Permissive ureter specification by TBX18-mediated repression of metanephric gene expression.

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.

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.

The establishment of the peristaltic machinery of the ureter is precisely controlled to cope with the onset of urine production in the fetal kidney. Retinoic acid (RA) has been identified as a signal that maintains the mesenchymal progenitors of the contractile smooth muscle cells (SMCs), while WNTs, SHH, and BMP4 induce their differentiation. How the activity of the underlying signalling pathways is controlled in time, space, and quantity to activate coordinately the SMC programme is poorly understood. Here, we provide evidence that the Zn-finger transcription factor GATA2 is involved in this crosstalk. In mice, Gata2 is expressed in the undifferentiated ureteric mesenchyme under control of RA signalling. Conditional deletion of Gata2 by a Tbx18cre driver results in hydroureter formation at birth, associated with a loss of differentiated SMCs. Analysis at earlier stages and in explant cultures revealed that SMC differentiation is not abrogated but delayed and that dilated ureters can partially regain peristaltic activity when relieved of urine pressure. Molecular analysis identified increased RA signalling as one factor contributing to the delay in SMC differentiation, possibly caused by reduced direct transcriptional activation of Cyp26a1, which encodes an RA-degrading enzyme. Our study identified GATA2 as a feedback inhibitor of RA signalling important for precise onset of ureteric SMC differentiation, and suggests that in a subset of cases of human congenital ureter dilatations, temporary relief of urine pressure may ameliorate the differentiation status of the SMC coat. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mesothelial mobilization in the developing lung and heart differs in timing, quantity, and pathway dependency.

The mesothelial lining of the lung, the visceral pleura, and of the heart, the epicardium, derive from a common multipotent precursor tissue, the mesothelium of the embryonic thoracic cavity that also contributes to organ-specific mesenchymal cell types. Insight into mesothelial mobilization and differentiation has prevailedin the developing heart while the mesenchymal transition and fate of the visceral pleura are poorly understood. Here, we use the fact that the early mesothelium of both the lung and the heart expresses the transcription factor gene Wt1, to comparatively analyze mesothelial mobilization in the two organs by a genetic cre-loxP-based conditional approach. We show that epicardial cells are mobilized in a large number between E12.5 and E14.5, whereas pleural mobilization occurs only sporadically and variably in few regions of the lung in a temporally highly confined manner shortly after E12.5. Mesothelium-specific inactivation of unique pathway components using a Wt1creERT2 line excluded a requirement for canonical WNT, NOTCH, HH, TGFB, PDGFRA, and FGFR1/FGFR2 signaling in the mesenchymal transition of the visceral pleura but indicated a deleterious effect of activated WNT, NOTCH, and HH signaling on lung development. Epicardial mobilization was negatively impacted on by loss of HH, PDGFRA, FGFR1/2 signaling. Epicardial overactivation of WNT, NOTCH, and HH disturbed epicardial and myocardial integrity. We conclude that mesothelial mobilization in the developing lung and heart differs in timing, quantity and pathway dependency, indicating the organ specificity of the program.

Retinoic acid signaling maintains epithelial and mesenchymal progenitors in the developing mouse ureter.

The differentiated cell types of the mature ureter arise from the distal ureteric bud epithelium and its surrounding mesenchyme. Uncommitted epithelial cells first become intermediate cells from which both basal and superficial cells develop. Mesenchymal progenitors give rise to separated layers of adventitial fibrocytes, smooth muscle cells and lamina propria fibrocytes. How progenitor expansion and differentiation are balanced is poorly understood. Here, we addressed the role of retinoic acid (RA) signaling in these programs. Using expression analysis of components and target genes, we show that pathway activity is restricted to the mesenchymal and epithelial progenitor pools. Inhibition of RA signaling in ureter explant cultures resulted in tissue hypoplasia with a relative expansion of smooth muscle cells at the expense of lamina propria fibroblasts in the mesenchyme, and of superficial cells at the expense of intermediate cells in the ureteric epithelium. Administration of RA led to a slight reduction of smooth muscle cells, and almost completely prevented differentiation of intermediate cells into basal and superficial cells. We identified cellular programs and transcriptional targets of RA signaling that may account for this activity. We conclude that RA signaling is required and sufficient to maintain mesenchymal and epithelial progenitors in early ureter development.

