On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells.

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.

Inborn errors of immunity with eosinophilia.

Monogenic diseases of the immune system, also known as inborn errors of immunity (IEIs), are caused by single-gene mutations and result in immune deficiency and dysregulation. More than 400 monogenic diseases have been described to date, and this number is rapidly expanding. The increasing availability of next-generation sequencing is now facilitating the diagnosis of IEIs. It is known that IEIs can predispose a person to not only infectious diseases but also cancer and immune disorders, such as inflammatory, autoimmune, and atopic diseases. IEIs with eosinophilia and atopic diseases can occur in several disorders. IEIs with eosinophilia have provided insights into human immunity and the pathogenesis of allergic diseases. Eosinophilia is not a rare finding in clinical practice, and it often poses problems in terms of etiologic research and differential diagnoses. Secondary eosinophilia is the most common form. The main underlying conditions are infectious diseases such as parasitic infections, allergic disorders, drug reactions, and of course IEIs. In clinical settings, the recognition of IEIs in the context of an allergic phenotype with eosinophilia is critical for prompt diagnosis and appropriate treatment aimed at modulating pathophysiological mechanisms and improving clinical symptoms.

High Th2 cytokine levels and upper airway inflammation in human inherited T-bet deficiency.

We have described a child suffering from Mendelian susceptibility to mycobacterial disease (MSMD) due to autosomal recessive, complete T-bet deficiency, which impairs IFN-γ production by innate and innate-like adaptive, but not mycobacterial-reactive purely adaptive, lymphocytes. Here, we explore the persistent upper airway inflammation (UAI) and blood eosinophilia of this patient. Unlike wild-type (WT) T-bet, the mutant form of T-bet from this patient did not inhibit the production of Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, when overexpressed in T helper 2 (Th2) cells. Moreover, Herpesvirus saimiri-immortalized T cells from the patient produced abnormally large amounts of Th2 cytokines, and the patient had markedly high plasma IL-5 and IL-13 concentrations. Finally, the patient's CD4+ αß T cells produced most of the Th2 cytokines in response to chronic stimulation, regardless of their antigen specificities, a phenotype reversed by the expression of WT T-bet. T-bet deficiency thus underlies the excessive production of Th2 cytokines, particularly IL-5 and IL-13, by CD4+ αß T cells, causing blood eosinophilia and UAI. The MSMD of this patient results from defective IFN-γ production by innate and innate-like adaptive lymphocytes, whereas the UAI and eosinophilia result from excessive Th2 cytokine production by adaptive CD4+ αß T lymphocytes.

An optimized workflow for CRISPR-Cas9 deletion of surface and intracellular factors in primary human T lymphocytes.

The appropriate regulation of T lymphocyte functions is key to achieve protective immune responses, while at the same time limiting the risks of tissue damage and chronic inflammation. Deciphering the mechanisms underpinning T cell responses in humans may therefore be beneficial for a range of infectious and chronic diseases. Recently, the development of methods based on CRISPR-Cas9 gene-editing has greatly expanded the available tool-box for the mechanistic studies of primary human T cell responses. While the deletion of a surface protein has become a relatively straightforward task, as long as an antibody for detection is available, the identification and selection of cells lacking an intracellular protein, a non-coding RNA or a protein for which no antibody is available, remain more problematic. Here, we discuss the options currently available to scientists interested in performing gene-editing in primary human T lymphocytes and we describe the optimization of a workflow for the screening and analysis of lymphocytes following gene-editing with CRISPR-Cas9 based on T cell cloning and T7 endonuclease I cleavage assay.

Th2 cells lacking T-bet suppress naive and memory T cell responses via IL-10.

Exacerbated immune responses and loss of self-tolerance lead to the development of autoimmunity and immunopathology. Novel therapies to target autoreactive T cells are still needed. Here, we report that Th2-polarized T cells lacking the transcription factor T-bet harbor strong immunomodulatory potential and suppress antigen-specific CD8+ T cells via IL-10. Tbx21-/- Th2 cells protected mice against virus-induced type 1 diabetes development and suppressed not only naive but also memory CD8+ T cell responses. IL-10-producing, but not IL-10-deficient Tbx21-/- Th2 cells down-regulated costimulatory molecules on dendritic cells and reduced their IL-12 production after lymphocytic choriomeningitis virus infection. Impaired dendritic cell activation hindered effector and cytotoxic CD8+ T cell development after infection. These findings indicate that Tbx21-/- Th2 cells strongly suppress proinflammatory responses of naive and memory T cells via IL-10. Thus, in vivo IL-10-secreting Th2 cells could harbor a therapeutic potential for the treatment of T cell-mediated inflammatory disorders.

