mutants (B) are much smaller than controls (A), and the ureter routing (white outline) is abnormal with the ureter entering the kidney from the lateral side, compared to a medial entry for controls. (C) Histological section through an E18.5 control kidney demonstrates organization of this stage kidney into three distinct regions, the outer cortex, OC, inner cortex, IC, and medulla, M. The zone of nephrogenesis is in the outer cortex and surrounds the kidney up to the point of ureter entry. (D) Histological section through Figure 2. Hox10 mutants exhibit ureter branching defects and kidneys do not detach from the body wall and urogenital ridge. (A–D) Visualization of the urogenital systems from E14.5 control (A, B) and Hox10 mutant (C, D) embryos by OPT. Embryos were stained with Pax2 (red) to label the nephrogenic mesenchyme and pan- cytokeratin (green) to label the epithelial structures. Overlay of background fluorescence delineates the organ surface (A, C brown), highlighting failure of the Hox10 mutant kidney to separate from the body wall. Ureter branching in Hox10 mutants (D) is reduced and branches are abnormally elongated compared to control animals (B). doi:10.1371/journal.pone.0023410.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23410