Hox10 Patterns Kidney Cortical Stroma Figure 3. Cultured Hox10 mutant kidneys exhibit ureter branching defects. (A–D) Control kidneys were dissected at E11.5 (A) and examined at 24 (B), 48 (C) and 72 (D) hour intervals. Pan-cytokeratin (green) was used to label the ureter epithelium and Pax2 (red) was used to label the nephrogenic mesenchyme. (E–H) Dissected E11.5 Hox10 mutant kidneys cultured and examined at identical time points as described for controls. Note that the Hox10 mutant kidneys exhibit aberrant ureter branching as compared to controls. (I–J) Hox10 mutant kidneys (J) have, on average, a 50% reduction in the number of branches as compared to controls (I). doi:10.1371/journal.pone.0023410.g003 these data demonstrate no defects in the early metanephric mesenchyme and suggest a unique function for Hox10 paralogous group genes in kidney organogenesis. Expression of Hox10 and Hox11 in the developing kidney To obtain a better understanding into the differences between Hox10 and Hox11 function in the kidney, we performed detailed expression analyses on both genes during multiple stages of development and carefully compared the two patterns. As previously described, in-situ hybridization analyses performed on tissue sections from E11.5 show that both Hox10 and Hox11 are expressed in the metanephric mesenchyme surrounding the ureter in similar patterns (Fig. 4A–B). However, in-situ hybridization experiments performed on dissected urogenital tissue from the same stage show that the anterior borders of Hox10 and Hox11 expression are clearly different (Fig. 5A, C arrowheads). As shown in Fig. 5C, the anterior limit of Hox11 expression is at the anterior border of the metanephric mesenchyme. In contrast, Hox10 expression is not limited to the metanephric mesenchyme, but extends into a more anterior region of the nephrogenic cord (Fig. 5A arrowhead). Thus, there are fundamental differences in the expression patterns of Hox10 and Hox11 genes even at these early stages of kidney organogenesis. In order to directly compare Hox10 and Hox11 expression patterns at later developmental stages, we performed Hox10 in situ hybridization analyses on tissue sections from a Hoxa11eGFP reporter line in which GFP fluorescence has been shown to accurately recapitulate Hox11 expression ([43], Figure S1). At E13.5, both Hox10 and Hox11 are expressed in the nephrogenic mesenchyme (Fig. 5B, B’, D, D’). However, Hox10 genes exhibit additional, unique expression in the thin layer of cells surrounding the nephrogenic mesenchyme known as the cortical stromal cells (Compare arrows in Fig. 5B’ to 5D’). Thus, Hox10 genes are expressed in both the stromal and nephrogenic mesenchyme compartments of the kidney at this stage, whereas the Hox11 genes are expressed exclusively in the nephrogenic mesenchyme. In addition, the difference in expression domains is consistent with the possibility that Hox10 genes play a unique role in cortical stromal cell development. PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23410