Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment.

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation.

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

EZH2 is required for parathyroid and thymic development through differentiation of the third pharyngeal pouch endoderm.

The Ezh2 gene encodes a histone methyltransferase of the polycomb repressive complex 2 that methylates histone H3 lysine 27. In this study, we investigated whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mouse ESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the third pharyngeal pouch (PP). We found that in conditionally deleted embryos the third PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer.

Chromatin and Transcriptional Response to Loss of TBX1 in Early Differentiation of Mouse Cells.

The T-box transcription factor TBX1 has critical roles in the cardiopharyngeal lineage and the gene is haploinsufficient in DiGeorge syndrome, a typical developmental anomaly of the pharyngeal apparatus. Despite almost two decades of research, if and how TBX1 function triggers chromatin remodeling is not known. Here, we explored genome-wide gene expression and chromatin remodeling in two independent cellular models of Tbx1 loss of function, mouse embryonic carcinoma cells P19Cl6, and mouse embryonic stem cells (mESCs). The results of our study revealed that the loss or knockdown of TBX1 caused extensive transcriptional changes, some of which were cell type-specific, some were in common between the two models. However, unexpectedly we observed only limited chromatin changes in both systems. In P19Cl6 cells, differentially accessible regions (DARs) were not enriched in T-BOX binding motifs; in contrast, in mESCs, 34% (n = 47) of all DARs included a T-BOX binding motif and almost all of them gained accessibility in Tbx1 -/- cells. In conclusion, despite a clear transcriptional response of our cell models to loss of TBX1 in early cell differentiation, chromatin changes were relatively modest.

TBX1 and Basal Cell Carcinoma: Expression and Interactions with Gli2 and Dvl2 Signaling.

Early events of basal cell carcinoma (BCC) tumorigenesis are triggered by inappropriate activation of SHH signaling, via the loss of Patched1 (Ptch1) or by activating mutations of Smoothened (Smo). TBX1 is a key regulator of pharyngeal development, mainly through expression in multipotent progenitor cells of the cardiopharyngeal lineage. This transcription factor is connected to several major signaling systems, such as FGF, WNT, and SHH, and it has been linked to cell proliferation and to the regulation of cell shape and cell dynamics. Here, we show that TBX1 was expressed in all of the 51 BCC samples that we have tested, while in healthy human skin it was only expressed in the hair follicle. Signal intensity and distribution was heterogeneous among tumor samples. Experiments performed on a cellular model of mouse BCC showed that Tbx1 is downstream to GLI2, a factor in the SHH signaling, and that, in turn, it regulates the expression of Dvl2, which encodes an adaptor protein that is necessary for the transduction of WNT signaling. Consistently, Tbx1 depletion in the cellular model significantly reduced cell migration. These results suggest that TBX1 is part of a core transcription network that promotes BCC tumorigenesis.

Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer.

The TBX1 gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription, and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here, we show that increased dosage of Tbx1 correlates with downregulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro-differentiated human induced pluripotent stem cells (hiPSCs) expressed higher levels of MEF2C and showed increased AHF acetylation, compared with hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c AHF enhancer by inducing histone deacetylation.

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells.

The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells.

Tbx1 regulates brain vascularization.

The transcription factor TBX1 is the major gene involved in 22q11.2 deletion syndrome (22q11.2DS). Using mouse models of these diseases, we have previously shown that TBX1 activates VEGFR3 in endothelial cells (EC), and that this interaction is critical for the development of the lymphatic vasculature. In this study, we show that TBX1 regulates brain angiogenesis. Using loss-of-function genetics and molecular approaches, we show that TBX1 regulates the VEGFR3 and DLL4 genes in brain ECs. In mice, loss of TBX1 causes global brain vascular defects, comprising brain vessel hyperplasia, enhanced angiogenic sprouting and vessel network disorganization. This phenotype is recapitulated in EC-specific Tbx1 conditional mutants and in an EC-only 3-dimensional cell culture system (matrigel), indicating that the brain vascular phenotype is cell autonomous. Furthermore, EC-specific conditional Tbx1 mutants have poorly perfused brain vessels and brain hypoxia, indicating that the expanded vascular network is functionally impaired. In EC-matrigel cultures, a Notch1 agonist is able to partially rescue microtubule hyperbranching induced by TBX1 knockdown. Thus, we have identified a novel transcriptional regulator of angiogenesis that exerts its effect in brain by negatively regulating angiogenesis through the DLL4/Notch1-VEGFR3 regulatory axis. Given the similarity of the phenotypic consequences of TBX1 mutation in humans and mice, this unexpected role of TBX1 in murine brain vascularization should stimulate clinicians to search for brain microvascular anomalies in 22q11.2DS patients and to evaluate whether some of the anatomical and functional brain anomalies in patients may have a microvascular origin.

