Lymphatic endothelial transcription factor Tbx1 promotes an immunosuppressive microenvironment to facilitate post-myocardial infarction repair.

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge syndrome disease gene that encodes a T-box transcription factor (TF). We found induced profound lymphangiogenic and immunomodulatory gene expression changes in lymphatic endothelial cells (LECs) after myocardial infarction (MI). The activated LECs penetrated the infarcted area and functioned as intramyocardial immune hubs to increase the numbers of tolerogenic dendritic cells (tDCs) and regulatory T (Treg) cells through the chemokine Ccl21 and integrin Icam1, thereby inhibiting the expansion of autoreactive CD8+ T cells and promoting reparative macrophage expansion to facilitate post-MI repair. Mimicking its timing and implementation may be an additional approach to treating autoimmunity-mediated cardiac diseases.

Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects.

Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.

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.

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.

Molecular Pathways and Animal Models of Truncus Arteriosus.

In normal cardiovascular development in birds and mammals, the outflow tract of the heart is divided into two distinct channels to separate the oxygenated systemic blood flow from the deoxygenated pulmonary circulation. When the process of outflow tract septation fails, a single common outflow vessel persists resulting in a serious clinical condition known as persistent truncus arteriosus or common arterial trunk. In this chapter, we will review molecular pathways and the cells that are known to play a role in the formation and development of the outflow tract and how genetic manipulation of these pathways in animal models can result in common arterial trunk.

Ventricular Septal Defects: Molecular Pathways and Animal Models.

Ventricular septation is a complex process which involves the major genes of cardiac development, acting on myocardial cells from first and second heart fields, and on mesenchymal cells from endocardial cushions. These genes, coding for transcription factors, interact with each other, and their differential expression conditions the severity of the phenotype. In this chapter, we will describe the formation of the ventricular septum in the normal heart, as well as the molecular mechanisms leading to the four main anatomic types of ventricular septal defects: outlet, inlet, muscular, and central perimembranous, resulting from failure of development of the different parts of the ventricular septum. Experiments on animal models, particularly transgenic mouse lines, have helped us to decipher the molecular determinants of ventricular septation. However, a precise description of the anatomic phenotypes found in these models is mandatory to achieve a better comprehension of the complex mechanisms responsible for the various types of VSDs.

Molecular Pathways and Animal Models of Semilunar Valve and Aortic Arch Anomalies.

The great arteries of the vertebrate carry blood from the heart to the systemic circulation and are derived from the pharyngeal arch arteries. In higher vertebrates, the pharyngeal arch arteries are a symmetrical series of blood vessels that rapidly remodel during development to become the asymmetric aortic arch arteries carrying oxygenated blood from the left ventricle via the outflow tract. At the base of the aorta, as well as the pulmonary trunk, are the semilunar valves. These valves each have three leaflets and prevent the backflow of blood into the heart. During development, the process of aortic arch and valve formation may go wrong, resulting in cardiovascular defects, and these may, at least in part, be caused by genetic mutations. In this chapter, we will review models harboring genetic mutations that result in cardiovascular defects affecting the great arteries and the semilunar valves.

Human Genetics of Truncus Arteriosus.

Integrated human genetics and molecular/developmental biology studies have revealed that truncus arteriosus is highly associated with 22q11.2 deletion syndrome. Other congenital malformation syndromes and variants in genes encoding TBX, GATA, and NKX transcription factors and some signaling proteins have also been reported as its etiology.

Human Genetics of Tetralogy of Fallot and Double-Outlet Right Ventricle.

Tetralogy of Fallot (TOF) and double-outlet right ventricle (DORV) are conotruncal defects resulting from disturbances of the second heart field and the neural crest, which can occur as isolated malformations or as part of multiorgan syndromes. Their etiology is multifactorial and characterized by overlapping genetic causes. In this chapter, we present the different genetic alterations underlying the two diseases, which range from chromosomal abnormalities like aneuploidies and structural mutations to rare single nucleotide variations affecting distinct genes. For example, mutations in the cardiac transcription factors NKX2-5, GATA4, and HAND2 have been identified in isolated TOF cases, while mutations of TBX5 and 22q11 deletion, leading to haploinsufficiency of TBX1, cause Holt-Oram and DiGeorge syndrome, respectively. Moreover, genes involved in signaling pathways, laterality determination, and epigenetic mechanisms have also been found mutated in TOF and/or DORV patients. Finally, genome-wide association studies identified common single nucleotide polymorphisms associated with the risk for TOF.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Complete Sequence of the 22q11.2 Allele in 1,053 Subjects with 22q11.2 Deletion Syndrome Reveals Modifiers of Conotruncal Heart Defects.

