FGFR2 signaling enhances the SHH-BMP4 signaling axis in early ureter development.

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.

Mesenchymal FGFR1 and FGFR2 control patterning of the ureteric mesenchyme by balancing SHH and BMP4 signaling.

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.

Notch signaling is a novel regulator of visceral smooth muscle cell differentiation in the murine ureter.

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.

Regulation of otocyst patterning by Tbx2 and Tbx3 is required for inner ear morphogenesis in the mouse.

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.

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

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.

Heparan Sulfate-Editing Extracellular Sulfatases Enhance VEGF Bioavailability for Ischemic Heart Repair.

RATIONALE: Mechanistic insight into the inflammatory response after acute myocardial infarction may inform new molecularly targeted treatment strategies to prevent chronic heart failure. OBJECTIVE: We identified the sulfatase SULF2 in an in silico secretome analysis in bone marrow cells from patients with acute myocardial infarction and detected increased sulfatase activity in myocardial autopsy samples. SULF2 (Sulf2 in mice) and its isoform SULF1 (Sulf1) act as endosulfatases removing 6-O-sulfate groups from heparan sulfate (HS) in the extracellular space, thus eliminating docking sites for HS-binding proteins. We hypothesized that the Sulfs have a role in tissue repair after myocardial infarction. METHODS AND RESULTS: Both Sulfs were dynamically upregulated after coronary artery ligation in mice, attaining peak expression and activity levels during the first week after injury. Sulf2 was expressed by monocytes and macrophages, Sulf1 by endothelial cells and fibroblasts. Infarct border zone capillarization was impaired, scar size increased, and cardiac dysfunction more pronounced in mice with a genetic deletion of either Sulf1 or Sulf2. Studies in bone marrow-chimeric Sulf-deficient mice and Sulf-deficient cardiac endothelial cells established that inflammatory cell-derived Sulf2 and endothelial cell-autonomous Sulf1 promote angiogenesis. Mechanistically, both Sulfs reduced HS sulfation in the infarcted myocardium, thereby diminishing Vegfa (vascular endothelial growth factor A) interaction with HS. Along this line, both Sulfs rendered infarcted mouse heart explants responsive to the angiogenic effects of HS-binding Vegfa164 but did not modulate the angiogenic effects of non-HS-binding Vegfa120. Treating wild-type mice systemically with the small molecule HS-antagonist surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide, 1 mg/kg/day) for 7 days after myocardial infarction released Vegfa from HS, enhanced infarct border-zone capillarization, and exerted sustained beneficial effects on cardiac function and survival. CONCLUSIONS: These findings establish HS-editing Sulfs as critical inducers of postinfarction angiogenesis and identify HS sulfation as a therapeutic target for ischemic tissue repair.

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).

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.

Dexamethasone improves therapeutic outcomes in a preclinical bacterial epididymitis mouse model.

