Protein kinase 7 regulating extracellular matrix assemblies and intracellular RAC1 and myosin II activities in the mesenchyme is important to Wolffian duct morphogenesis.

Wolffian/epididymal duct morphogenesis highly coordinates with its specialized function of providing microenvironment for sperm maturation. Without normal development of Wolffian duct, male infertility will result. Therefore, it is important to understand the cellular and molecular mechanisms that regulated Wolffian duct morphogenesis. Our previous study showed that mediolateral intercalation of epithelial cells was a major driver of ductal elongation. In addition, radial intercalation of mesenchymal cells surrounding the duct also played roles in Wolffian duct morphogenesis, and all of these cell re-arrangements were regulated by protein tyrosine kinase 7 (PTK7), a member of the planar cell polarity (PCP) non-canonical Wnt pathway. In this study, we showed that PTK7 regulated assemblies of extracellular matrix (ECM) at the basement membrane and throughout the mesenchyme. Abnormal assembly of laminin, collagen IV, and nephronectin at the basement membrane and fibrosis-like deposition of fibrilla collagen in the interstitium were observed in Ptk7 knockout Wolffian ducts. Meanwhile, PTK7 regulated activity levels of small GTPase RAC1 and myosin II in the mesenchyme of the Wolffian duct. Activity levels of RAC1 and myosin II decreased in the Ptk7 knockout mesenchyme compared to controls. When in-vitro-cultured Wolffian ducts were treated with collagenase IV, cross-link of fibrilla collagen was reduced, Wolffian duct elongation and coiling was reduced significantly, and cyst-like bulge was developed in the epithelial duct. When Wolffian ducts were treated with RAC1 inhibitor NSC23766, fibrilla collagen in the mesenchyme was disassembled, and Wolffian duct elongation was suppressed significantly. Our finding suggested that PTK7 regulated ECM assembly in the mesenchyme likely through regulating RAC1 and myosin II activities. Dynamic assembly and remodeling of ECM were important to Wolffian duct morphogenesis. Examination of gene expression in PTK7-/- Wolffian ducts吴弗管（Wolffian duct）/附睾管的形态发生与其为精子成熟提供微环境的特化功能高度协同。若吴弗管发育异常，将导致雄性不育。因此，阐明调控吴弗管形态发生的细胞与分子机制具有重要意义。既往研究表明，上皮细胞的中外侧插层是导管伸长的主要驱动因素。此外，导管周围间充质细胞的径向插层也参与吴弗管的形态发生，而上述所有细胞重排过程均受蛋白酪氨酸激酶7（protein tyrosine kinase 7, PTK7）调控——该蛋白属于平面细胞极性（planar cell polarity, PCP）非经典Wnt通路家族成员。本研究发现，PTK7可调控基底膜及整个间充质区域的细胞外基质（extracellular matrix, ECM）组装。在Ptk7基因敲除的吴弗管中，可观察到层粘连蛋白、IV型胶原与肾连蛋白在基底膜处的组装异常，以及间质内出现类纤维化的原纤维胶原沉积。同时，PTK7可调控吴弗管间充质中小GTP酶RAC1与肌球蛋白II的活性水平。与对照组相比，Ptk7基因敲除的间充质中RAC1与肌球蛋白II的活性均有所下降。当对体外培养的吴弗管施加IV型胶原酶处理时，原纤维胶原的交联作用减弱，吴弗管的伸长与卷曲程度显著降低，且上皮导管出现囊状膨出。若用RAC1抑制剂NSC23766处理吴弗管，则间充质内的原纤维胶原发生解聚，吴弗管的伸长过程亦受到显著抑制。本研究结果提示，PTK7或可通过调控RAC1与肌球蛋白II的活性，实现对间充质区域ECM组装的调控。细胞外基质的动态组装与重塑对吴弗管的形态发生至关重要。对PTK7敲除（PTK7-/-）吴弗管的基因表达检测