<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

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

<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

<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

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

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

<div><p>Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. <em>Bmp4</em> is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of <em>Bmp4</em> in caudal body formation, we utilized a conditional <em>Bmp4</em> mouse allele (Bmp4^flox/flox) and the <em>Isl1 (Islet1)</em>-Cre mouse line. <em>Isl1</em>-Cre is expressed in the peri-cloacal region and the developing hindimb field. <em>Isl1Cre;Bmp4^flox/flox</em> conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that <em>Isl1</em>-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show <em>Bmp4</em> is essential for the aPCM formation independently with <em>Shh</em> signaling. Furthermore, we show <em>Bmp4</em> is a major BMP ligand for caudal body formation as shown by compound genetic analyses of <em>Bmp4</em> and <em>Bmp7</em>. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in <em>Isl1</em>-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.</p> </div

Hindlimb fusion of <i>Bmp4</i> cKO mice.

<p>(A) Diagram of peri-cloacal regions. Peri-cloacal regions (PC-regions) including the base of the umbilical cord and anterior cloacal mesenchyme. hl, hindlimb field. (B, C) In situ hybridization (ISH) analysis reveals <i>Bmp4</i> expression in the peri-cloacal regions (square in C) and hindlimb field at E9.5. (D, E) The <i>R26R-lacZ</i> Cre reporter shows that the <i>Isl1</i>-Cre expressing cells are present in the peri-cloacal regions and the developing hindimb field at E9.5 (D, yellow circle, E). (F–J) Hindlimb fusion shown by gross appearance and by skeletal preparations of <i>Bmp4</i> cKO mice. The fibulae are aberrantly located medially (I) or are absent (J) in the mutants. Red arrowheads indicate the ossified fibula.</p

Defective aPCM formation of <i>Bmp4</i> cKO mice.

<p>(A) <i>Bmp4</i> is expressed in the aPCM at E10.5. (B) Immunohistochemical analysis of pSMAD in the aPCM at E10.5. (C–J) Section in situ hybridization analysis with the aPCM and cloacal marker genes for wild type (C, E, G, I) and mutant embryos (D, F, H, J) at E10.5. (K–N) Immunohistochemical analysis of pSMAD in the aPCM of wild types (K) and mutant embryos (L) at E10.5. (M, N) High magnification images of square region in K and L. The squares in A–L indicate the aPCM.</p

Tissue contribution of <i>Isl1</i>-expressing cells to the caudal body.

<p>(A–H) Expression pattern of <i>Isl1</i> mRNA during caudal body development. (A, E) <i>Isl1</i> is expressed in the lateral plate mesoderm adjacent to the future hindlimb bud and the base of the allantois at E8.5 (bracket in A and E). (B, F) <i>Isl1</i> is expressed in the caudal body region and hindlimb bud at E9.5 (square in B). Its expression is detected in the peri-cloacal regions (square in F). (C, G) <i>Isl1</i> expression is detected in the cloacal region at E10.5 (arrow in C). It is expressed in the URS (arrowhead in G) and cloacal mesenchyme including aPCM (square in G). <i>Isl1</i> expression in the hindlimb bud is reduced at E10.5. (D, H) Its expression is maintained in the developing GT at E11.5 (arrow in D). Its expression is detected in GT mesenchyme and URS (arrowhead in H). Asterisk indicates cloaca. (I–P) The <i>R26R-lacZ</i> Cre reporter shows LacZ staining of caudal body regions in <i>Isl1</i>-mER-Cre-mER embryos at E15.5 after administration of tamoxifen at E8.5–E11.5. Whole-mount view of stained embryos of hindlimb and external genitalia (I–L) and pelvic organs (M–P). <i>Isl1</i>-expressing cells contribute to the hindlimb, external genitalia and bladder. Insets in I–L are high magnification of GT. Ventral GT is located at the bottom. t, tail; hl, hindlimb bud.</p

Deletion of both Nesprin 1 and 2 causes decreased cardiac wall thickness and impaired cardiac function.

<p>(A–D) Left ventricular anterior wall during diastole and systole (LVAWd and LVAWs), Left ventricular posterior wall during diastole and systole (LVPWd and LVPWs) at 5, 10, 25 and 52 weeks. (E) % Fractional Shortening (%FS) at 5, 10, 25, 52 weeks. (F) Heart Rate (HR) at 5, 10, 25, 52 weeks. N = 6–10 mice per condition/timepoint. ANOVA with a post hoc Bonferroni test *p<0.05, N/C = No significant change vs. Wild Type.</p