Figure 6. Nephrogenic mesenchyme markers become restrict- ed to regions of Foxd1 cell integration. (A–F) Serial sections through control (A, C, E) and Hox10 mutants (B, D, F) at E13.5. (A) Foxd1 expression is observed in the cortical stromal cells fully integrated into the periphery of the kidney at E13.5 in controls. (B) Foxd1 signal is regionally restricted in Hox10 mutants. (C–D) Expression of the nephrogenic marker Pax2 in control (C) and Hox10 mutant (D) embryos. In Hox10 mutants, Pax2 (D) is restricted to the domain in the kidney that is selectively expressing Foxd1. Red asterisks depict nephrogenic mesenchymal expression of Pax2 in (C, D). Similar patterns are observed with both Eya1(E, F) and Six2 (G,H) in which the normal expression in the mesenchymal condensations in the in the nephrogenic zone (E, G) becomes positionally restricted to regions of Foxd1-expressing cells in the Hox10 mutant (F, H). doi:10.1371/journal.pone.0023410.g006 mutants. It is not until later stages of organogenesis that the primary defect of Hox10 triple mutants is observed. The cortical stromal cells fail to properly differentiate and integrate into the Hox10 Patterns Kidney Cortical Stroma developing kidney. The defect in cortical stromal cell development in Hox10 mutants leads to additional phenotypes including aberrant ureter branching and decreased nephrogenesis (Fig. 9). Further support for a role for Hox10 genes in cortical stromal cell function comes from the fact that many of the structural and molecular defects in Hox10 mutants are highly reminiscent of those that have been reported for Foxd1. Specifically, loss of Foxd1 function results in hypoplastic kidneys, impaired branching morphogenesis and reduction of nephron number [34,36]. In addition, Foxd1 mutant kidneys fail to detach from the body wall during embryogenesis and as a result, do not ascend from the pelvic to lumbar region. All of these characteristics are phenocopied in Hox10 triple mutants and therefore provide further support for Hox10 genes playing a critical role in the development and integration of cortical stromal cells. It is interesting to note however, that the initial stromal cell precursor population is retained in both Foxd1 and Hox10 mutants. Additionally, we show that Hox10 does not regulate Foxd1 expression in the cortical stromal cells. Thus, Hox10 does not regulate the initial formation of the cortical stromal cell population, but rather, governs the proper integration and subsequent differentiation of this lineage in the developing metanephric kidney. A previous study has shown that the cortical stromal cells play an important role in regulating the formation of the kidney capsule [36]. Both Hox10 and Foxd1 mutants demonstrate defects in capsule development, as evidenced by the loss Sfrp1 and Tenascin- C expression in the periphery of the developing kidney [34,36]. Discerning the mechanisms by which Hox10 genes regulate the differentiation and integration of this population will be the focus of future studies. Another key question regards the cellular origins of the cortical stromal cell lineage. Previous studies have suggested that stromal cells may be derived from the metanephric blastema, either as cells that do not undergo nephron differentiation like the cap mesenchyme, or from a unique cell lineage present in the blastema [32,62,63]. More recently, a lineage trace with an Osr1-Cre provided evidence that the initial population of Foxd1-expressing cells are derived from the intermediate mesoderm [64]. Alterna- tively, a recent fate map study in chick suggests that the kidney stromal cells may be derived from paraxial mesoderm progenitors in this species [65]. We report here that Foxd1-expressing cells are initially observed as a highly concentrated cap-like pattern that is localized just anterior to the metanephric mesenchyme. As development progresses, these cells are observed in progressively posterior positions, becoming integrated into the periphery of the kidney to form the cortical stroma. These data are consistent with the cortical stromal cell lineage arising from the nephrogenic mesenchyme in a region just anterior to the metanephric mesenchyme. The more anterior border of Hox10 expression as compared to Hox11 is consistent with a unique role for Hox10 in the formation of this cell type. In support of this hypothesis, Mugford et al (2008b) showed that while the stromal cell lineage was labeled strongly when Osr1-Cre was activated at early developmental stages, while only the nephrogenic mesenchyme and not the cortical stroma was labeled when Osr1-Cre was activated at later stages. This result provides additional support for our hypothesis that the cortical stromal cell lineage arises from a population of cells that is anterior to the metanephric mesen- chyme. Hence, the combined data suggest that the general AP patterning functions of Hox genes directly translates into the differentiation of the distinct cell populations in the kidney. Future experiments removing Hox10 (or Foxd1) in specific cell populations will allow for a more definitive determination of when and where PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23410