STUDY QUESTION: Can dexamethasone improve infertility-related cauda epididymidal tissue damage caused by bacterial epididymitis? SUMMARY ANSWER: Dexamethasone in addition to anti-microbial treatment effectively reduces long-term deleterious epididymal tissue damage by dampening the host's adaptive immune response. WHAT IS KNOWN ALREADY: Despite effective anti-microbial treatment, ~40% of patients with epididymitis experience subsequent sub- or infertility. An epididymitis mouse model has shown that the host immune response is mainly responsible for the magnitude of epididymal tissue damage that is fundamentally causative of the subsequent fertility issues. STUDY DESIGN, SIZE, DURATION: Bacterial epididymitis was induced in male mice by using uropathogenic Escherichia coli (UPEC). From Day 3 after infection onwards, mice were treated with daily doses of levofloxacin (20 mg/kg, total n = 12 mice), dexamethasone (0.5 mg/kg, total n = 9) or both in combination (total n = 11) for seven consecutive days. Control animals were left untreated, i.e. given no interventional treatment following UPEC infection (total n = 11). Half of the animals from each group were killed either at 10 or 31 days post-infection. PARTICIPANTS/MATERIALS, SETTING, METHODS: A mouse model of induced bacterial epididymitis was applied to adult male C57BL/6J mice. At the respective endpoints (10 or 31 days post-infection), epididymides were collected. Effectiveness of antibiotic treatment was assessed by plating of epididymal homogenates onto lysogeny broth agar plates. Overall tissue morphology and the degree and nature of tissue damage were assessed histologically. Quantitative RT-PCR was used to assess local cytokine transcript levels. Blood was drawn and serum analysed for systemic IgG and IgM levels by ELISA. In addition, correlation analyses of clinical data and serum-analyses of IgG and IgM levels in patients with epididymitis were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The addition of dexamethasone to the standard anti-microbial treatment did not further worsen epididymal tissue integrity. In fact, an obviously dampened immune response and reduced tissue reaction/damage was observed at both 10 and 31 days post-infection following combined treatment. More specifically, epididymal duct continuity was preserved, enabling sperm transit. In contrast, in untreated or antibiotic-treated animals, damage of the epididymal duct and duct constrictions were observed, associated with a lack of cauda spermatozoa. In line with the bacteriostatic/bactericidal effect of levofloxacin (alone as well as in combination), local cytokine transcript levels were significantly and similarly reduced in animals treated with levofloxacin alone (P < 0.01) or in combination with dexamethasone (P < 0.05) compared to UPEC-infected untreated animals. Interestingly, the addition of dexamethasone to the anti-microbial treatment induced a unique dampening effect on adaptive immunity, since systemic IgG and IgM levels as well as the pan-T cell marker CD3 were reduced at both 10 and 31 days post-infection. LIMITATIONS, REASONS FOR CAUTION: Breeding studies to address the fertility-protecting effect of the combined treatment were not possible in the experimental animals because the vas deferens was ligated (model specific). WIDER IMPLICATIONS OF THE FINDINGS: Whereas innate immunity is necessary and involved in acute bacterial clearance, adaptive immunity seems to be responsible for long-term, subclinical immunological activities that may negatively affect the pathogenesis of bacterial epididymitis even after effective bacterial eradication. These effects can be reduced in mice by the additional treatment with dexamethasone. This immunological characteristic of bacterial epididymitis shows similarities to the Jarisch-Herxheimer reaction known from other types of bacterial infection. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Deutsche Forschungsgemeinschaft, Monash University and the Medical Faculty of Justus-Liebig University to the International Research Training Group on 'Molecular pathogenesis of male reproductive disorders' (GRK 1871). R.W., K.L.L. and M.P.H. were supported by grants from the National Health and Medical Research Council of Australia (ID1079646, ID1081987, ID1020269 and ID1063843) and by the Victorian Government's Operational Infrastructure Support Program. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

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.

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.

Tbx2 controls lung growth by direct repression of the cell cycle inhibitor genes Cdkn1a and Cdkn1b.

Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.

Tbx18 and the fate of epicardial progenitors.

Uncovering the origins of myocardial cells is important for understanding and treating heart diseases. Cai et al. suggest that Tbx18-expressing epicardium provides a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Here we show that the T-box transcription factor gene 18 (Tbx18) itself is expressed in the myocardium, showing that their genetic lineage tracing system does not allow conclusions of an epicardial origin of cardiomyocytes in vivo to be drawn.

Wt1 and epicardial fate mapping.

RATIONALE: The embryonic epicardium is a crucial cell source of the cardiac fibrous skeleton as well as of the coronary system. Genetic lineage tracing systems based on Wt1 regulatory sequences provided evidence that epicardium-derived cells also adopt a myocardial fate in the mouse. OBJECTIVE: To define the adequacy of Wt1-based lineage tracing systems for epicardial fate mapping. METHODS AND RESULTS: Using in situ hybridization analysis and immunofluorescence on tissue sections, we detected endogenous expression of Wt1 mRNA and Wt1 protein in the proepicardium and epicardium and also in endothelial cells throughout cardiogenesis. Expression analysis of a sensitive GFP reporter showed that recombination mediated by cre recombinase in the Wt1(creEGFP) line occurs randomly and sporadically in all cells of the embryo. Recombination in cardiomyocytes was found in the linear heart tube before establishment of a (pro)epicardium. In contrast, the tamoxifen-inducible Wt1(creERT2) mouse line mediated poor and variable recombination in the epicardium. Recombination in cardiomyocytes was not detected in this case. CONCLUSIONS: Frequently used Wt1 based cre-mediated lineage tracing systems are not suitable for epicardial fate mapping because of endogenous endothelial expression of Wt1, ectopic recombination (Wt1(creEGFP)), and poor recombination efficiency (Wt1(creERT2)) in the developing heart. We conclude that claims of a cardiomyocyte fate of epicardial cells in the mouse are not substantiated.