TBX5-encoded T-box transcription factor 5 variant T223M is associated with long QT syndrome and pediatric sudden cardiac death.

Long QT syndrome (LQTS) is a genetic disease resulting in a prolonged QT interval on a resting electrocardiogram, predisposing affected individuals to polymorphic ventricular tachycardia and sudden death. Although a number of genes have been implicated in this disease, nearly one in four individuals exhibiting the LQTS phenotype are genotype-negative. Whole-exome sequencing identified a missense T223M variant in TBX5 that cosegregates with prolonged QT interval in a family with otherwise genotype-negative LQTS and sudden death. The TBX5-T223M variant was absent among large ostensibly healthy populations (gnomAD) and predicted to be pathogenic by in silico modeling based on Panther, PolyPhen-2, Provean, SIFT, SNAP2, and PredictSNP prediction tools. The variant was located in a highly conserved region of TBX5 predicted to be part of the DNA-binding interface. A luciferase assay identified a 57.5% reduction in the ability of TBX5-T223M to drive expression at the atrial natriuretic factor promotor compared to wildtype TBX5 in vitro. We conclude that the variant is pathogenic in this family, and we put TBX5 forward as a disease susceptibility allele for genotype-negative LQTS. The identification of this familial variant may serve as a basis for the identification of previously unknown mechanisms of LQTS with broader implications for cardiac electrophysiology.

Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria.

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αß T and non-classic CD4+ αß TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αß T, and CD4+ αß TH1∗ cells unable to compensate for this deficit.

Antigen-Specific CD4+ T Cells Exhibit Distinct Kinetic and Phenotypic Patterns During Primary and Secondary Responses to Infection.

Although CD4+ T cell memory is a critical component of adaptive immunity, antigen-specific CD4+ T cell recall responses to secondary infection have been inadequately studied. Here we examine the kinetics of the secondary response in an important immunological model, infection with attenuated Listeria monocytogenes (Lm). We identify CD4+ T cell subsets that preferentially expand during a secondary response and highlight the importance of prime-boost strategies in expanding and maintaining antigen-specific, tissue-resident memory CD4+ T cells. Following intravenous infection with an attenuated strain of Lm, we found that total antigen-specific CD4+ T cells responded more robustly in secondary compared with primary infection, reaching near-peak levels in secondary lymphoid organs (SLOs) and the liver by three days post-infection. During the secondary response, CD4+ T cells also contracted more quickly. Primary Lm infection generated two main classes of effector cells: Th1 cells that assist macrophages and T follicular helper (Tfh) cells that aid B cells in antibody production. We found that during the secondary response, a population of Ly6C+ Tfh cells emerged in SLOs and was the basis for the skewing of this response to a Tfh phenotype. Deletion of T-bet in T cells precluded development of Ly6C+ Tfh cells, but did not alter anti-Lm antibody responses. Moreover, during recall responses, CD49a+ Th1 cells preferentially expanded and accumulated in the liver, achieving a new set point. Parabiosis experiments indicated that, in contrast to Tfh cells and most splenic Th1 cells, the majority of CD49a+ Th1 cells in the liver were tissue resident. Overall, these data demonstrate a robust secondary CD4+ T cell response that differs in kinetics and composition from the primary response and provide insight into targets to enhance both peripheral and tissue-resident CD4+ T cell responses.

Mice Deficient in T-bet Form Inducible NO Synthase-Positive Granulomas That Fail to Constrain Salmonella.

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ-/- mice succumb rapidly to STm infections, T-bet-/- mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ-/- and T-bet-/- mice. In IFN-γ-/- mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet-/- mice induce significant levels of IFN-γ- after challenge. Moreover, T-bet-/- mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet-/- mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS+ granuloma formation. In wild-type mice, most bacteria are within iNOS+ granulomas, but in T-bet-/- mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS+ granulomas and prevent dissemination.

Stage-Specific Requirement for Eomes in Mature NK Cell Homeostasis and Cytotoxicity.