Transcriptional control in cardiac progenitors: Tbx1 interacts with the BAF chromatin remodeling complex and regulates Wnt5a.

Mutations of the Wnt5a gene, encoding a ligand of the non-canonical Wnt pathway, and the Ror2 gene, encoding its receptor, have been found in patients with cardiac outflow tract defects. We found that Wnt5a is expressed in the second heart field (SHF), a population of cardiac progenitor cells destined to populate the cardiac outflow tract and the right ventricle. Because of cardiac phenotype similarities between Wnt5a and Tbx1 mutant mice, we tested potential interactions between the two genes. We found a strong genetic interaction in vivo and determined that the loss of both genes caused severe hypoplasia of SHF-dependent segments of the heart. We demonstrated that Wnt5a is a transcriptional target of Tbx1 and explored the mechanisms of gene regulation. Tbx1 occupies T-box binding elements within the Wnt5a gene and interacts with the Baf60a/Smarcd1 subunit of a chromatin remodeling complex. It also interacts with the Setd7 histone H3K4 monomethyltransferase. Tbx1 enhances Baf60a occupation at the Wnt5a gene and enhances its H3K4 monomethylation status. Finally, we show that Baf60a is required for Tbx1-driven regulation of target genes. These data suggest a model in which Tbx1 interacts with, and probably recruits a specific subunit of, the BAF complex as well as histone methylases to activate or enhance transcription. We speculate that this may be a general mechanism of T-box function and that Baf60a is a key component of the transcriptional control in cardiac progenitors.

Tbx1 is a negative modulator of Mef2c.

The developmental role of the T-box transcription factor Tbx1 is exquisitely dosage-sensitive. In this study, we performed a microarray-based transcriptome analysis of E9.5 embryo tissues across a previously generated Tbx1 mouse allelic series. This analysis identified several genes whose expression was affected by Tbx1 dosage. Interestingly, we found that the expression of the gene encoding the cardiogenic transcription factor Mef2c was negatively correlated to Tbx1 dosage. In vivo data revealed Mef2c up-regulation in the second heart field (SHF) of Tbx1 null mutant embryos compared with wild-type littermates at E9.5. Conversely, Mef2c expression was decreased in the SHF and in somites of Tbx1 gain-of-function mutants. These results are consistent with the described role of Tbx1 in suppressing cardiac progenitor cell differentiation and indicate also a negative effect of Tbx1 on Mef2c during skeletal muscle differentiation. We show that Tbx1 occupies conserved regulatory regions of the Mef2c locus, suggesting a direct effect on Mef2c transcription. However, we also show that Tbx1 interferes with the Gata4→ Mef2c regulatory pathway. Overall, our study uncovered a target of Tbx1 with critical developmental roles, so highlighting the power of the dosage gradient approach that we used.

Lack of Mid1, the mouse ortholog of the Opitz syndrome gene, causes abnormal development of the anterior cerebellar vermis.

Opitz G/BBB syndrome (OS) is a genetic disorder characterized by midline developmental defects. Male patients with the X-linked form of OS, caused by loss-of-function mutations in the MID1 gene, show high variability of the clinical signs. MID1 encodes a ubiquitin ligase that controls phosphatase 2A, but its role in the pathogenesis of the disease is still unclear. Here, we report a mouse line carrying a nonfunctional ortholog of the human MID1 gene, Mid1. Mid1-null mice show the brain anatomical defect observed in patients (i.e., hypoplasia of the anterior portion of the medial cerebellum, the vermis). We found that the presence of this defect correlates with motor coordination and procedural and nonassociative learning impairments. The defect is limited to the most anterior lobes of the vermis, the region of the developing cerebellum adjacent to the dorsal midbrain. Analyses at midgestation reveal that lack of Mid1 causes the shortening of the posterior dorsal midbrain, the rostralization of the midbrain/cerebellum boundary, and the downregulation of a key player in the development of this region, Fgf17. Thus, lack of Mid1 causes a misspecification of the midbrain/cerebellar boundary that results in an abnormal development of the most anterior cerebellar lobes. This animal model provides a tool for additional in vivo studies of the physiological and pathological role of the Mid1 gene and a system to investigate the development and function of anterior cerebellar domains.