Notch signaling regulates smooth muscle differentiation of epicardium-derived cells.

RATIONALE: The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. OBJECTIVE: To define the role of Notch signaling in murine epicardial development. METHODS AND RESULTS: Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor ß signaling in SM differentiation of EPDCs. CONCLUSIONS: Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.

Wnt/ß-catenin signaling maintains the mesenchymal precursor pool for murine sinus horn formation.

RATIONALE: Canonical (ß-catenin [Ctnnb1]-dependent) wingless-related MMTV integration site (Wnt) signaling plays an important role in the development of second heart field-derived structures of the heart by regulating precursor cell proliferation. The signaling pathways that regulate the most posterior elongation of the heart, that is, the addition of the systemic venous return from a Tbx18(+) precursor population, have remained elusive. OBJECTIVE: To define the role of Ctnnb1-dependent Wnt signaling in the development of the cardiac venous pole. METHODS AND RESULTS: We show by in situ hybridization analysis that Wnt pathway components are expressed and canonical Wnt signaling is active in the developing sinus horns. We analyzed sinus horn (Tbx18(cre))-specific Ctnnb1 loss- and gain-of-function mutant embryos. In Ctnnb1-deficient embryos, the dorsal part of the sinus horns is not myocardialized but consists of cells with at least partial fibroblast identity; the sinoatrial node is unaffected. Stabilization of Ctnnb1 in this domain results in the formation of undifferentiated cell aggregates. Analysis of cellular changes revealed a role of canonical Wnt signaling in proliferation of the Tbx18(+) mesenchymal progenitor cell population. CONCLUSIONS: Wnt/ß-catenin signaling maintains the Tbx18(+)Nkx2-5(-) mesenchymal precursor pool for murine sinus horn formation.

TBX2 specifies and maintains inner hair and supporting cell fate in the Organ of Corti.

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.

The transcription factor GATA4 promotes myocardial regeneration in neonatal mice.

Heart failure is often the consequence of insufficient cardiac regeneration. Neonatal mice retain a certain capability of myocardial regeneration until postnatal day (P)7, although the underlying transcriptional mechanisms remain largely unknown. We demonstrate here that cardiac abundance of the transcription factor GATA4 was high at P1, but became strongly reduced at P7 in parallel with loss of regenerative capacity. Reconstitution of cardiac GATA4 levels by adenoviral gene transfer markedly improved cardiac regeneration after cryoinjury at P7. In contrast, the myocardial scar was larger in cardiomyocyte-specific Gata4 knockout (CM-G4-KO) mice after cryoinjury at P0, indicative of impaired regeneration, which was accompanied by reduced cardiomyocyte proliferation and reduced myocardial angiogenesis in CM-G4-KO mice. Cardiomyocyte proliferation was also diminished in cardiac explants from CM-G4-KO mice and in isolated cardiomyocytes with reduced GATA4 expression. Mechanistically, decreased GATA4 levels caused the downregulation of several pro-regenerative genes (among them interleukin-13, Il13) in the myocardium. Interestingly, systemic administration of IL-13 rescued defective heart regeneration in CM-G4-KO mice and could be evaluated as therapeutic strategy in the future.

Lack of Genetic Interaction between Tbx18 and Tbx2/Tbx20 in Mouse Epicardial Development.

The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.

Tbx2 and Tbx3 Act Downstream of Shh to Maintain Canonical Wnt Signaling during Branching Morphogenesis of the Murine Lung.

Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. Here, we show by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 act together in the lung mesenchyme to maintain branching morphogenesis. Expression of both genes depends on epithelially derived Shh signaling, with additional modulation by Bmp, Wnt, and Tgfß signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with our previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b, we conclude that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.