Performance of a targeted cell-free DNA prenatal test for 22q11.2 deletion in a large clinical cohort.

OBJECTIVE: 22q11.2 deletion is more common than trisomies 18 and 13 combined, yet no routine approach to prenatal screening for this microdeletion has been established. This study evaluated the clinical sensitivity and specificity of a targeted cell-free DNA (cfDNA) test to screen for fetal 22q11.2 deletion in a large cohort, using blinded analysis of prospectively enrolled pregnancies and stored clinical samples. METHODS: In order to ensure that the analysis included a meaningful number of cases with fetal 22q11.2 deletion, maternal plasma samples were obtained by prospective, multicenter enrolment of pregnancies with a fetal cardiac abnormality and from stored clinical samples from a research sample bank. Fetal genetic status, as evaluated by microarray analysis, karyotyping with fluorescence in-situ hybridization or a comparable test, was available for all cases. Samples were processed as described previously for the Harmony prenatal test, with the addition of DANSR (Digital Analysis of Selected Regions) assays targeting the 3.0-Mb region of 22q11.2 associated with 22q11.2 deletion syndrome. Operators were blinded to fetal genetic status. Sensitivity and specificity of the cfDNA test for 22q11.2 deletion were calculated based on concordance between the cfDNA result and fetal genotype. RESULTS: The final study group consisted of 735 clinical samples, including 358 from prospectively enrolled pregnancies and 377 stored clinical samples. Of 46 maternal plasma samples from pregnancies with a 22q11.2 deletion, ranging in size from 1.25 to 3.25 Mb, 32 had a cfDNA result indicating a high probability of 22q11.2 deletion (sensitivity, 69.6% (95% CI, 55.2-80.9%)). All 689 maternal plasma samples without a 22q11.2 deletion were classified correctly by the cfDNA test as having no evidence of a 22q11.2 deletion (specificity, 100% (95% CI, 99.5-100%)). CONCLUSIONS: The results of this large-scale prospective clinical evaluation of the sensitivity and specificity of a targeted cfDNA test for fetal 22q11.2 deletion demonstrate that this test can detect the common and smaller, nested 22q11.2 deletions with a low (0-0.5%) false-positive rate. Although the positive predictive value (PPV) observed in this study population was 100%, the expected PPV in the general pregnant population is estimated to be 12.2% at 99.5% specificity and 41.1% at 99.9% specificity. The use of this cfDNA test to screen for 22q11.2 deletion could enhance identification of pregnancies at risk for 22q11.2 deletion syndrome without significantly increasing the likelihood of maternal anxiety and unnecessary invasive procedures related to a false-positive result. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Prenatal diagnosis of familial 22q11.2 deletion syndrome in a pregnancy with concomitant cardiac and urinary tract abnormalities in the fetus and the mother.

OBJECTIVE: We present prenatal diagnosis of familial 22q11.2 deletion syndrome in a pregnancy with concomitant cardiac and urinary tract abnormalities in the fetus and the mother. CASE REPORT: A 28-year-old woman primigravid underwent amniocentesis at 23 weeks of gestation because of fetal ultrasound findings of aortic stenosis, interrupted aortic arch (IAA), left multicystic kidney, right hydronephrosis and ureterocele. Amniocentesis revealed a karyotype of 46,XX. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed the result of arr 22q11.21 (18,894,835-21,505,417) × 1.0 [GRCh37 (hg19)] with a 2.611-Mb 22q11.21 deletion encompassing 41 Online Mendelian Inheritance in Man (OMIM) genes including UFD1L, TBX1, GNB1L, COMT and MED15. aCGH analysis on the DNAs extracted from parental bloods confirmed that the mother carried the same 22q11.21 microdeletion. Level II ultrasound additionally found ventricular septal defect (VSD) and persistent left superior vena cava (PLSVC). Examination of the woman showed short stature, malar hypoplasia, hypertelorism, bulbous nasal tip, prominent nasal root, hypoplasia of nasal wings, right renal agenesis, left ureterovesical reflux and VSD with repair, but normal intelligence and normal neuropsychiatric development. The woman decided to continue the pregnancy, and a 2903-g female baby was delivered at 38 weeks of gestation with left multicystic kidney, right hydronephrosis, dysgenesis of corpus callosum, IAA, VSD, PLSVC, patent ductus arteriosus, patent foramen ovale, atrial septal defect, dilated main pulmonary artery and tricuspid regurgitation. The neonate died at the age of one month. CONCLUSION: Prenatal diagnosis of concomitant congenital heart defects and urinary tract abnormalities in the fetus and the parent should raise a suspicion of familial 22q11.2 deletion syndrome.

