Hox10 Patterns Kidney Cortical Stroma Figure 8. Hox10 kidneys demonstrate aberrant branching defects and are exposed to elevated levels of Bmp4. (A, D) Bmp4 is normally down-regulated in the body wall around the kidney by E12.5 (A), but this expression is ectopically maintained in Hox10 mutants (D). (B, C) Ret is normally expressed in the tips of the UB, providing an inductive signal that stimulates the growth of new ureter branches. (E, F) Ectopic Ret expression is observed in extended regions along the stalks of the ampulla (arrowheads) in Hox10 mutants. doi:10.1371/journal.pone.0023410.g008 50 mM NH4Cl in PBS at room temperature for 20 minutes, followed by a 20 minute incubation with GSP solution (0.2% gelatin+0.075% saponin (S4521, Sigma) in PBS) at 37uC. Slides were then incubated with 1:250 rhodamine labeled DBA (Vector Labs) and 1:400 fluorescein labeled LTA (Vector Labs) at 4uC overnight in the humidifying chamber. Slides were then incubated with GSP solution at 37uC two times for 20 minutes and rinsed in Figure 9. Model depicting the role of the Hox10 genes in the proper differentiation and integration of the cortical stromal cells during mammalian kidney development. In control animals, the cortical stromal cells are initially concentrated in a cap-like region just anterior to the metanephric kidney. As development progresses, these cells become integrated into the periphery of the kidney. Once integrated, the cortical stromal cells promote proper ureter branching and nephron differentiation. In Hox10 mutants, while the formation of the initial cortical stromal population is unaffected, these cells fail to properly integrate into the kidney periphery. The absence of cortical stromal cells in parts of the developing kidney creates distinct areas of signaling defects that secondarily results in aberrant ureter branching and decreased nephrogenesis. doi:10.1371/journal.pone.0023410.g009 PBS+0.05% tween at room temperature. After washing for 20 minutes with PBS, slides were mounted with Pro-Long Gold, antifade reagent (Invitrogen). OPT imaging of embryonic kidneys E14.5 kidneys were collected in ice cold PBS and fixed in 4% PFA for 10 minutes. They were washed two times in cold PBS and then washed in TBS for 20 minutes. After washing the kidneys in TBS+1% triton (TBSTr) for 20 minutes, they were blocked in TBSTr+1% BSA at room temperature for one hour. They were then incubated overnight at 4uC rocking with anti-pan-cytokeratin (1:100) and anti-Cadherin-6 (1:200) in TBSTr+1% donkey serum. The next day they were washed in TBSTr three to four times at room temperature and one time overnight at 4uC. Kidneys were then incubated overnight at 4uC rocking with donkey anti-mouse Alexafluor 555 (Invitrogen, 1:100) and goat anti-rabbit Alexafluor 488 (Invitrogen, 1:200) diluted in TBSTr+1% donkey serum. After washing four to five times at room temperature and one time overnight at 4uC in TBSTr, the tissue was fixed for 15 minutes in 4% PFA and washed three times 10 minutes in TBS. Kidneys were then stored in TBS+0.1% sodium azide prior to embedding. Stained kidneys were embedded in warm 1% low melting point agarose and left until set. Slices containing the specimen were excised, and glued to aluminium-magnetic mounts. Specimens were then dehydrated in 100% methanol for 6 hours with 3 changes of methanol, and then cleared overnight in Benzyl Alcohol Benzyl Benzoate mixed at a ratio of 1:2. Once clear, samples were imaged in a Bioptonics 3001 OPT scanner (Bioptonics, UK), at maximum resolution of 3.2 mm per pixel zoom. Images were acquired at 0.9 degree intervals, with each frame averaged over 4 images. Quantification/Skeletonisation was done using a Kidney Analysis Application [72]. Rendering/ Visualization was performed using Drishti http://anusf.anu.edu. au/Vizlab/drishti/. Supporting Information Figure S1 Hox11 mRNA expression overlaps with Hoxa11eGFP reporter expression. Hox11 in situ analysis (A) was done on tissue sections from an E13.5 Hoxa11eGFP PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23410