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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., (© 2024. The Author(s).)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1 -/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract., (© 2022. The Author(s).)

The auditory function of the mammalian cochlea relies on two types of mechanosensory hair cells and various non-sensory supporting cells. Recent studies identified the transcription factors INSM1 and IKZF2 as regulators of outer hair cell (OHC) fate. However, the transcriptional regulation of the differentiation of inner hair cells (IHCs) and their associated inner supporting cells (ISCs) has remained enigmatic. Here, we show that the expression of the transcription factor TBX2 is restricted to IHCs and ISCs from the onset of differentiation until adulthood and examine its function using conditional deletion and misexpression approaches in the mouse. We demonstrate that TBX2 acts in prosensory progenitors as a patterning factor by specifying the inner compartment of the sensory epithelium that subsequently gives rise to IHCs and ISCs. Hair cell-specific inactivation or misexpression causes transdifferentiation of hair cells indicating a cell-autonomous function of TBX2 in inducing and maintaining IHC fate., (© 2022. The Author(s).)

The coordinated development of the mesenchymal and epithelial progenitors of the murine ureter depends on a complex interplay of diverse signaling activities. We have recently shown that epithelial FGFR2 signaling regulates stratification and differentiation of the epithelial compartment by enhancing epithelial Shh expression, and mesenchymal SHH and BMP4 activity. Here, we show that FGFR1 and FGFR2 expression in the mesenchymal primordium impinges on the SHH/BMP4 signaling axis to regulate mesenchymal patterning and differentiation. Mouse embryos with conditional loss of Fgfr1 and Fgfr2 in the ureteric mesenchyme exhibited reduced mesenchymal proliferation and prematurely activated lamina propria formation at the expense of the smooth muscle cell program. They also manifested hydroureter at birth. Molecular profiling detected increased SHH, WNT and retinoic acid signaling, whereas BMP4 signaling in the mesenchyme was reduced. Pharmacological activation of SHH signaling in combination with inhibition of BMP4 signaling recapitulated the cellular changes in explant cultures of wild-type ureters. Additional experiments suggest that mesenchymal FGFR1 and FGFR2 act as a sink for FGF ligands to dampen activation of Shh and BMP receptor gene expression by epithelial FGFR2 signaling., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The patterned array of basal, intermediate and superficial cells in the urothelium of the mature ureter arises from uncommitted epithelial progenitors of the distal ureteric bud. Urothelial development requires signaling input from surrounding mesenchymal cells, which, in turn, depend on cues from the epithelial primordium to form a layered fibro-muscular wall. Here, we have identified FGFR2 as a crucial component in this reciprocal signaling crosstalk in the murine ureter. Loss of Fgfr2 in the ureteric epithelium led to reduced proliferation, stratification, intermediate and basal cell differentiation in this tissue, and affected cell survival and smooth muscle cell differentiation in the surrounding mesenchyme. Loss of Fgfr2 impacted negatively on epithelial expression of Shh and its mesenchymal effector gene Bmp4. Activation of SHH or BMP4 signaling largely rescued the cellular defects of mutant ureters in explant cultures. Conversely, inhibition of SHH or BMP signaling in wild-type ureters recapitulated the mutant phenotype in a dose-dependent manner. Our study suggests that FGF signals from the mesenchyme enhance, via epithelial FGFR2, the SHH-BMP4 signaling axis to drive urothelial and mesenchymal development in the early ureter., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

All epithelial components of the inner ear, including sensory hair cells and innervating afferent neurons, arise by patterning and differentiation of epithelial progenitors residing in a simple sphere, the otocyst. Here, we identify the transcriptional repressors TBX2 and TBX3 as novel regulators of these processes in the mouse. Ablation of Tbx2 from the otocyst led to cochlear hypoplasia, whereas loss of Tbx3 was associated with vestibular malformations. The loss of function of both genes (Tbx2/3cDKO) prevented inner ear morphogenesis at midgestation, resulting in indiscernible cochlear and vestibular structures at birth. Morphogenetic impairment occurred concomitantly with increased apoptosis in ventral and lateral regions of Tbx2/3cDKO otocysts around E10.5. Expression analyses revealed partly disturbed regionalisation, and a posterior-ventral expansion of the neurogenic domain in Tbx2/3cDKO otocysts at this stage. We provide evidence that repression of FGF signalling by TBX2 is important to restrict neurogenesis to the anterior-ventral otocyst and implicate another T-box factor, TBX1, as a crucial mediator in this regulatory network., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: In the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Whether the embryonic pulmonary mesenchyme is a homogenous precursor pool and how it diversifies into different cell lineages is poorly understood. We have previously shown that the T-box transcription factor gene Tbx2 is expressed in the pulmonary mesenchyme of the developing murine lung and is required therein to maintain branching morphogenesis., Methods: We determined Tbx2/TBX2 expression in the developing murine lung by in situ hybridization and immunofluorescence analyses. We used a genetic lineage tracing approach with a Cre line under the control of endogenous Tbx2 control elements (Tbx2 cre ), and the R26 mTmG reporter line to trace TBX2-positive cells in the murine lung. We determined the fate of the TBX2 lineage by co-immunofluorescence analysis of the GFP reporter and differentiation markers in normal murine lungs and in lungs lacking or overexpressing TBX2 in the pulmonary mesenchyme., Results: We show that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung. In differentiated smooth muscle cells and in fibroblasts, expression of TBX2 is still widespread but strongly reduced. In mesothelial and endothelial cells expression is more variable and scattered. All fetal smooth muscle cells, endothelial cells and fibroblasts derive from TBX2 + progenitors, whereas half of the mesothelial cells have a different descent. The fate of TBX2-expressing cells is not changed in Tbx2-deficient and in TBX2-constitutively overexpressing mice but the distribution and abundance of endothelial and smooth muscle cells is changed in the overexpression condition., Conclusion: The fate of pulmonary mesenchymal progenitors is largely independent of TBX2. Nevertheless, a successive and precisely timed downregulation of TBX2 is necessary to allow proper differentiation and functionality of bronchial smooth muscle cells and to limit endothelial differentiation. Our work suggests expression of TBX2 in an early pulmonary mesenchymal progenitor and supports a role of TBX2 in maintaining the precursor state of these cells.