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

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).

BMP4 uses several different effector pathways to regulate proliferation and differentiation in the epithelial and mesenchymal tissue compartments of the developing mouse ureter.

Heterozygous loss of Bmp4 results both in humans and mice in severe malformation of the urinary tract. These defects have at least partially been attributed to loss of expression of Bmp4 in the ureteric mesenchyme, yet the cellular and molecular function of this signal as well as its effector pathways in this tissue have remained incompletely resolved. Here, we show that mice with a conditional deletion of Bmp4 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional and physical ureter obstruction. Proliferation in both the mesenchymal and epithelial progenitor pools was severely reduced and smooth muscle cell and urothelial differentiation programs were not activated. Epithelial expression of P-ERK1/2, P-AKT and P-P38, and mesenchymal expression of P-SMAD1/5/9, P-P38 and P-AKT were abrogated. Pharmacological inhibition and activation experiments in ureter cultures defined AKT as the most relevant downstream effector for epithelial and mesenchymal proliferation as well as for epithelial differentiation. Epithelial proliferation and differentiation were also influenced by P-38 and ERK1/2, while SMAD signaling, together with AKT and P-38, were required for smooth muscle cell differentiation. Our analysis suggests that BMP4 is the signal that couples the proliferation and differentiation programs in the epithelial and mesenchymal tissue compartments of the developing ureter by different downstream effectors, most importantly AKT and SMAD.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Diversification of Cell Lineages in Ureter Development.

The mammalian ureter consists of a mesenchymal wall composed of smooth muscle cells and surrounding fibrocytes of the tunica adventitia and the lamina propria and an inner epithelial lining composed of layers of basal, intermediate, and superficial cells. How these cell types arise from multipotent progenitors is poorly understood. Here, we performed marker analysis, cell proliferation assays, and genetic lineage tracing to define the lineage relations and restrictions of the mesenchymal and epithelial cell types in the developing and mature mouse ureter. At embryonic day (E) 12.5, the mesenchymal precursor pool began to subdivide into an inner and outer compartment that began to express markers of smooth muscle precursors and adventitial fibrocytes, respectively, by E13.5. Smooth muscle precursors further diversified into lamina propria cells directly adjacent to the ureteric epithelium and differentiated smooth muscle cells from E16.5 onwards. Uncommitted epithelial progenitors of the ureter differentiated into intermediate cells at E14.5. After stratification into two layers at E15.5 and three cell layers at E18.5, intermediate cells differentiated into basal cells and superficial cells. In homeostasis, proliferation of all epithelial and mesenchymal cell types remained low but intermediate cells still gave rise to basal cells, whereas basal cells divided only into basal cells. These studies provide a framework to further determine the molecular mechanisms of cell differentiation in the tissues of the developing ureter.

Fgfr2 is required for the expansion of the early adrenocortical primordium.

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from intermediate mesoderm of the anterior urogenital ridge. Previous work has pinpointed a minor role of the FGFR2IIIb isoform in expansion and differentiation of the fetal adrenal cortex in mice but did not address the complete role of FGFR2 and FGFR1 signaling in adrenocortical development. Here, we show that a Tbx18(cre) line mediates specific recombination in the coelomic epithelium of the anterior urogenital ridge which gives rise by a delamination process to the adrenocortical primordium. Mice with conditional (Tbx18(cre)-mediated) deletion of all isoforms of Fgfr2 exhibited severely hypoplastic adrenal glands around birth. Cortical cells were dramatically reduced in number but showed steroidogenic differentiation and zonation. Neuroendocrine chromaffin cells were also reduced and formed a cell cluster adjacent to but not encapsulated by steroidogenic cells. Analysis of earlier time points revealed that the adrenocortical primordium was established in the intermediate mesoderm at E10.5 but that it failed to expand at subsequent stages. Our further experiments show that FGFR2 signaling acts as early as E11.5 to prevent apoptosis and enhance proliferation in adrenocortical progenitor cells. FGFR1 signaling does not contribute to early adrenocortical development. Our work suggests that FGFR2IIIb and IIIc isoforms largely act redundantly to promote expansion of the adrenocortical primordium.