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.

Transcriptional Patterning of the Ventricular Cardiac Conduction System.

RATIONALE: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. OBJECTIVE: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. METHODS AND RESULTS: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. CONCLUSIONS: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells.

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.

Tbx15 is required for adipocyte browning induced by adrenergic signaling pathway.

OBJECTIVE: The T-box gene Tbx15 is abundantly expressed in adipose tissues, especially subcutaneous and brown fat. Although its expression is correlated with obesity, its precise biological role in adipose tissue is poorly understood in vivo. Here we investigated the function of Tbx15 in brown adipose thermogenesis and white adipose browning in vivo. METHODS: In the present study, we generated adipose-specific Tbx15 knockout (AKO) mice by crossing Tbx15 floxed mice with adiponectin-Cre mice to delineate Tbx15 function in adipose tissues. We systematically investigated the influence of Tbx15 on brown adipose thermogenesis and white adipose browning in mice, as well as the possible underlying molecular mechanism. RESULTS: Upon cold exposure, adipocyte browning in inguinal adipose tissue was significantly impaired in Tbx15 AKO mice. Furthermore, ablation of Tbx15 blocked adipocyte browning induced by ß3 adrenergic agonist CL 316243, which did not appear to alter the expression of Tbx15. Analysis of DNA binding sites using chromatin-immunoprecipitation (ChIP) revealed that TBX15 bound directly to a key region in the Prdm16 promoter, indicating it regulates transcription of Prdm16, the master gene for adipocyte thermogenesis and browning. Compared to control mice, Tbx15 AKO mice displayed increased body weight gain and decreased whole body energy expenditure in response to high fat diets. CONCLUSION: Taken together, these findings suggest that Tbx15 regulates adipocyte browning and might be a potential target for the treatment of obesity.

Downregulation of Transcription Factor T-Bet as a Protective Strategy in Monosodium Urate-Induced Gouty Inflammation.

Gout is sterile joint inflammation triggered by the damaging effects of monosodium urate (MSU) crystals accumulation. Previous studies suggest transcription factor T-bet plays an important role in inflammatory arthritis. Notably, mice lacking T-bet markedly reduced joint inflammation of rheumatoid arthritis models, however, the involvement of T-bet in gouty inflammation has yet to be clarified. Here, we took advantage of T-bet knockout (KO) mice to investigate the role of T-bet in the pathogenesis of MSU-induced gout inflammation. T-bet KO and wild type (WT) mice were used for models of acute inflammation induced with MSU crystals, including footpad, air pouch and peritonitis models. Inflammatory cytokines and phagocytosis were detected in bone-marrow-derived macrophages (BMDMs) from T-bet KO and WT mice treated with MSU crystals in vitro. In addition, T-bet expression in peripheral blood mononuclear cells (PBMCs) from gout patients was measured, as well as plasma inflammatory cytokines. We found that the levels of interleukin (IL)-17, IL-23, and interferon-γ were reduced, but tumor necrosis factor-α was not, in BMDMs from T-bet KO compared with WT mice after MSU challenge in vitro, as well as MSU phagocytosis. In comparison with WT mice in vivo, the swelling index of T-bet KO mice was significantly decreased in the footpad model. T-bet deficiency also dramatically relieved MSU-induced inflammatory cell infiltration in peritonitis and air pouch models in vivo, and as well as the IL-1ß levels of air pouch lavage fluid (APLF). In addition, plasma IL-17 and IL-23 levels were elevated in acute gout, whereas protein levels of T-bet were downregulated in PBMCs from acute gout patients and intercritical gout treated with MSU crystals in vitro as well. Transcription factor T-bet deficiency protects against MSU-induced gouty inflammation, suggesting that downregulation of T-bet could be a protective strategy and contribute to spontaneous remission of inflammation in acute gout.

Eomes transcription factor is required for the development and differentiation of invariant NKT cells.