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 Tbx18 cre 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., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

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 Wt1 creERT2 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.

The organized array of smooth muscle cells (SMCs) and fibroblasts in the walls of visceral tubular organs arises by patterning and differentiation of mesenchymal progenitors surrounding the epithelial lumen. Here, we show that the TBX2 and TBX3 transcription factors have novel and required roles in regulating these processes in the murine ureter. Co-expression of TBX2 and TBX3 in the inner mesenchymal region of the developing ureter requires canonical WNT signaling. Loss of TBX2/TBX3 in this region disrupts activity of two crucial drivers of the SMC program, Foxf1 and BMP4 signaling, resulting in decreased SMC differentiation and increased extracellular matrix. Transcriptional profiling and chromatin immunoprecipitation experiments revealed that TBX2/TBX3 directly repress expression of the WNT antagonists Dkk2 and Shisa2 , the BMP antagonist Bmper and the chemokine Cxcl12 These findings suggest that TBX2/TBX3 are effectors of canonical WNT signaling in the ureteric mesenchyme that promote SMC differentiation by maintaining BMP4 and WNT signaling in the inner region, while restricting CXCL12 signaling to the outer layer of fibroblast-fated mesenchyme., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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.

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).

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., (Copyright © 2017 by the American Society of Nephrology.)

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2014. Published by The Company of Biologists Ltd.)

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 β-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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tbx2 and Tbx3 are two highly related members of the T-box transcription factor gene family that regulate patterning and differentiation of a number of tissue rudiments in the mouse. Both genes are partially co-expressed in the ventral diencephalon and the infundibulum; however, a functional requirement in murine pituitary development has not been reported. Here, we show by genetic lineage tracing that Tbx2(+) cells constitute the precursor population of the neurohypophysis. However, Tbx2 is dispensable for neurohypophysis development as revealed by normal formation of this organ in Tbx2-deficient mice. By contrast, loss of Tbx3 from the ventral diencephalon results in a failure to establish the Tbx2(+) domain in this region, and a lack of evagination of the infundibulum and formation of the neurohypophysis. Rathke's pouch is severely hypoplastic, exhibits defects in dorsoventral patterning, and degenerates after E12.5. In Tbx3-deficient embryos, the ventral diencephalon is hyperproliferative and displays an abnormal cellular architecture, probably resulting from a failure to repress transcription of Shh. We further show that Tbx3 and Tbx2 repress Shh by sequestering the SRY box-containing transcription factor Sox2 away from a Shh forebrain enhancer (SBE2), thus preventing its activation. These data suggest that Tbx3 is required in the ventral diencephalon to establish a Shh(-) domain to allow formation of the infundibulum.

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.

In the cochlea, sensory transduction depends on the endocochlear potential (EP) and the unique composition of the endolymph, both of which are maintained by a highly specialized epithelium at the cochlear lateral wall, the stria vascularis. The generation of the EP by the stria vascularis, in turn, relies on the insulation of an intrastrial extracellular compartment by epithelial basal cells. Despite the physiological importance of basal cells, their cellular origin and the molecular pathways that lead to their differentiation are unclear. Here, we show by genetic lineage tracing in the mouse that basal cells exclusively derive from the otic mesenchyme. Conditional deletion of E-cadherin in the otic mesenchyme and its descendants does not abrogate the transition from mesenchymal precursors to epithelial basal cells. Rather, dedifferentiation of intermediate cells, altered morphology of basal and marginal cells and hearing impairment due to decreased EP in E-cadherin mutant mice demonstrate an essential role of E-cadherin in terminal basal cell differentiation and their interaction with other strial cell types to establish and maintain the functional architecture of the stria vascularis., (2011 Elsevier Inc. All rights reserved.)

