Biology subjects

Bilio, M.

Publications and source records attributed to Bilio, M..

8 recordsLinked to original sources

bioRxiv2026

Regulatory mutants of the Tbx1 gene alter transcription programs of lineage determination and patterning in early mesoderm.

The Tbx1 gene is haploinsufficient in mice and in humans, where it causes a DiGeorge syndrome phenotype characterized by developmental deficits of the pharyngeal apparatus. TBX1 plays a critical role in the differentiation and regionalization of the cardiopharyngeal mesoderm lineage and its derivatives. Nevertheless, its regulation is incompletely understood. Here we used a combination of computational and wet-lab approaches to identify regulatory sequences of the Tbx1 gene, and we use single-cell molecular analysis as a read-out and to establish the consequences of their deletion. Results revealed a cluster of regulatory sequences with at least three distinct elements. Elimination of the entire cluster caused a near shut down of the gene, while individual deletions had milder, quantitative effects. Transcriptomic analyses of the deletion mutants revealed the down regulation of genes related to cardiopharyngeal lineage specification and, more surprisingly, up regulation and anteriorization of genes related to embryonic patterning, thereby providing a rationale for the severe dysmorphogenesis of the posterior pharyngeal apparatus observed in Tbx1 mutant mice.

developmental biology↗

bioRxiv2026

Vegfr3 is required in the Tbx1 expression domain for cardiac outflow tract development.

Gene inactivation in model organisms has identified numerous genes and signaling pathways involved in mammalian cardiac OFT development. Human genetics data have implicated the VEGFR3 gene in OFT development but when and where it is required is unknown. In this study we determined the sensitivity of the developing murine cardiac OFT to reduced Vegfr3 gene dosage and we tested whether its requirement is dependent upon TBX1, a known regulator of Vegfr3 expression in cardiac and lymphatic endothelial cells. We found that in the mouse, a single copy if the Vegfr3 gene was sufficient for normal cardiac OFT development in most cases. Mutation of a single copy of the Tbx1 gene greatly enhanced the sensitivity of OFT development to Vegfr3 dosage reduction and led to the formation of severe OFT anomalies. In addition, deletion of Vegfr3 in the Tbx1 expression domain also led to OFT abnormalities. We used RNAscope to reveal the location of Vegfr3 and Tbx1 transcripts in midterm mouse embryos. This revealed co-localization of these transcripts that was restricted to the aortic sac endothelium, suggesting that the distal OFT is a potential site of genetic interaction between Vegfr3 and TBX1 that is critical for normal OFT development.

developmental biology↗

bioRxiv2024

Tbx1 stabilizes differentiation of the cardiopharyngeal mesoderm and drives morphogenesis in the pharyngeal apparatus

BackgroundTBX1 is required for the development of the pharyngeal apparatus. In the mouse, fish, and ascidian, Tbx1 is a marker of cardiopharyngeal mesoderm (CPM), a cell population that provides progenitors to the heart and branchiomeric muscles. However, in mammals: a) the molecular cascade that drives the diversification of this multipotent cell population, and b) the role of Tbx1 therein, are not well defined. Material and methodsWe used in vitro differentiation of WT and Tbx1-/- mouse embryonic stem cells into precardiac mesoderm, and performed single cell RNA-seq and ATAC-seq at two differentiation stages. We then used WT and Tbx1-/- mouse embryos for in vivo validation of the key findings. Results and conclusionsWe found that the response to loss of TBX1 is cell sub-population-specific, both in terms of gene expression and chromatin remodeling. We show that Tbx1 regulates chromatin accessibility and gene expression of an ancient transcriptional module that orchestrates the development of the trunk, pharynx and heart across evolution. This module is co-regulated and includes genes encoding the conserved transcription factor families of Tea Shirt (Tshz), Sine Oculis (Six), Eye absent (Eya), and Ebf/Collier. Analysis of putative regulatory regions of these genes, which were selected using a machine-learning computational procedure, predicted a feed-forward regulatory relationship between TBX1 and SIX factors that drives or stabilizes the module. Most surprisingly, we found a drift in the differentiation trajectory of the Tbx1 mutant CPM that led to a relative expansion of cells with epithelial-like transcriptional features in the cell culture model and in mouse embryos. We conclude that TBX1 is a critical factor for maintaining the transcriptional profile of the CPM.

developmental biology↗

bioRxiv2022

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

The loss of a single copy of TBX1 accounts for most of the clinical signs and symptoms of 22q11.2 deletion syndrome (22q11.2DS), a common genetic disorder that is characterized by multiple congenital anomalies and brain-related clinical problems, some of which likely have vascular origins. Tbx1 mutant mice have brain vascular anomalies, thus making them a useful model to gain insights into the brain disorders associated with the human disease. Here, we found that Tbx1 has a dynamic expression pattern in brain endothelial cells (ECs), including tip cells, during early vascularization, but it is not expressed in EC progenitors. Its main morphogenetic function in the brain is to regulate negatively filopodia biogenesis and vessel branching. Because of similar phenotypes reported for Vegfr3 loss of function, we pursued a mouse genetic approach to test TBX1-VEGFR3 interaction through gain and loss of function experiments. Vegfr3 is expressed in brain ECs with extensive overlap with Tbx1 expression. We demonstrate that inactivating Vegfr3 in the Tbx1 expression domain in a Tbx1 mutant background enhances vessel branching and filopodia formation to a greater extent than that observed in the individual mutants. Furthermore, using a mouse transgenic line, we show that increasing Vegfr3 expression in the Tbx1 expression domain fully rescued the vessel branching and filopodia phenotypes caused by Tbx1 loss of function. Similar results were obtained using an in vitro model of endothelial tubulogenesis. Overall, these results provide genetic evidence that Vegfr3 is a regulator of early vessel branching and filopodia formation in the brain, and is a likely critical effector of the brain vascular phenotype caused by Tbx1 loss of function.

developmental biology↗

bioRxiv2021

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 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 molecular level, the in vivo vB12 treatment let us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that 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, SLUG+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos and vB12 treatment restore 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 core mesoderm and the arrangement of multiple lineages within the PhAp.

developmental biology↗

bioRxiv2019

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

Introduction and HypothesisPatients 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.\n\nWe 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.\n\nMethodsWe 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.\n\nResultsThe 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).\n\nMouse 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.\n\nConclusionsChildren 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.

developmental biology↗

bioRxiv2018

Tbx1 regulates extracellular matrix- and cell-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 (CPs) 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 extra cellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos showed that this function is non-cell autonomous and, in cultured cells, loss of TBX1 impairs cell migration and focal adhesions. Additionally, we found that ECM-mediated outside-in 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.

developmental biology↗

bioRxiv2017

Tbx1 represses Mef2c gene expression by inducing histone 3 deacetylation of the anterior heart field enhancer

The TBX1 gene is haploinsufficient in the 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 is associated with down regulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro differentiated induced pluripotent stem cells (hiPSCs) expressed higher levels of Mef2c and showed increased AHF acetylation, compared to 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.

developmental biology↗