Andreas Kispert

Red pulp macrophages (RPM) and bone marrow macrophages (BMM) are iron-recycling cells, involved in iron homeostasis and erythropoiesis. Here we show, by conditional deletion strategies using Cx3cr1Cre-mediated targeting to inactivate Notch signalling components in mice, that canonical Notch2 signalling regulates the development of RPM and BMM. Loss of functional Notch2, or its nuclear mediator Rbpj, impair RPM and BMM development and cause iron overload in the spleen and bone marrow. In the absence of Notch2, prototypic RPM genes are downregulated, which is accompanied by splenic extramedullary haematopoiesis and changes in splenic microarchitecture. Treatment of mice with an anti-Notch2 blocking antibody recapitulates the impaired erythrophagocyte and de novo extramedullary hematopoiesis phenotypes characteristic to the genetic model. Furthermore, early postnatal transfer of bone marrow and fetal liver progenitors rescues the defects in RPM and BMM in a Notch2-dependent manner, demonstrating the potential to restore defective tissue resident macrophage niches by Notch-competent progenitors. Thus, our study demonstrates that canonical Notch2 signalling is required for the development, maintenance, and function of iron-recycling macrophages.

Background: The coordinated development of the mesenchymal and epithelial primordia of the murine ureter requires intense tissue communication. Previous studies have revealed the crucial role of the FGFR2-SHH-FOXF1-BMP4 signaling axis in the proliferation and differentiation programs of both the epithelium and the mesenchyme. Canonical (β-Catenin/CTNNB1-dependent) WNT signaling in the mesenchymal primordium has been reported to influence mesenchymal development by enhancing proliferation and favoring the smooth muscle cell fate over adventitial fibrocytes. Methods: To investigate the role of mesenchymal WNT signaling in early ureter development further, we analyzed the ureters of Ctnnb1 -deficient mouse mutants for cellular and molecular changes during embryogenesis. Using pharmacological inhibition and activation approaches in explant cultures of embryonic ureters, we assessed the contribution of altered signaling activities to differentiation changes in Ctnnb1 -deficient ureters. Transcriptional profiling of embryonic ureters with short-term WNT signaling inhibition allowed us to identify the primary targets of this signaling pathway in the ureteric mesenchyme. Results: We demonstrate that both mesenchymal and epithelial differentiation are impaired in ureters lacking mesenchymal Ctnnb1 . This defect is linked to an inability to activate pro-differentiation transcription factors in either tissue primordia. Epithelial changes can be attributed, at least in part, to the loss of BMP4 signaling and the gain of canonical WNT signaling in the epithelial primordium. Mesenchymal WNT signaling directly activates transcription of multiple components of the SHH-FOXF1-BMP4 signaling axis, thereby enhancing this module in the inner region of the ureteric mesenchyme. Conclusions: Our work improves the understanding of the signaling network that coordinates cytodifferentiation in the early ureter. Mesenchymal WNT signaling plays a central role in mesenchymal fate decisions but also in promoting epithelial differentiation through enhancement of the SHH-FOXF1-BMP4 axis and inhibition of epithelial WNT-signaling.

The urothelium is a stratified epithelium that lines the inner surfaces of organs in the urinary drainage system. It has a layered composition of basal, intermediate, and large superficial cells. The process, by which this cytoarchitecture develops from uncommitted precursor cells is not well understood. In this study, we analyzed the cellular and molecular functions of the transcription factor ΔNP63 in urothelial development in the murine ureter. ΔNP63 expression begins in epithelial progenitors of this organ at embryonic day (E)14.5 and is confined to basal and intermediate cells from late fetal stages onwards. In mice with a conditional loss of ΔNp63 in the epithelial progenitors, the ureteric urothelium presented as a folded monolayer composed predominantly of partially differentiated superficial cells around birth. At E15.5, the proliferation and division planes of epithelial cells remained unaffected. However, genes essential for cell adhesion and division in other epithelial stratification programs were downregulated, as were genes involved in B and I cell differentiation. In contrast, Ihh expression in the epithelium increased concomitantly with the premature formation of the lamina propria. Our results suggest that a conserved ΔNP63 gene regulatory module is responsible for epithelial stratification and the differentiation of basal and intermediate cells, as well as the timely differentiation of the lamina propria in fetal ureter development. The urothelial cytoarchitecture was progressively reconstituted in ΔNp63-deficient ureters after birth. This suggests that a few NP63 ⁺ epithelial progenitors, which have escaped recombination, can undergo rapid proliferative expansion and differentiation into I and B cells.