[Phenotypic and genotypic analysis of a fetus carrying an intermediate 22q11.2 deletion encompassing the CRKL gene].

OBJECTIVE: To delineate the phenotypic characteristics of 22q11.2 deletion syndrome and the role of CRKL gene in the pathogenesis of cardiac abnormalities. METHODS: G-banded karyotyping, single nucleotide polymorphism (SNP) array and fluorescence in situ hybridization (FISH) were performed on a fetus with tetralogy of Fallot detected by ultrasound. Correlation between the genotype and phenotype was explored after precise mapping of the breakpoints on chromosome 22q11.2. SNP array was also performed on peripheral blood samples from both parents to clarify its origin. RESULTS: The fetus showed a normal karyotype of 46,XY. SNP array performed on fetal blood sample revealed a 749 kb deletion (chr22: 20 716 876-21 465 659) at 22q11.21, which encompassed the CRKL gene but not TBX1, HIRA, COMT and MAPK1. Precise mapping of the breakpoints suggested that the deleted region has overlapped with that of central 22q11.2 deletion syndrome. SNP array analysis of the parental blood samples suggested that the 22q11.21 deletion has a de novo origin. The presence of 22q11.21 deletion in the fetus was also confirmed by FISH analysis. CONCLUSION: Central 22q11.21 deletion probably accounts for the cardiac abnormalities in the fetus, for which the CRKL gene should be considered as an important candidate.

[Prenatal diagnosis of 22q11 microdeletion syndrome].

OBJECTIVE: To establish a method for the prenatal diagnosis of 22q11 microdeletion syndrome. METHODS: BACs-on-Beads (BoBs) and fluorescence in situ hybridization (FISH) were performed on a fetus for whom amniotic chromosomal culturing has failed and a pair of twin fetuses suspected for 22q11 deletion syndrome. RESULTS: 22q11 microdeletion was detected in all 3 fetuses by prenatal BoBs as well as FISH, with only one red signal detected at the DiGeorge/VCFS N25 site and two green signals on the 22q13.3 ARSA site. CONCLUSION: The combination of prenatal BoBs and FISH can provide a method for the prenatal diagnosis of 22q11 microdeletion.

Congenital Heart Defects and Measures of Fetal Growth in Newborns with Down Syndrome or 22q11.2 Deletion Syndrome.

OBJECTIVES: To estimate the association between congenital heart defects (CHD) and indices of fetal growth in Down and 22q11.2 deletion syndromes. STUDY DESIGN: We established 2 Danish nationwide cohorts of newborn singletons with either Down syndrome (n = 670) or 22q11.2 deletion syndrome (n = 155), born 1997-2011. In both cohorts, we analyzed the association between CHD, CHD severity, and indices of fetal growth by multivariable linear regression adjusted for potential confounders. We report mean differences in gestational age specific z-scores compared with newborns without CHD. RESULTS: Down syndrome and 22q11.2 deletion syndrome were both associated with lower mean birth weight and head circumference z-scores. We found no association between CHD or CHD severity and indices of fetal growth. In Down syndrome, the association between any CHD and the mean difference in head circumference z-score was 0.03 (95% CI -0.12, 0.18), and the estimate regarding birth weight z-score was 0.09 (95% CI -0.08, 0.25). The corresponding estimates in 22q11.2 deletion syndrome were 0.00 (95% CI -0.33, 0.32) and -0.09 (95% CI -0.45, 0.26). CONCLUSIONS: We found no association between CHD and fetal growth measures in newborns with Down syndrome or 22q11.2 deletion syndrome. Thus, in certain subtypes of CHD, the contribution of genetic factors to prenatal growth impairment may be more important than circulatory disturbances.