Congenital ureter anomalies, including hydroureter, affect up to 1% of the newborn children. Despite the prevalence of these developmental abnormalities in young children, the underlying molecular causes are only poorly understood. Here, we show that the high mobility group domain transcription factor Sox9 plays an important role in ureter development in the mouse. Transient Sox9 expression was detected in the undifferentiated ureteric mesenchyme and inactivation of Sox9 in this domain resulted in strong proximal hydroureter formation due to functional obstruction. Loss of Sox9 did not affect condensation, proliferation and apoptosis of the undifferentiated mesenchyme, but perturbed cyto-differentiation into smooth muscle cells (SMCs). Expression of genes encoding extracellular matrix (ECM) components was strongly reduced, suggesting that deficiency in ECM composition and/or signaling may underlie the observed defects. Prolonged expression of Sox9 in the ureteric mesenchyme led to increased deposition of ECM components and SMC dispersal. Furthermore, Sox9 genetically interacts with the T-box transcription factor 18 gene (Tbx18) during ureter development at two levels--as a downstream mediator of Tbx18 function and in a converging pathway. Together, our results argue that obstructive uropathies in campomelic dysplasia patients that are heterozygous for mutations in and around SOX9 arise from a primary requirement of Sox9 in the development of the ureteric mesenchyme.

The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

Sox9 encodes an HMG-domain transcription factor that is critically required in numerous developmental processes such as chondrogenesis and otic placode formation. Here, we show that Sox9 is expressed in the mesenchyme surrounding the developing cochlea in the mouse suggesting that Sox9 may also control development of the otic fibrocyte compartment and the surrounding otic capsule. Tissue-specific inactivation of Sox9 in the periotic mesenchyme using a Tbx18(Cre) mouse line results in arrest of early chondrogenesis and consequently, in a lack of cochlear otic capsule formation. Furthermore, loss of Sox9 severely compromises expansion, differentiation and remodeling of the otic fibrocyte compartment. Early cell proliferation defects in the entire periotic mesenchyme of Sox9-deficient inner ears suggest a cell-autonomous function of Sox9 for the development of the inner mesenchymal compartment. Abnormal cochlear duct morphogenesis in Sox9 mutants including disruption of the coiling process is tightly associated with the onset of mesenchymal defects whereas the absence of major differentiation defects in the otic epithelium suggests that Sox9-dependent mesenchymal signals primarily control epithelial morphogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

In the cochlea, fibrocytes play important physiological roles, including the maintenance of the ionic composition of the endolymph. Human deafness upon fibrocyte alterations witnesses their crucial role for hearing. We demonstrate that differentiation of otic fibrocytes requires the T-box transcription factor gene Tbx18. Tbx18 expression during inner ear development is restricted to the sub-region of otic mesenchyme that is fated to differentiate into fibrocytes. We rescued the somitic defect that underlies the perinatal lethality of Tbx18-mutant mice by a transgenic approach, and measured auditory brainstem responses. Adult Tbx18-deficient mice showed profound deafness and a complete disruption of the endocochlear potential that is essential for the transduction of sound by sensory hair cells. The differentiation of otic fibrocytes of the spiral ligament was severely compromised. Tissue architecture of the stria vascularis of the lateral wall was disrupted, exhibiting an almost complete absence of the basal cell layer, and a reduction and changes of intermediate and marginal cells, respectively. Stria vascularis defects resulted from the failure of Tbx18-mutant otic fibrocytes to generate the basal cell layer by a mesenchymal-epithelial transition. Defects in otic fibrocyte differentiation may be subordinate to a primary role of Tbx18 in early compartmentalization of the otic mesenchyme, as lineage restriction and boundary formation between otic fibrocytes and the surrounding otic capsule were severely affected in the mutant. Our study sheds light on the genetic control of patterning and differentiation of the otic mesenchyme, uncovers distinct steps of stria vascularis formation and illuminates the importance of non-epithelially-derived otic cell types for normal hearing and the etiology of deafness.

The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias.

Tbx20, a member of the T-box family of transcriptional regulators, shows evolutionary conserved expression in the developing heart. In the mouse, Tbx20 is expressed in the cardiac crescent, then in the endocardium and myocardium of the linear and looped heart tube before it is restricted to the atrioventricular canal and outflow tract in the multi-chambered heart. Here, we show that Tbx20 is required for progression from the linear heart tube to a multi-chambered heart. Mice carrying a targeted mutation of Tbx20 show early embryonic lethality due to hemodynamic failure. A linear heart tube with normal anteroposterior patterning is established in the mutant. The tube does not elongate, indicating a defect in recruitment of mesenchyme from the secondary heart field, even though markers of the secondary heart field are not affected. Furthermore, dorsoventral patterning of the tube, formation of working myocardium, looping, and further differentiation and morphogenesis fail. Instead, Tbx2, Bmp2 and vinexin alpha (Sh3d4), genes normally restricted to regions of primary myocardium and lining endocardium, are ectopically expressed in the linear heart tube of Tbx20 mutant embryos. Because Tbx2 is both necessary and sufficient to repress chamber differentiation (Christoffels et al., 2004a; Harrelson et al., 2004), Tbx20 may ensure progression to a multi-chambered heart by repressing Tbx2 in the myocardial precursor cells of the linear heart tube destined to form the chambers.