<i>Tbx18</i> expression in the urogenital sinus mesenchyme.

<p>(A) Quantitative reverse transcription PCR detects <i>Tbx18</i> mRNA as early as E14.5, with expression peaking around embryonic day 16.5 (for each stage n ≥ 4). (B, D, F) TBX18 immunohistochemistry shows TBX18 is expressed in the dorsal aspect of the UGS in the region of the forming anterior prostate buds at E16.5 (B), P0 (D), and P3 (F). (C, E, G) Smooth Muscle Actin (SMA) IHC on sections adjacent to TBX18 stains showing the proximity of these two expression domains; TBX18 is detected in only a small subset of SMA-positive cells. (H-K) Lineage tracing analysis of <i>Tbx18</i> expressing cells using the <i>Gt(ROSA)26Sor</i>^{<i>tm4(ACTB-tdTomato</i>,<i>-EGFP)Luo</i>}/J reporter. Green cells express or are descended from <i>Tbx18</i> expressing cells (in the images presented here, the <i>Tomato</i> signal was excluded for clarity). (H-I) P0. (J-K) P35 sections show <i>Tbx18</i> descendants primarily occupy the anterior prostate stroma (J), and dorsal prostate stroma (K). M is Mesenchyme, <b>and E is Epithelium. Scale bars are 100μm B and C, 50μm in D-K.</b></p

<i>Tbx18</i> LOF phenotype in P0 UGS.

<p>(A-I) H&E stains of P0 urogenital sinuses. (A-C) Wild type histology shows high cell density in the mesenchyme surrounding the epithelial prostate buds (A, B), and urethral epithelium is composed of 4–6 cell layers with a smooth apical surface (C). (D-F) 12Gso/<i>Tbx18</i>^<i>LacZ</i> compound heterozygotes present an intermediate phenotype in the UGS mesenchyme and the urethral epithelium. (G-I) <i>Tbx18</i>^{<i>GFP/GFP</i>} mutants have very low mesenchymal cell density surrounding epithelial prostate buds. The urethral epithelium in these mutants is increased in thickness with larger cell volumes (compare arrows in C, F, and I). (J) Measurements of the epithelial thickness in the urethral epithelium. The epithelium on the dorsal side is significantly increased in thickness compared to wild type littermates.</p

Immunohistochemical analysis with stromal and epithelial markers.

<p>We used immunohistochemistry to examine the distribution of stromal and epithelial markers in sections of 5 week-old (A-C) wild-type, (D-I) 12Gso/<i>Tbx18</i>^<i>LacZ</i>, and (J-L) PB-Cre4^<i>+</i>; <i>Tbx18</i>^{<i>flox/flox</i>} anterior prostates. IHC was carried out on slides adjacent to those imaged in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154413#pone.0154413.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154413#pone.0154413.g004" target="_blank">4</a> and include the same regions of the tissue. (A) Luminal epithelial cells stained with an antibody detecting Cytokeratins 8 and 18 (CK8/CK18, green) form a continuous layer on the apical surface of the epithelial compartment in wild type prostate and (D) in distal ductal regions with normal pathology in the 12Gso/<i>Tbx18</i>^<i>LacZ</i> compound heterozygotes. (B, E) Basal epithelial cells stained with Cytokeratin 5 (CK5, green) also stain similarly in the wild type and distal regions of the mutant ducts, forming a discontinuous layer between the apical and basement membrane. (G, H, J, K) In contrast, CK8/CK18 and CK5 staining is significantly reduced in the epithelial compartments of the proximal anterior prostate regions in both 12Gso/<i>Tbx18</i>^<i>LacZ</i> (G, H) and PB-Cre4⁺; <i>Tbx18</i>^{<i>flox/flox</i>} mice (J, K). SMA (red) was co-stained with the Cytokeratin markers to define the smooth muscle layer of the stroma in each section. (C, F) Smooth muscle cells, identified with SMA (red), and fibroblasts, stained with the VIM antibody (green) form apposed but separate layers around the epithelial compartments in wild type animals and in distal regions of normal histology in the anterior prostate of 12Gso/<i>Tbx18</i>^<i>LacZ</i> mice. (I, L) However, in both types of <i>Tbx18</i> mutants we found cells positive for both the VIM and SMA markers (arrow in Fig I, L), indicative of myofibroblasts. In addition, compared to wild type littermates (C), the mutant anterior lobes contained larger numbers of VIM+ cells (green; arrowheads in I, L). Scale bars correspond to 100μm.</p

<i>Tbx18</i> LOF in 12Gso/<i>Tbx18</i>^<i>LacZ</i> mutants leads to prostate abnormalities in adults.

<p>The anterior prostates of five week-old 12Gso/<i>Tbx18</i>^<i>LacZ</i> mutants include stromal hypertrophy and epithelial disorganization, as revealed by Hematoxylin and Eosin (H&E) stains. Sectioned anterior prostates of 12Gso/<i>Tbx18</i>^<i>LacZ</i> mutants (panel A, with areas highlighted in black, blue, and white boxes shown at high resolution in B, E, F respectively) include distal regions with relatively normal morphology (B) indistinguishable from that of wild type littermates (C, D). However, the same sections of mutant prostates show significant stromal hypertrophy (E) and epithelial disorganization and lack a clear boundary between stromal and epithelial compartments (F) in regions proximal to the urethra. (G) Measurement of stromal thickness and numbers of nuclei within the stromal regions confirm that both measurements are significantly different in mutant proximal anterior prostates than in wild type littermate controls. S is stroma; E is epithelium. Scale bars correspond to 100μm in all panels.</p

Functional categories enriched in RNA-seq comparison of E16.5 and E18.5 <i>Tbx18</i> mutant compared to wild type UGS.

<p>Functional categories enriched in RNA-seq comparison of E16.5 and E18.5 <i>Tbx18</i> mutant compared to wild type UGS.</p