Figure 4. Ureteric bud formation and early expression of key nephrogenic mesenchymal markers are unaltered in Hox10 mutants. At E11.5, both Hox11 (A) and Hox10 (B) are expressed in the condensed mesenchyme surrounding the ureter. (C, D) Hox11 expression in Hox10 mutant embryos (D) is identical to controls (C). Six2 expression is observed similarly throughout the condensed metanephric mesenchyme of E11.5 control (E) and E11.5 mutant (F) embryos. At E11.5, the expression patterns of Eya1 and WT1 are also unaltered in control (G, I) compared to Hox10 mutants (H, J). doi:10.1371/journal.pone.0023410.g004 Hox10 is required for cortical stromal cell function Previous studies have shown that the stromal cell population in the developing kidney is critical for proper branching and differentiation of the kidney capsule [35,36]. The earliest known marker for this cell lineage is the transcription factor Foxd1, which is exclusively expressed in the cortical stromal cells as early as E11.5 [34]. Consistent with these data, whole mount in situ hybridization analyses performed on dissected urogenital tissue show that Foxd1 cannot be detected until E11.5. At E11.5, Foxd1- Hox10 Patterns Kidney Cortical Stroma expressing cells are observed in a highly concentrated cap-like pattern that is localized just anterior to the metanephric mesenchyme (Fig 5E). Over approximately the next 12 hours of development, Foxd1-expressing cells are observed in progressively posterior positions, (Fig. 5F, G), and by E13.5, the cortical stromal cells surround and are fully integrated into the developing kidney (Fig. 6A). While Foxd1-expressing cells initiate normally in Hox10 mutants, they do not fully integrate into the kidney in Hox10 mutants (Fig. 6B). Instead, they are observed only in a restricted subset of cells at the periphery of the kidney (Figs. 6A, B). These data demonstrate that Hox10 has a critical function in promoting the proper integration of these cells into the developing kidney. Nephrogenic mesenchyme markers become restricted to regions where Foxd1-expressing cells integrate We next examined whether the restricted expression domain of Foxd1 in the periphery of the developing kidney in Hox10 mutants might lead to a localized signaling environment in which proper UB branching and nephrogenesis are also restricted. While the ureteric epithelial expression of Pax2 remains intact at E13.5 in Hox10 triple mutants, Pax2 expression in the nephrogenic mesenchyme becomes restricted to the domain in the periphery of the kidney adjacent to the remaining Foxd1-expressing cells (Fig. 6C, D asterisks). Similarly, the expression of the nephrogenic markers Eya1 and Six2 also become restricted to the region immediately adjacent to the Foxd1-expressing cells in the kidney periphery in Hox10 mutants (Fig. 6F, H). These results imply that the continued expressions of these key nephrogenic mesenchymal markers in Hox10 mutants are both dependent upon and reacting to the proper integration of stromal cells. We next examined the expression patterns of several genes that have reported functions in cortical cell differentiation in both control and Hox10 triple mutant kidneys. At E15.5, Foxd1 expression continues to be restricted in the developing kidney in Hox10 mutants (Fig. 7A, B). Other important markers for stromal cell differentiation include Pbx1 and Raldh2 [39,54,55,56]. Normally, these stromal markers are also expressed around the entire periphery in the developing kidney at E15.5 (Fig. 7C, E). However, in Hox10 mutant kidneys, the expression patterns of both of these markers become restricted to the same regions in the periphery of the kidney as Foxd1 (Fig 7D, F). These data provide further evidence that Hox10 plays an important role in the proper integration of the cortical stromal cells during kidney organogen- esis. Kidney capsule maturation is disrupted in Hox10 mutants Tenascin-C is a marker for differentiated stromal cells and is normally detected in the kidney periphery within the population of cells that has been postulated to play a role in renal capsule formation (Fig 7G; [34]. In contrast to the peripheral expression of Tenascin-C in differentiated cortical stromal cells in control embryos, this signal is completely lost in Hox10 mutants and indicates that this population of cells fails to properly differentiate (Fig. 7G, H). In addition to Tenascin-C, previous studies have shown that the secreted frizzled-related protein Sfrp1 is strongly expressed in the renal capsule [36,57,58,59]. Consistent with these reports, we also find that Sfrp1 is expressed at a high level throughout the kidney capsule cell layer and surrounds the periphery of the developing kidney at E14.5 in control embryos (Fig. 7I). However, there is a complete down-regulation of this gene in Hox10 mutants (Fig. 7J arrowheads). Thus, it appears that Hox10 plays in role in the regulation of kidney capsule formation that is similar to what has recently reported for Foxd1 [36]. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23410