Mutations in BMP4 have been associated with malformations of the urinary tract in human patients. Genetic studies in mice have shown that these defects are linked to the expression of Bmp4 in the mesenchymal primordium of the ureter, where it acts as a critical signal for coordinated cytodifferentiation of the mesenchymal and epithelial tissues. Here, we used unbiased transcriptional profiling of ureters with genetic depletion of Bmp4 and pharmacological inhibition of BMP4 signaling to decipher the gene regulatory network controlled by BMP4 in the early ureter, focusing on transcription factors as possible drivers of cytodifferentiation. We show that in Bmp4-deficient ureters, expression of Grhl3, Msx2, Pparg, Trp63, and Foxa1 in the epithelial compartment and of Gata6, Hopx, Id2, Id4, Myocd, Snai1, and Tbx18 in the mesenchymal primordium is reduced. Expression of Msx2, Pparg, Gata6, Id genes, Tbx18, and Snai1 requires direct BMP4 signaling input, whereas reduced expression of the other genes is likely due to secondary changes, including increased retinoic acid signaling. Conditional gene targeting of Smad4 revealed that BMP4-dependent activation of transcription factor genes is mediated in part by SMAD effectors in both ureteral tissues. Thus, our work links BMP4 (signaling) to known transcriptional regulators of ureteral cytodifferentiation and uncovers additional factors that may be relevant to this program.

The urothelium is a stratified epithelium with an important barrier function in the urinary drainage system. The differentiation and maintenance of the three major urothelial cell types (basal, intermediate and superficial cells) is incompletely understood. Here, we show that mice with a conditional deletion of the transcription factor gene peroxisome proliferator activated receptor gamma (Pparg) in the ureteric epithelium have a dilated ureter at postnatal stages with a urothelium consisting of a layer of undifferentiated luminal cells and a layer of proliferating basal cells. Molecular analysis of fetal stages revealed that the expression of a large number of genes is not activated in superficial cells and that of a few genes, including Shh, is not activated in intermediate and basal cells. Pharmacological activation of SHH signaling in explant cultures of perinatal Pparg-deficient ureters reduced ureteral width and urothelial cell number to normal levels, increased the number of intermediate cells and slightly reduced basal cell proliferation. Our data suggest that PPARG independently activates the expression of structural genes in superficial cells and of Shh in basal and intermediate cells, and that both functions contribute to urothelial integrity.

Malformations of the ureter are among the most common birth defects in humans. Although some of these anomalies are asymptomatic, others are clinically relevant, causing perinatal lethality or progressing to kidney failure in childhood. The genetic causes and developmental aetiology of ureteral anomalies are difficult to study in humans; however, embryological and genetic analyses in the mouse have provided insights into the complex developmental programmes that govern ureter formation from simple tissue primordia, and the pathological consequences that result from disruption of these programmes. Abnormalities in the formation of the nephric duct and ureteric bud lead to changes in the number of ureters (and kidneys), whereas the formation of ectopic ureteric buds, failure of the nephric duct to target the cloaca or failure of the distal ureter to mature underlie vesicoureteral reflux, ureter ectopia, ureterocoele and subsequent hydroureter. Alterations in ureter specification, early growth or cyto-differentiation programmes have now also been associated with various forms of perinatal hydroureter and hydronephrosis as a consequence of functional obstruction. The characterization of cellular processes and molecular drivers of ureterogenesis in the mouse may not only aid understanding of the aetiology of human ureteral anomalies, improve prognostication and benefit the development of therapeutic strategies, but may also prove important for efforts to generate a bioartificial organ.

