exclusively expressed in these cells as early as E11.5 [34]. Functional analyses have shown that the loss of Foxd1 results in a reduction of nephron number, defects in ureter branching and loss of kidney capsule formation [34,36]. However, much remains to be determined regarding the origin of stromal cells, how they become integrated into the kidney and the signaling networks involved in their function. Hox genes are expressed along the anterior-posterior (AP) body axis and are necessary for patterning many mesodermal organs. While 28 of the 39 Hox genes are expressed in the kidney [40], only the Hox11 paralogous group genes have been shown to play functional roles during mammalian kidney development [18,22]. Prior to UB invasion, the Hox11 genes are expressed in the condensed metanephric mesenchyme and then later become localized to the nephrogenic cap mesenchyme as nephrogenesis proceeds [41,42,43]. Functional studies have further shown that Hox11 proteins activate Six2 and Gdnf expression in the metanephric mesenchyme [18,22,44]. As a result of this regulatory role, the UB fails to form in Hox11 mutant mice and the metanephric mesenchyme subsequently undergoes apoptosis [22]. Hox10 genes are also strongly expressed in the developing kidney at multiple stages of embryogenesis. Interestingly, while the expression patterns of both Hox10 and Hox11 are largely overlapping, we observe that the anterior boundary of Hox10 expression in the nephrogenic cord is anterior to that of Hox11. Both Hox10 and Hox11 are similarly expressed in the nephrogenic mesenchyme at E13.5, however, we find that Hox10 exhibits additional expression in the cortical stromal cells and suggests that these genes may be playing a unique role in the stroma. The functional data reported herein confirms this novel role and shows that the cortical stromal cells fail to properly differentiate and integrate into the kidney in Hox10 mutants. This cellular defect results in aberrant ureter branching, decreased nephrogenesis and a loss of kidney capsule formation, phenotypes reminiscent of those reported for Foxd1 [34,36]. In summary, our results indicate that the Hox10 genes play a critical role in regulating cortical stromal cell differentiation and integration in the mammalian kidney. Results Kidney morphology in Hox10 mutants There are three Hox10 paralogs in mammals, Hoxa10, Hoxc10 and Hoxd10. While a previous study has shown that Hox10 triple mutants develop severe skeletal defects [45], the potential role(s) these genes play in kidney organogenesis has not been reported. In order to address this, mice with combinations of mutations for Hoxa10, Hoxc10, and Hoxd10 were generated. Our data indicate that mice with any combination of three mutant alleles within the Hox10 paralogous group have no discernable kidney phenotype. This lack of mutant phenotype is likely due to the functional redundancy that exists among paralogous groups [22,45,46,47,48,49,50]. Consistent with this, combinations of four mutant alleles results in kidney abnormalities and these animals die two to six months after birth. Five-allele mutant mice demonstrate stronger defects and die between zero and eight weeks of age. Finally, Hox10 triple mutants display the most severe phenotypes and die within 24 hours of birth. Overtly, Hox10 triple mutant kidneys are hypoplastic and morphologically underdeveloped as compared to controls (Fig. 1). Histologically, H/E staining at E18.5 reveals that the outer cortex of Hox10 mutant kidneys extends around only a portion of the kidney, the inner cortex is expanded, and the medulla is greatly reduced (Fig. 1C, D). Notably, the medio-lateral position of the kidneys is reversed, which results in the mispositioning of the Hox10 Patterns Kidney Cortical Stroma ureter-pelvic junction (UPJ) to the lateral instead of medial side of the kidney and aberrant ureter routing from the UPJ to the bladder (Fig. 1B). The latter phenotype appears to be secondary to the failure of Hox10 mutant kidneys to detach from the body wall during embryogenesis (Fig. 1F and 2C and data not shown). As a result, the kidneys cannot properly rotate and ascend from the pelvic into the lumbar region as normally occurs in wild-type animals. Lotus Tetragonolobus Lectin (LTL) and Dolichos Biflorus Agglutinin (DBA) staining in control and Hox10 mutant kidneys sectioned through the same frontal plane further demonstrate that Hox10 triple mutant kidneys exhibit multiple organizational defects by E16.5 (Fig. 1E, F). Normally, LTL labels the proximal tubules of mature nephrons that are confined to the cortical region, while DBA labels differentiated collecting ducts that extend radially into the medullary region (Fig. 1E). However, as depicted in Fig. 1F, both LTL and DBA staining are reduced and mislocalized in Hox10 mutants, highlighting the significant patterning defects in these mutants. Hox10 triple mutants exhibit severe ureter branching defects Subsequent to ureteric bud invasion, the ureter begins to branch within the metanephric mesenchyme and normally undergoes six to seven branching events by E14.5 [51]. Additionally, the kidney detaches from the body wall and ascends within the body cavity. In Hox10 mutants, the kidney fails to detach completely from the body wall and the reproductive tract (asterisks in Figure 1F, and Figure 2A, C). To compare the number of branching events and the number of early nephrons present we performed optical projection tomography (OPT) on dissected kidneys from E14.5 control and Hox10 mutant embryos stained with pan-cytokeratin, to label the ureteric tree, and Cadherin-6 to label early nephrons (Fig. 2B, D, Movies S1 and S2, Table S1) [52]. By E14.5, Hox10 mutant kidneys (n of 4) demonstrate a five-fold reduction in the number of ureteric branches compared to the total number of branches observed in controls (n of 3). There is also a three-fold reduction in the number of Cadherin-6-positive bodies, indicating reduced nephron formation in mutant kidneys. The aberrant ureter branching in Hox10 mutants is due to intrinsic signaling defects in the developing kidney In order to test whether the aberrant ureter branching observed in Hox10 mutants might be secondary to its physical inability to properly detach from the body wall and ascend or intrinsic to signaling defects in the kidney, we isolated kidneys from control and Hox10 mutant embryos at E11.5 and cultured them for 72 hours. Initially, the overall size and morphology of the tissue isolated was comparable between control and mutant kidneys (Fig 3A, E). However, the mutant kidneys were noticeably smaller and underdeveloped as compared to controls at the 24- and 48-hour time intervals (Fig 3B–C, F–G). After 72 hours in culture, the kidneys were stained for pan-cytokeratin to label the ureter epithelium and Pax2 to label the nephrogenic mesenchyme. Hox10 mutants develop a distinct morphological patterning defect as compared to controls, similar to what is observed in vivo, with increased length of each ureteric branch and less bifurcations (Fig 3D, H). Hox10 mutants have, on average, a 50% reduction in the number of branches compared to controls (Fig 3I, J). Hence, these results support that the branching defects observed in Hox10 mutants is due to signaling defects in the kidney and not secondary to the failure to detach from the body wall. PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23410