MID1 mutation screening in a large cohort of Opitz G/BBB syndrome patients: twenty-nine novel mutations identified.

Opitz G/BBB Syndrome (OS) is a multiple congenital anomaly disorder characterized by defects along the body midline. The disease is characterized by variable expressivity of signs that include hypertelorism, cleft lip and/or palate, laryngo-tracheo-esophageal abnormalities, cardiac defects, and hypospadias. OS patients also present with mental retardation and brain anatomical abnormalities. An autosomal dominant form mapping to chromosome 22 and an X-linked form of OS are known. The gene responsible for the X-linked form of OS, MID1, codes for a member of the Tripartite Motif family of E3 ubiquitin ligases. Here we report 29 novel mutations in 29 unrelated patients of a cohort of 140 male OS cases. These mutations are found in both familial and sporadic cases. They are scattered along the entire length of the gene and are represented by missense and nonsense mutations, insertions and deletions causing frame shift mutations, and deletion of either single exons or the entire gene. The variety of the mutations found confirms that loss-of-function is the mechanism underlying the OS phenotype. Moreover, the low percentage of MID1-mutated OS patients, 47% of the familial and 13% of the sporadic cases, suggests a wider genetic heterogeneity underlying the OS phenotype.

Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules.

BACKGROUND: Opitz G/BBB syndrome is a genetic disorder characterized by developmental midline abnormalities, such as hypertelorism, cleft palate, and hypospadias. The gene responsible for the X-linked form of this disease, MID1, encodes a TRIM/RBCC protein that is anchored to the microtubules. The association of Mid1 with the cytoskeleton is regulated by dynamic phosphorylation, through the interaction with the alpha4 subunit of phosphatase 2A (PP2A). Mid1 acts as an E3 ubiquitin ligase, regulating PP2A degradation on microtubules. RESULTS: In spite of these findings, the biological role exerted by the Opitz syndrome gene product is still unclear and the presence of other potential interacting moieties in the Mid1 structure prompted us to search for additional cellular partners. Through a yeast two-hybrid screening approach, we identified a novel gene, MIG12, whose protein product interacts with Mid1. We confirmed by immunoprecipitation that this interaction occurs in vivo and that it is mediated by the Mid1 coiled-coil domain. We found that Mig12 is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development. Transiently expressed Mig12 is found diffusely in both nucleus and cytoplasm, although it is enriched in the microtubule-organizing center region. Consistently with this, endogenous Mig12 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization. When co-transfected with Mid1, Mig12 is massively recruited to thick filamentous structures composed of tubulin. These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays. CONCLUSIONS: Our findings suggest that Mig12 co-operates with Mid1 to stabilize microtubules. Mid1-Mig12 complexes might be implicated in cellular processes that require microtubule stabilization, such as cell division and migration. Impairment in Mig12/Mid1-mediated microtubule dynamic regulation, during the development of embryonic midline, may cause the pathological signs observed in Opitz syndrome patients.

X-linked Opitz syndrome: novel mutations in the MID1 gene and redefinition of the clinical spectrum.

Opitz (or G/BBB) syndrome is a pleiotropic genetic disorder characterized by hypertelorism, hypospadias, and additional midline defects. This syndrome is heterogeneous with an X-linked (XLOS) and an autosomal dominant (ADOS) form. The gene implicated in the XLOS form, MID1, encodes a protein containing a RING-Bbox-Coiled-coil motif belonging to the tripartite motif (TRIM) family. To further clarify the molecular basis of XLOS, we have undertaken mutation analysis of the MID1 gene in patients with Opitz syndrome (OS). We found novel mutations in 11 of 63 male individuals referred to us as sporadic or familial X-linked OS cases. The mutations are scattered throughout the gene, although more are represented in the 3' region. By reviewing all the MID1-mutated OS patients so far described, we confirmed that hypertelorism and hypospadias are the most frequent manifestations, being present in almost every XLOS individual. However, it is clear that laryngo-tracheo-esophageal (LTE) defects are also common anomalies, being manifested by all MID1-mutated male patients. Congenital heart and anal abnormalities are less frequent than reported in literature. In addition, we can include limb defects in the OS clinical synopsis as we found a MID1-mutated patient showing syndactyly. The low frequency of mutations in MID1 and the high variability of the phenotype suggest the involvement of other genes in the OS phenotype.