In mammalian ureters, the lamina propria presents as a prominent layer of connective tissue underneath the urothelium. Despite its important structural and signaling functions, little is known how the lamina propria develops. Here, we show that in the murine ureter the lamina propria arises at late fetal stages and massively increases by fibrocyte proliferation and collagen deposition after birth. WNT, SHH, BMP4 and retinoic acid signaling are all active in the common mesenchymal progenitor of smooth muscle cells and lamina propria fibrocytes. However, around birth, the lamina propria becomes a target for epithelial WNT and SHH signals and a source of BMP4 and retinoic acid. SHH and WNT signaling promote lamina propria and smooth muscle cell differentiation and proliferation at fetal and early postnatal stages, whereas BMP4 signaling is required for early smooth muscle cell differentiation but not for its later maintenance. Our findings suggest that, in the presence of SHH and WNT signaling, it is the modulation of BMP4 signaling which is the major determinant for the segregation of lamina propria and smooth muscle cells.

Around 180 genes have been associated with congenital anomalies of the kidney and urinary tract (CAKUT) in mice, and represent promising novel candidate genes for human CAKUT. In whole-exome sequencing data of two siblings with genetically unresolved multicystic dysplastic kidneys (MCDK), prioritizing variants in murine CAKUT-associated genes yielded a rare variant in the teashirt zinc finger homeobox 3 (TSHZ3) gene. Therefore, the role of TSHZ3 in human CAKUT was assessed. Twelve CAKUT patients from 9/301 (3%) families carried five different rare heterozygous TSHZ3 missense variants predicted to be deleterious. CAKUT patients with versus without TSHZ3 variants were more likely to present with hydronephrosis, hydroureter, ureteropelvic junction obstruction, MCDK, and with genital anomalies, developmental delay, overlapping with the previously described phenotypes in Tshz3-mutant mice and patients with heterozygous 19q12-q13.11 deletions encompassing the TSHZ3 locus. Comparable with Tshz3-mutant mice, the smooth muscle layer was disorganized in the renal pelvis and thinner in the proximal ureter of the nephrectomy specimen of a TSHZ3 variant carrier compared to controls. TSHZ3 was expressed in the human fetal kidney, and strongly at embryonic day 11.5-14.5 in mesenchymal compartments of the murine ureter, kidney, and bladder. TSHZ3 variants in a 5′ region were more frequent in CAKUT patients than in gnomAD samples (p < 0.001). Mutant TSHZ3 harboring N-terminal variants showed significantly altered SOX9 and/or myocardin binding, possibly adversely affecting smooth muscle differentiation. Our results provide evidence that heterozygous TSHZ3 variants are associated with human CAKUT, particularly MCDK, hydronephrosis, and hydroureter, and, inconsistently, with specific extrarenal features, including genital anomalies.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell–specific overexpression of Sox9 , which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell–specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

The murine kidney and ureter develop in a regionalized fashion from the ureteric bud and its surrounding mesenchyme. Whereas the factors that establish the metanephric cell lineages have been well characterized, much less is known about the molecular cues that specify the ureter. Here, we have identified a crucial patterning function in this process for Tbx18, a T-box transcription factor gene specifically expressed in the mesenchymal primordium of the ureter. Using misexpression and loss-of-function mice combined with molecular profiling approaches, we show that Tbx18 is required and sufficient to repress metanephric mesenchymal gene programs. We identify Wt1 as a functional target of TBX18. Our work suggests that TBX18 acts as a permissive factor in ureter specification by generating a mesenchymal domain around the distal ureteric bud where SHH and BMP4 signaling can occur.