Overactivity or blockade of transforming growth factor-β each generate a specific ureter malformation

Abstract

Transforming growth factor-β (TGFβ) has been reported to be dysregulated in malformed ureters. There exists, however, little information on whether altered TGFβ levels actually perturb ureter development. We therefore hypothesised that TGFβ has functional effects on ureter morphogenesis. Tgfb1, Tgfb2 and Tgfb3 transcripts coding for TGFβ ligands, as well as Tgfbr1 and Tgfbr2 coding for TGFβ receptors, were detected by quantitative polymerase chain reaction in embryonic mouse ureters collected over a wide range of stages. As assessed by in situ hybridisation and immunohistochemistry, the two receptors were detected in embryonic urothelia. Next, TGFβ1 was added to serum-free cultures of embryonic day 15 mouse ureters. These organs contain immature smooth muscle and urothelial layers and their in vivo potential to grow and acquire peristaltic function can be replicated in serum-free organ culture. Such organs therefore constitute a suitable developmental stage with which to define roles of factors that affect ureter growth and functional differentiation. Exogenous TGFβ1 inhibited growth of the ureter tube and generated cocoon-like dysmorphogenesis. RNA sequencing suggested that altered levels of transcripts encoding certain fibroblast growth factors (FGFs) followed exposure to TGFβ. In serum-free organ culture exogenous FGF10 but not FGF18 abrogated certain dysmorphic effects mediated by exogenous TGFβ1. To assess whether an endogenous TGFβ axis functions in developing ureters, embryonic day 15 explants were exposed to TGFβ receptor chemical blockade; growth of the ureter was enhanced, and aberrant bud-like structures arose from the urothelial tube. The muscle layer was attenuated around these buds, and peristalsis was compromised. To determine whether TGFβ effects were limited to one stage, explants of mouse embryonic day 13 ureters, more primitive organs, were exposed to exogenous TGFβ1, again generating cocoon-like structures, and to TGFβ receptor blockade, again generating ectopic buds. As for the mouse studies, immunostaining of normal embryonic human ureters detected TGFβRI and TGFβRII in urothelia. Collectively, these observations reveal unsuspected regulatory roles for endogenous TGFβ in embryonic ureters, fine-tuning morphogenesis and functional differentiation. Our results also support the hypothesis that the TGFβ up-regulation reported in ureter malformations impacts on pathobiology. Further experiments are needed to unravel the intracellular signalling mechanisms involved in these dysmorphic responses. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.