VEGFR3 modulates brain microvessel branching in a mouse model of 22q11.2 deletion syndrome.

The loss of a single copy of <i>TBX1</i> accounts for most of the clinical signs and symptoms of 22q11.2 deletion syndrome, a common genetic disorder that is characterized by multiple congenital anomalies and brain-related clinical problems, some of which likely have vascular origins. <i>Tbx1</i> mutant mice have brain vascular anomalies, thus making them a useful model to gain insights into the human disease. Here, we found that the main morphogenetic function of TBX1 in the mouse brain is to suppress vessel branching morphogenesis through regulation of <i>Vegfr3</i> We demonstrate that inactivating <i>Vegfr3</i> in the <i>Tbx1</i> expression domain on a <i>Tbx1</i> mutant background enhances brain vessel branching and filopodia formation, whereas increasing <i>Vegfr3</i> expression in this domain fully rescued these phenotypes. Similar results were obtained using an in vitro model of endothelial tubulogenesis. Overall, the results of this study provide genetic evidence that <i>VEGFR3</i> is a regulator of early vessel branching and filopodia formation in the mouse brain and is a likely mediator of the brain vascular phenotype caused by <i>Tbx1</i> loss of function.

A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome.

TBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 (vB12) treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here, we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At the molecular level, in vivo vB12 treatment enabled us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SNAI2, also known as SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with, but distinct from, ISL1+ and TBX1+ populations. In addition, SNAI2+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos, and vB12 treatment restored cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in the core mesoderm and the arrangement of multiple lineages within the PhAp.

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.

A dual role for Tbx1 in cardiac lymphangiogenesis through genetic interaction with Vegfr3.

The transcription factor TBX1 is the major gene implicated in 22q11.2 deletion syndrome (22q11.2DS). The complex clinical phenotype includes vascular anomalies and a recent report presented new cases of primary lymphedema in 22q11.2DS patients. We have previously shown that TBX1 is required for systemic lymphatic vessel development in prenatal mice and it is critical for their survival postnatally. Using loss-of-function genetics and transgenesis in the mouse, we show here a strong genetic interaction between Tbx1 and Vegfr3 in cardiac lymphangiogenesis. Intriguingly, we found that different aspects of the cardiac lymphatic phenotype in Tbx1-Vegfr3 compound heterozygotes were regulated independently by the two genes, with Tbx1 primarily regulating vessel numbers and Vegfr3 vessel morphology. Consistent with this observation, Tbx1Cre -activated expression of a Vegfr3 transgene rescued partially the cardiac lymphatic abnormalities in compound heterozygotes. Through time-controlled genetic experiments, we show that Tbx1 is activated and required in cardiac lymphatic endothelial cell (LEC) progenitors between E10.5 and E11.5. Furthermore, we found that it is also required later in development for the growth of the cardiac lymphatics. Finally, our study revealed a differential sensitivity between ventral and dorsal cardiac lymphatics to the effects of altered Tbx1 and Vegfr3 gene dosage, and we show that this likely results from an earlier requirement for Tbx1 in ventral cardiac LEC progenitors.

Cardiopharyngeal mesoderm origins of musculoskeletal and connective tissues in the mammalian pharynx.

Cardiopharyngeal mesoderm (CPM) gives rise to muscles of the head and heart. Using genetic lineage analysis in mice, we show that CPM develops into a broad range of pharyngeal structures and cell types encompassing musculoskeletal and connective tissues. We demonstrate that CPM contributes to medial pharyngeal skeletal and connective tissues associated with both branchiomeric and somite-derived neck muscles. CPM and neural crest cells (NCC) make complementary mediolateral contributions to pharyngeal structures, in a distribution established in the early embryo. We further show that biallelic expression of the CPM regulatory gene Tbx1, haploinsufficient in 22q11.2 deletion syndrome patients, is required for the correct patterning of muscles with CPM-derived connective tissue. Our results suggest that CPM plays a patterning role during muscle development, similar to that of NCC during craniofacial myogenesis. The broad lineage contributions of CPM to pharyngeal structures provide new insights into congenital disorders and evolution of the mammalian pharynx.

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 regulates extracellular matrix-cell interactions in the second heart field.