Ureter growth and differentiation.

The mammalian ureter is a slender tube that connects the renal pelvis with the bladder. It allows the unidirectional movement of urine by means of a peristaltically active smooth muscle layer that together with fibroelastic material ensheathes a water-impermeable multilayered urothelium. The ureteric urothelium as well as the outer mesenchymal coat arise from undifferentiated precursor tissues, the distal ureteric bud and its surrounding mesenchyme, respectively. Specification, growth and differentiation of these ureteric precursor tissues are tightly linked to each other, and are highly integrated with those of the adjacent rudiments of kidney and bladder. Here, we review the current knowledge on the cellular mechanisms as well as the molecular players that guide development of the tissue architecture of the ureter and its peristalsis.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.

Canonical Wnt signaling regulates the proliferative expansion and differentiation of fibrocytes in the murine inner ear.

Otic fibrocytes tether the cochlear duct to the surrounding otic capsule but are also critically involved in maintenance of ion homeostasis in the cochlea, thus, perception of sound. The molecular pathways that regulate the development of this heterogenous group of cells from mesenchymal precursors are poorly understood. Here, we identified epithelial Wnt7a and Wnt7b as possible ligands of Fzd-mediated ß-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated periotic mesenchyme (POM). Mice with a conditional deletion of Ctnnb1 in the POM exhibited a complete failure of fibrocyte differentiation, a severe reduction of mesenchymal cells surrounding the cochlear duct, loss of pericochlear spaces, a thickening and partial loss of the bony capsule and a secondary disturbance of cochlear duct coiling shortly before birth. Analysis at earlier stages revealed that radial patterning of the POM in two domains with highly condensed cartilaginous precursors and more loosely arranged inner mesenchymal cells occurred normally but that proliferation in the inner domain was reduced and cytodifferentiation failed. Cells with mis/overexpression of a stabilized form of Ctnnb1 in the entire POM mesenchyme sorted to the inner mesenchymal compartment and exhibited increased proliferation. Our analysis suggests that Wnt signals from the cochlear duct epithelium are crucial to induce differentiation and expansion of fibrocyte precursor cells. Our findings emphasize the importance of epithelial-mesenchymal signaling in inner ear development.

Tbx18 expression demarcates multipotent precursor populations in the developing urogenital system but is exclusively required within the ureteric mesenchymal lineage to suppress a renal stromal fate.

The mammalian urogenital system derives from multipotent progenitor cells of different germinal tissues. The contribution of individual sub-populations to specific components of the mature system, and the spatiotemporal restriction of the respective lineages have remained poorly characterized. Here, we use comparative expression analysis to delineate sub-regions within the developing urogenital system that express the T-box transcription factor gene Tbx18. We show that Tbx18 is transiently expressed in the epithelial lining and the subjacent mesenchyme of the urogenital ridge. At the onset of metanephric development Tbx18 expression occurs in a band of mesenchyme in between the metanephros and the Wolffian duct but is subsequently restricted to the mesenchyme surrounding the distal ureter stalk. Genetic lineage tracing reveals that former Tbx18(+) cells of the urogenital ridge and the metanephric field contribute substantially to the adrenal glands and gonads, to the kidney stroma, the ureteric and the bladder mesenchyme. Loss of Tbx18 does not affect differentiation of the adrenal gland, the gonad, the bladder and the kidney. However, ureter differentiation is severely disturbed as the mesenchymal lineage adopts a stromal rather than a ureteric smooth muscle fate. DiI labeling and tissue recombination experiments show that the restriction of Tbx18 expression to the prospective ureteric mesenchyme does not reflect an active condensation process but is due to a specific loss of Tbx18 expression in the mesenchyme out of range of signals from the ureteric epithelium. These cells either contribute to the renal stroma or undergo apoptosis aiding in severing the ureter from its surrounding tissues. We show that Tbx18-deficient cells do not respond to epithelial signals suggesting that Tbx18 is required to prepattern the ureteric mesenchyme. Our study provides new insights into the molecular diversity of urogenital progenitor cells and helps to understand the specification of the ureteric mesenchymal sub-lineage.