Hox10 Genes Function in Kidney Development in the Differentiation and Integration of the Cortical Stroma Alisha R. Yallowitz1¤, Steven M. Hrycaj1, Kieran M. Short2, Ian M. Smyth2,3, Deneen M. Wellik1* 1 Department of Internal Medicine, Division of Molecular Medicine and Genetics University of Michigan, Ann Arbor, Michigan, United States of America, 2 Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia, 3 Department of Anatomy and Developmental Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia Abstract Organogenesis requires the differentiation and integration of distinct populations of cells to form a functional organ. In the kidney, reciprocal interactions between the ureter and the nephrogenic mesenchyme are required for organ formation. Additionally, the differentiation and integration of stromal cells are also necessary for the proper development of this organ. Much remains to be understood regarding the origin of cortical stromal cells and the pathways involved in their formation and function. By generating triple mutants in the Hox10 paralogous group genes, we demonstrate that Hox10 genes play a critical role in the developing kidney. Careful examination of control kidneys show that Foxd1-expressing stromal precursor cells are first observed in a cap-like pattern anterior to the metanephric mesenchyme and these cells subsequently integrate posteriorly into the kidney periphery as development proceeds. While the initial cap-like pattern of Foxd1-expressing cortical stromal cells is unaffected in Hox10 mutants, these cells fail to become properly integrated into the kidney, and do not differentiate to form the kidney capsule. Consistent with loss of cortical stromal cell function, Hox10 mutant kidneys display reduced and aberrant ureter branching, decreased nephrogenesis. These data therefore provide critical novel insights into the cellular and genetic mechanisms governing cortical cell development during kidney organogenesis. These results, combined with previous evidence demonstrating that Hox11 genes are necessary for patterning the metanephric mesenchyme, support a model whereby distinct populations in the nephrogenic cord are regulated by unique Hox codes, and that differential Hox function along the AP axis of the nephrogenic cord is critical for the differentiation and integration of these cell types during kidney organogenesis. Citation: Yallowitz AR, Hrycaj SM, Short KM, Smyth IM, Wellik DM (2011) Hox10 Genes Function in Kidney Development in the Differentiation and Integration of the Cortical Stroma. PLoS ONE 6(8): e23410. doi:10.1371/journal.pone.0023410 Editor: Mai Har Sham, The University of Hong Kong, China Received March 3, 2011; Accepted July 16, 2011; Published August 16, 2011 Copyright: 2011 Yallowitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institutes of Health grant DK071929 (DW), by the University of Michigan’s Training Program in Organogenesis T32-HD007505 (AY), by University of Michigan’s Pediatric Nephrology fellowship T32-DK065517 (AY AND SH), and in part by the National Institutes of Health through the University of Michigan’s Cancer Center Support Grant (5 P30 CA46592). This work was also supported in part by fellowship support from the National Health and Medical Research Council (NH&MRC) and Monash University and Project Grant funding from the NH&MRC (IS and KS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dwellik@umich.edu ¤ Current address: Stony Brook University Medical Center, Department of Pathology, Stony Brook, New York, United States of America Introduction Metanephric kidney development initiates at approximately E10.5 in mice, when the metanephric mesenchyme condenses at the posterior end of the nephrogenic cord adjacent to the hindlimbs. These cells signal to the Wolffian duct to promote evagination of the ureteric bud, which invades the metanephric mesenchyme. Subsequent reciprocal inductive interactions between the nephro- genic mesenchyme and the ureter ultimately lead to the formation of the mature branched kidney [1,2,3]. Initially, Gdnf expression in the metanephric mesenchyme is recognized by the receptors Ret and Gfra1 on the ureteric epithelium and causes both the formation and invasion of the UB into the mesenchyme [4,5,6,7,8,9,10,11, 12,13,14]. After UB invasion, a number of transcription factors including Pax2, Eya1, Wt1, Sall1, and the Hox11 are necessary to maintain Gdnf expression in the mesenchyme, promote continued proliferation and expansion of the mesenchyme and to control the further budding and branching of the epithelial ureteric tree [15,16,17,18,19,20,21,22,23]. In turn, the ureteric tree expresses Fgf2 and Bmp7 to promote survival and proliferation of the mesenchyme and Wnt9b to initiate the formation of cap mesenchyme around the branch tips [24,25,26,27,28]. Undifferen- tiated cap mesenchyme expresses Six2, which regulates nephrogenic progenitor cell renewal [29]. Finally, a portion of the cap mesenchyme expresses Wnt4 and subsequently undergoes epitheli- alization to form nephrons [30,31]. In addition to the nephrogenic mesenchyme and ureter epithelium, a third cell type is present during early kidney organogenesis, the stromal cell population. Stromal cells are first observed just after ureteric bud invasion as a group of cells that surround the condensed mesenchyme [32]. A number of genetic studies have shown that stromal cells play critical roles during kidney organogenesis including producing signals that maintain outer and inner zones of differentiation, regulating pathways involved in the differentiation of nephrons, ureter branching morphogenesis, and in regulating the formation of the kidney capsule [32,33,34,35,36,37,38,39]. At present, the transcription factor Foxd1 is the earliest known marker of stromal cells and is PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23410