Tbx1, the major candidate gene for DiGeorge or 22q11.2 deletion syndrome, is required for efficient incorporation of cardiac progenitors of the second heart field (SHF) into the heart. However, the mechanisms by which TBX1 regulates this process are still unclear. Here, we have used two independent models, mouse embryos and cultured cells, to define the role of TBX1 in establishing morphological and dynamic characteristics of SHF in the mouse. We found that loss of TBX1 impairs extracellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos suggested that this function is non-cell autonomous, and, in cultured cells, loss of TBX1 impairs cell migration and FAs. Additionally, we found that ECM-mediated integrin signaling is disrupted upon loss of TBX1. Finally, we show that interfering with the ECM-integrin-FA axis between E8.5 and E9.5 in mouse embryos, corresponding to the time window within which TBX1 is required in the SHF, causes outflow tract dysmorphogenesis. Our results demonstrate that TBX1 is required to maintain the integrity of ECM-cell interactions in the SHF and that this interaction is critical for cardiac outflow tract development. More broadly, our data identifies a novel TBX1 downstream pathway as an important player in SHF tissue architecture and cardiac morphogenesis.

Left pulmonary artery in 22q11.2 deletion syndrome. Echocardiographic evaluation in patients without cardiac defects and role of Tbx1 in mice.

INTRODUCTION AND HYPOTHESIS: Patients with 22q11 deletion syndrome (22q11.2DS) present, in about 75% of cases, typical patterns of cardiac defects, with a particular involvement on the ventricular outflow tract and great arteries. However, in this genetic condition the dimensions of the pulmonary arteries (PAs) never were specifically evaluated. We measured both PAs diameter in patients with 22q11.2DS without cardiac defects, comparing these data to a normal control group. Moreover, we measured the PAs diameter in Tbx1 mutant mice. Finally, a cell fate mapping in Tbx1 mutants was used to study the expression of this gene in the morphogenesis of PAs. METHODS: We evaluated 58 patients with 22q11.2DS without cardiac defects. The control group consisted of 54 healthy subjects, matched for age and sex. All cases underwent a complete transthoracic echocardiography. Moreover, we crossed Tbx1+/- mice and harvested fetuses. We examined the cardiovascular phenotype of 8 wild type (WT), 37 heterozygous (Tbx1+/-) and 6 null fetuses (Tbx1-/-). Finally, we crossed Tbx1Cre/+mice with R26RmT-mG Cre reporter mice to study Tbx1 expression in the pulmonary arteries. RESULTS: The echocardiographic study showed that the mean of the LPA/RPA ratio in 22q11.2DS was smaller (0.80 ± 0.12) than in controls (0.97 ± 0.08; p < 0.0001). Mouse studies resulted in similar data as the size of LPA and RPA was not significantly different in WT embryos, but in Tbx1+/- and Tbx1-/- embryos the LPA was significantly smaller than the RPA in both mutants (P = 0.0016 and 0.0043, respectively). We found that Tbx1 is expressed near the origin of the PAs and in their adventitia. CONCLUSIONS: Children with 22q11.2DS without cardiac defects show smaller LPA compared with healthy subjects. Mouse studies suggest that this anomaly is due to haploinsufficiency of Tbx1. These data may be useful in the clinical management of children with 22q11.2DS and should guide further experimental studies as to the mechanisms underlying PAs development.

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.

Cortical Development Requires Mesodermal Expression of Tbx1, a Gene Haploinsufficient in 22q11.2 Deletion Syndrome.

In mammals, proper temporal control of neurogenesis and neural migration during embryonic development ensures correct formation of the cerebral cortex. Changes in the distribution of cortical projection neurons and interneurons are associated with behavioral disorders and psychiatric diseases, including schizophrenia and autism, suggesting that disrupted cortical connectivity contributes to the brain pathology. TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS), a chromosomal deletion disorder characterized by a greatly increased risk for schizophrenia. We have previously shown that Tbx1 heterozygous mice have reduced prepulse inhibition, a behavioral abnormality that is associated with 22q11.2DS and nonsyndromic schizophrenia. Here, we show that loss of Tbx1 disrupts corticogenesis in mice by promoting premature neuronal differentiation in the medio-lateral embryonic cortex, which gives rise to the somatosensory cortex (S1). In addition, we found altered polarity in both radially migrating excitatory neurons and tangentially migrating inhibitory interneurons. Together, these abnormalities lead to altered lamination in the S1 at the terminal stages of corticogenesis in Tbx1 null mice and similar anomalies in Tbx1 heterozygous adult mice. Finally, we show that mesoderm-specific inactivation of Tbx1 is sufficient to recapitulate the brain phenotype indicating that Tbx1 exerts a cell nonautonomous role in cortical development from the mesoderm.

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.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

miRNeye: a microRNA expression atlas of the mouse eye.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. RESULTS: In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina. CONCLUSIONS: This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.