The 22q11.2 deletion syndrome (22q11.2DS) results from non-allelic homologous recombination between low-copy repeats termed LCR22. About 60%-70% of individuals with the typical 3 megabase (Mb) deletion from LCR22A-D have congenital heart disease, mostly of the conotruncal type (CTD), whereas others have normal cardiac anatomy. In this study, we tested whether variants in the hemizygous LCR22A-D region are associated with risk for CTDs on the basis of the sequence of the 22q11.2 region from 1,053 22q11.2DS individuals. We found a significant association (FDR p < 0.05) of the CTD subset with 62 common variants in a single linkage disequilibrium (LD) block in a 350 kb interval harboring CRKL. A total of 45 of the 62 variants were associated with increased risk for CTDs (odds ratio [OR) ranges: 1.64-4.75). Associations of four variants were replicated in a meta-analysis of three genome-wide association studies of CTDs in affected individuals without 22q11.2DS. One of the replicated variants, rs178252, is located in an open chromatin region and resides in the double-elite enhancer, GH22J020947, that is predicted to regulate CRKL (CRK-like proto-oncogene, cytoplasmic adaptor) expression. Approximately 23% of patients with nested LCR22C-D deletions have CTDs, and inactivation of Crkl in mice causes CTDs, thus implicating this gene as a modifier. Rs178252 and rs6004160 are expression quantitative trait loci (eQTLs) of CRKL. Furthermore, set-based tests identified an enhancer that is predicted to target CRKL and is significantly associated with CTD risk (GH22J020946, sequence kernal association test (SKAT) p = 7.21 × 10-5) in the 22q11.2DS cohort. These findings suggest that variance in CTD penetrance in the 22q11.2DS population can be explained in part by variants affecting CRKL expression.

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.

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

22q11.2 duplications: Expanding the clinical presentation.

22q11.2 duplication syndrome has a frequency of ~1/700 in the intellectual disability population. Despite this frequency, there is limited information on the variable clinical presentation. Although the phenotype and incidence of congenital anomalies are well described for 22q11.2 deletion syndrome, they are not as well understood for individuals with 22q11.2 duplication syndrome. This study is a single-center, retrospective review of patients diagnosed with 22q11.2 duplication syndrome designed to categorize the variable phenotype seen in these individuals. The data suggest that the incidence of congenital anomalies may be higher than previously reported for this syndrome. Affected individuals are at increased risk for a variety of problems including gastrointestinal complications, endocrine dysfunction, ophthalmologic abnormalities, palatal anomalies, congenital heart disease, musculoskeletal differences, and neurologic abnormalities. Individuals with 22q11.2 duplication syndrome would benefit from care coordinated by a multidisciplinary team and managed according to the 22q11.2 deletion syndrome guidelines.

To roll the eyes and snap a bite - function, development and evolution of craniofacial muscles.

Craniofacial muscles, muscles that move the eyes, control facial expression and allow food uptake and speech, have long been regarded as a variation on the general body muscle scheme. However, evidence has accumulated that the function of head muscles, their developmental anatomy and the underlying regulatory cascades are distinct. This article reviews the key aspects of craniofacial muscle and muscle stem cell formation and discusses how this differs from the trunk programme of myogenesis; we show novel RNAseq data to support this notion. We also trace the origin of head muscle in the chordate ancestors of vertebrates and discuss links with smooth-type muscle in the primitive chordate pharynx. We look out as to how the special properties of head muscle precursor and stem cells, in particular their competence to contribute to the heart, could be exploited in regenerative medicine.

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.

TBX1 is required for normal stria vascularis and semicircular canal development.

Little is known about the role of TBX1 in post-otocyst stages of inner ear development. Here, we report on mice with a missense mutation of Tbx1 that are viable with fully developed but abnormally formed inner ears. Mutant mice are deaf due to an undeveloped stria vascularis and show vestibular dysfunction associated with abnormal semicircular canal formation. We show that TBX1 is expressed in endolymph-producing strial marginal cells and vestibular dark cells of the inner ear and is an upstream regulator of Esrrb, which previously was shown to control the developmental fate of these cells. We also show that TBX1 is expressed in sensory cells of the crista ampullaris, which may relate to the semicircular canal abnormalities observed in mutant mice. Inner ears of mutant embryos have a non-resorbed fusion plate in the posterior semicircular canal and a single ampulla connecting anterior and lateral canals. We hypothesize that the TBX1 missense mutation prevents binding with specific co-regulatory proteins. These findings reveal previously unknown functions of TBX1 during later stages of inner ear development.

Decrease in the T-box1 gene expression in embryonic brain and adult hippocampus of down syndrome mouse models.

Down syndrome (DS, Trisomy 21) is the most common genetic cause of delayed fetal brain development and postnatal intellectual disability. Although delayed fetal brain development might be involved in intellectual disability, no evidence of an association between these abnormal phenotypes has been shown. To identify molecules differentially expressed in both the prenatal forebrain and adult hippocampus of Ts1Cje mice, a mouse model of DS, we employed a transcriptomic analysis. In the present study, we conducted transcriptomic profiling of the hippocampus of adult Ts1Cje mice and compared the results with the previously obtained transcriptomic profile of the prenatal forebrain at embryonic day 14.5. Results showed that the Tbx1 mRNA expression was decreased at both life stages. In addition, the decreased expression of Tbx1 mRNA was confirmed in other DS mouse models, Dp(16)1Yey/+ and Ts1Rhr mice, which carry longer and shorter trisomic regions, respectively. Taken together, these findings suggest that Tbx1 may link the delayed fetal brain development and intellectual disability in DS.