TBX18 regulates the differentiation of periductal smooth muscle stroma and the maintenance of epithelial integrity in the prostate

The T-box transcription factor TBX18 is essential to mesenchymal cell differentiation in several tissues and Tbx18 loss-of-function results in dramatic organ malformations and perinatal lethality. Here we demonstrate for the first time that Tbx18 is required for the normal development of periductal smooth muscle stromal cells in prostate, particularly in the anterior lobe, with a clear impact on prostate health in adult mice. Prostate abnormalities are only subtly apparent in Tbx18 mutants at birth; to examine postnatal prostate development we utilized a relatively long-lived hypomorphic mutant and a novel conditional Tbx18 allele. Similar to the ureter, cells that fail to express Tbx18 do not condense normally into smooth muscle cells of the periductal prostatic stroma. However, in contrast to ureter, the periductal stromal cells in mutant prostate assume a hypertrophic, myofibroblastic state and the adjacent epithelium becomes grossly disorganized. To identify molecular events preceding the onset of this pathology, we compared gene expression in the urogenital sinus (UGS), from which the prostate develops, in Tbx18-null and wild type littermates at two embryonic stages. Genes that regulate cell proliferation, smooth muscle differentiation, prostate epithelium development, and inflammatory response were significantly dysregulated in the mutant urogenital sinus around the time that Tbx18 is first expressed in the wild type UGS, suggesting a direct role in regulating those genes. Together, these results argue that Tbx18 is essential to the differentiation and maintenance of the prostate periurethral mesenchyme and that it indirectly regulates epithelial differentiation through control of stromal-epithelial signaling. Embryos were collected from timed matings of Tbx18Gfp/+ knock-in mutants at E16.5 and E18.5, and genotyped to identify Tbx18Gfp/Gfp null mutants and wild-type (WT) littermates. The urogenital sinus (UGS) was dissected and used to extract RNA from each of three animals of each genotype. The RNA samples were pooled to generate libraries for sequencing.T-box转录因子TBX18（T-box transcription factor TBX18）在多种组织的间充质细胞（mesenchymal cell）分化过程中发挥不可或缺的作用，Tbx18功能丧失会引发严重的器官畸形并导致围产期致死（perinatal lethality）。本研究首次证实，Tbx18对于前列腺导管周平滑肌基质细胞的正常发育至关重要，尤其在前列腺前叶，这对成年小鼠的前列腺健康具有显著影响。Tbx18突变体在出生时仅表现出轻微的前列腺异常；为探究出生后前列腺的发育情况，我们使用了一种存活期较长的低功能等位基因突变体（hypomorphic mutant）以及一种新型的条件性Tbx18等位基因（conditional Tbx18 allele）。与输尿管（ureter）类似，无法表达Tbx18的细胞无法正常凝聚形成前列腺基质的导管周平滑肌细胞。但与输尿管不同的是，突变体前列腺的导管周基质细胞会呈现肥大性肌成纤维细胞（myofibroblastic）状态，且相邻的上皮组织会出现明显的结构紊乱。为明确该病理发生前的分子事件，我们在两个胚胎发育阶段，对前列腺的起源组织——尿生殖窦（urogenital sinus, UGS）——的基因表达情况进行了比较，实验对象为Tbx18纯合缺失突变体（Tbx18-null）与野生型（wild-type, WT）同窝幼崽。在野生型UGS首次表达Tbx18的时期前后，突变体UGS中调控细胞增殖、平滑肌分化、前列腺上皮发育以及炎症反应的基因均出现显著表达失调，这提示Tbx18对这些基因具有直接调控作用。综上，上述结果表明Tbx18对于前列腺尿道周间充质的分化与维持至关重要，且它可通过调控基质-上皮信号通路，间接调节上皮细胞的分化。我们通过对Tbx18Gfp/+敲入突变体（Tbx18Gfp/+ knock-in mutant）进行定时交配，在胚胎发育第16.5天（E16.5）与第18.5天（E18.5）收集胚胎，并通过基因分型鉴定出Tbx18Gfp/Gfp纯合缺失突变体与野生型同窝幼崽。随后解剖分离尿生殖窦，从每种基因型的3只个体中提取RNA，将RNA样本混合以构建测序文库。