A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development

Abstract

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme

“…Because expression of both genes was restricted to the inner layer of the UM, we questioned whether their expression depends on SHH or WNT signals emanating from the UE (Trowe et al, 2012;Yu et al, 2002). To address this question, we used a conditional pathway deletion approach, with a Tbx18 cre line, which mediates recombination in the entire UM starting from E11.5 (Airik et al, 2010;Bohnenpoll et al, 2013), and floxed alleles of the unique mediator of SHH signaling, Smo (Long et al, 2001), and of canonical WNT signaling, Ctnnb1 (Brault et al, 2001), as previously reported (Bohnenpoll et al, 2017b;Trowe et al, 2012). Loss of Smo had no effect on Tbx2 and Tbx3 expression in the UM at E12.5 (Fig.…”

“…At E11.5, SHH activates a SMO-dependent pathway in the UM that is required for survival of cells in the outer layer, for proliferation and SMC differentiation of cells of the inner layer, and also for urothelial proliferation and differentiation. The proliferation and differentiation functions of SHH signaling are mediated by the forkhead transcription factor FOXF1, which, in turn, induces expression of and synergizes with BMP4 in SMC differentiation (Bohnenpoll et al, 2017b;Mamo et al, 2017). At E12.5, WNT7B and WNT9B activate the canonical WNT pathway in cells of the inner mesenchymal layer, disruption of which results in reduced proliferation and failed SMC differentiation of these cells, and expansion of the adventitial fate to the inner layer (Trowe et al, 2012).…”

Delayed onset of smooth muscle cell differentiation leads to hydroureter formation in mice with conditional loss of the zinc finger transcription factor gene Gata2 in the ureteric mesenchyme

“…To identify molecular changes that may cause defective SMC differentiation in Gata2cKO ureters, we screened (using in situ hybridisation) for activity of pathways and expression of genes that have been implicated in this programme: Ptch1 , target of SHH signalling ; Bmp4 ; Id2 , target of BMP signalling ; Axin2 , target of WNT signalling ; Rarb , target of RA signalling ; and the transcription factor genes Foxf1 , Tbx18 , Tszh3 , and Sox9 . At E14.5, all of these genes were expressed in the inner UM of mutant ureters as in the control except for Tbx18 , Tshz3 , and Rarb , which were up‐regulated (supplementary material, Figure S9).…”

“…Ureters were explanted on 0.4 μm polyester membrane Transwell supports (#3450; Corning Inc, Lowell, MA, USA) and cultured at the air–liquid interface as previously described . BMS493 (#3509; Tocris BioScience, Minneapolis, MN, USA) and RA (#0695; Tocris) were dissolved in DMSO and added to the medium at a final concentration of 1 μ m .…”

“…The onset of SMC differentiation is controlled by a complex interplay of signalling modules and transcription factor activities. Sonic hedgehog (SHH) from the UE acts in the UM via the transcription factor FOXF1, which, in turn, induces the secreted signalling molecule BMP4 with which it cooperates in SMC activation . Epithelial WNT signals restrict via mesenchymal TBX2 and TBX3 adventitial fates to the outer layer and induce SMC differentiation in the inner layer .…”