Hox10 Patterns Kidney Cortical Stroma Figure 7. Cortical stromal cells fail to properly differentiate and the kidney capsule does not form in Hox10 mutants. (A) In control embryos, Foxd1 is expressed in the cortical stromal cells that surround the kidney at E15.5. (B) Foxd1 signal in Hox10 mutants is restricted peripherally in the developing kidney (arrowheads). (C–F) Pbx1 and Raldh2 are known markers for stromal cell differentiation that are normally expressed in the nephrogenic zone and cortical stroma of the developing kidney in control embryos (C, E). The expression of both of these genes is restricted (D, F arrowheads) in a pattern similar to what is observed for Foxd1 in Hox10 mutants (B). (G) Tenascin-C is expressed in differentiated cortical stromal cells in controls. (H) Tenascin-C expression is lost in the kidney periphery in Hox10 mutants (arrowheads). (I–J) Sfrp1 expression in control (I) and Hox10 mutant (J) E14.5 embryos. Sfrp1 is strongly expressed in the kidney capsule of control animals (J) and is missing in the periphery (arrowheads) of Hox10 mutant kidneys (J). doi:10.1371/journal.pone.0023410.g007 these early regulatory signals are required for the appropriate differentiation and integration of the kidney cortical stroma cells. Materials and Methods Animals and histology Generation of Hox10 mutant embryos was previously described [45]. Embryos and kidneys were dissected in PBS, fixed in formalin for one to three hours, and dehydrated through graded alcohols and stored in 70% ethanol at 4uC. Embryos were vacuum-embedded in paraffin, sectioned at 5 mm and stained with hematoxylin and eosin. A piece of tissue from the embryo was used for genotyping. All animal experiments performed in this report were reviewed and approved by the University of Michigan’s Committee on Use and Care of Animals, Protocol #08787, under Animal Welfare Assurance #A3114-01 on file with the NIH Office of Laboratory Animal Welfare. In situ hybridization Whole mount in situ hybridization was performed as previously described [22,66]. For section in situ hybridization, embryos were collected in PBS and fixed overnight in 4% paraformaldehyde in PBS (PFA) at 4uC. Embryos were then rinsed in PBS and immersed in 30% sucrose at 4uC overnight prior to embedding into OCT media. 20 to 30 mm frozen sections were cut and slides were stored at 280uC. In situ hybridization (ISH) was performed as previously described [37,67]. Prior to ISH on sections of Hoxa11eGFP tissue, slides were rinsed in PBS and fluorescent images were taken on an Olympus BX-51 upright light microscope with an Olympus DP70 camera. Hoxa10, Hoxc10, and Hoxd10 in situ probes were previously described [68,69,70]. Immunohistochemistry (IHC) For IHC on sections, embryos were processed and sectioned as described above for section ISH. IHC using antibodies to E- cadherin (R&D Systems) and smooth muscle actin (Cy3-conjugat- ed mouse monoclonal, Sigma) were performed as previously described [71]. Anti-E-cadherin was used in a dilution of 1:100 and detected by a 1:500 dilution of donkey anti-goat Alexafluor 488 (Invitrogen). Anit-smooth muscle actin was used in a dilution of 1:400. Immunohistochemical (IHC) localization of pan- cytokeratin (Sigma) on whole embryonic kidneys at E12.5 and E13.5 was performed as previously described [17,52]. Anti-pan- cytokeratin was used in a dilution of 1:200 and detected by a 1:300 dilution of goat anti-mouse FITC (Jackson Immunolabs). Lectin immunohistochemistry was performed as previously described [37]. After washing in PBS, slides were incubated in PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23410