Eomes regulates the differentiation of CD8+ T cells into effector and memory phases. However, its role in invariant (i)NKT cells remains unknown. Here, we show the impact of Eomes on iNKT cells in the thymus and peripheral tissue using conditional knockout (Eomes-cKO) mice. In the thymus, CD1d-tetramer+CD24+CD44-NK1.1-CD69+stage 0 iNKT cells express higher levels of Eomes than the other iNKT stages. We also found that Eomes regulates NKT1 cell differentiation predominantly. Interestingly, the expression of Eomes in the steady state is low, but can be upregulated after TCR stimulation. We also showed epigenetic changes in the Eomes locus after activation. In addition, vaccination of C57BL/6, but not Eomes-cKO mice with iNKT ligand-loaded dendritic cells generated KLRG1+iNKT cells in lung, characterized as effector memory phenotype by transcriptome profiling. Thus, Eomes regulates not only the differentiation of NKT1 cells in the thymus, but also their differentiation into memory-like KLRG1+iNKT cells in the periphery.

Transcription Factor T-bet Attenuates the Development of Elastase-induced Emphysema in Mice.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by peripheral airways inflammation and emphysema. Emerging evidence indicates a contribution of both innate and adaptive immune cells to the development of COPD. Transcription factor T-bet modulates the function of immune cells and therefore might be involved in the pathogenesis of COPD. To elucidate the role for T-bet in elastase-induced emphysema, pathological phenotypes were compared between wild-type and T-bet-/- mice. T-bet-/- mice demonstrated enhanced emphysema development on histological analyses, with higher values of mean linear intercept and dynamic compliance relative to wild-type mice. The number of neutrophils in BAL fluids, lung IL-6 and IL-17 expression, and the proportion of CD4+ T cells positive for IL-17 or retinoic acid receptor-related orphan receptor-γt were higher in T-bet-/- mice than in wild-type mice. Although T-bet downregulates cytokine expression in bone marrow-derived macrophages and MH-S cells, a murine alveolar cell line, depending on the surrounding environment, IL-6 expression in alveolar macrophages isolated from elastase-treated mice was not dependent on T-bet. Coculture of bone marrow-derived macrophages and CD4+ T cells revealed that T-bet regulation of IL-17 expression was dependent on CD4+ T cells. Neutralizing antibodies against IL-6R or IL-17 ameliorated the development of emphysema in T-bet-/- mice. In conclusion, we demonstrate that T-bet ameliorates elastase-induced emphysema formation by modulating the host immune response in the lungs.

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.

Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency.

The CXCL12-CXCR4 pathway has crucial roles in stem cell homing and maintenance, neuronal guidance, cancer progression, inflammation, remote-conditioning, cell migration and development. Recently, work in chick suggested that signalling via CXCR4 in neural crest cells (NCCs) has a role in the 22q11.2 deletion syndrome (22q11.2DS), a disorder where haploinsufficiency of the transcription factor TBX1 is responsible for the major structural defects. We tested this idea in mouse models. Our analysis of genes with altered expression in Tbx1 mutant mouse models showed down-regulation of Cxcl12 in pharyngeal surface ectoderm and rostral mesoderm, both tissues with the potential to signal to migrating NCCs. Conditional mutagenesis of Tbx1 in the pharyngeal surface ectoderm is associated with hypo/aplasia of the 4th pharyngeal arch artery (PAA) and interruption of the aortic arch type B (IAA-B), the cardiovascular defect most typical of 22q11.2DS. We therefore analysed constitutive mouse mutants of the ligand (CXCL12) and receptor (CXCR4) components of the pathway, in addition to ectodermal conditionals of Cxcl12 and NCC conditionals of Cxcr4. However, none of these typical 22q11.2DS features were detected in constitutively or conditionally mutant embryos. Instead, duplicated carotid arteries were observed, a phenotype recapitulated in Tie-2Cre (endothelial) conditional knock outs of Cxcr4. Previous studies have demonstrated genetic interaction between signalling pathways and Tbx1 haploinsufficiency e.g. FGF, WNT, SMAD-dependent. We therefore tested for possible epistasis between Tbx1 and the CXCL12 signalling axis by examining Tbx1 and Cxcl12 double heterozygotes as well as Tbx1/Cxcl12/Cxcr4 triple heterozygotes, but failed to identify any exacerbation of the Tbx1 haploinsufficient arch artery phenotype. We conclude that CXCL12 signalling via NCC/CXCR4 has no major role in the genesis of the Tbx1 loss of function phenotype. Instead, the pathway has a distinct effect on remodelling of head vessels and interventricular septation mediated via CXCL12 signalling from the pharyngeal surface ectoderm and second heart field to endothelial cells.