Vitamin B12 ameliorates the phenotype of a mouse model of DiGeorge syndrome.

Abstract: Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome.

Clinical experience with single-nucleotide polymorphism-based non-invasive prenatal screening for 22q11.2 deletion syndrome.

OBJECTIVES: To evaluate the performance of a single-nucleotide polymorphism (SNP)-based non-invasive prenatal test (NIPT) for the detection of fetal 22q11.2 deletion syndrome in clinical practice, assess clinical follow-up and review patient choices for women with high-risk results. METHODS: In this study, 21 948 samples were submitted for screening for 22q11.2 deletion syndrome using a SNP-based NIPT and subsequently evaluated. Follow-up was conducted for all cases with a high-risk result. RESULTS: Ninety-five cases were reported as high risk for fetal 22q11.2 deletion. Diagnostic testing results were available for 61 (64.2%) cases, which confirmed 11 (18.0%) true positives and identified 50 (82.0%) false positives, resulting in a positive predictive value (PPV) of 18.0%. Information regarding invasive testing was available for 84 (88.4%) high-risk cases: 57.1% (48/84) had invasive testing and 42.9% (36/84) did not. Ultrasound anomalies were present in 81.8% of true-positive and 18.0% of false-positive cases. Two additional cases were high risk for a maternal 22q11.2 deletion; one was confirmed by diagnostic testing and one had a positive family history. There were three pregnancy terminations related to screening results of 22q11.2 deletion, two of which were confirmed as true positive by invasive testing. CONCLUSIONS: Clinical experience with this SNP-based non-invasive screening test for 22q11.2 deletion syndrome indicates that these deletions have a frequency of approximately 1 in 1000 in the referral population with most identifiable through this test. Use of this screening method requires the availability of counseling and other management resources for high-risk pregnancies.

TBX1 protein interactions and microRNA-96-5p regulation controls cell proliferation during craniofacial and dental development: implications for 22q11.2 deletion syndrome.

T-box transcription factor TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS, DiGeorge syndrome/Velo-cardio-facial syndrome), whose phenotypes include craniofacial malformations such as dental defects and cleft palate. In this study, Tbx1 was conditionally deleted or over-expressed in the oral and dental epithelium to establish its role in odontogenesis and craniofacial developmental. Tbx1 lineage tracing experiments demonstrated a specific region of Tbx1-positive cells in the labial cervical loop (LaCL, stem cell niche). We found that Tbx1 conditional knockout (Tbx1(cKO)) mice featured microdontia, which coincides with decreased stem cell proliferation in the LaCL of Tbx1(cKO) mice. In contrast, Tbx1 over-expression increased dental epithelial progenitor cells in the LaCL. Furthermore, microRNA-96 (miR-96) repressed Tbx1 expression and Tbx1 repressed miR-96 expression, suggesting that miR-96 and Tbx1 work in a regulatory loop to maintain the correct levels of Tbx1. Cleft palate was observed in both conditional knockout and over-expression mice, consistent with the craniofacial/tooth defects associated with TBX1 deletion and the gene duplication that leads to 22q11.2DS. The biochemical analyses of TBX1 human mutations demonstrate functional differences in their transcriptional regulation of miR-96 and co-regulation of PITX2 activity. TBX1 interacts with PITX2 to negatively regulate PITX2 transcriptional activity and the TBX1 N-terminus is required for its repressive activity. Overall, our results indicate that Tbx1 regulates the proliferation of dental progenitor cells and craniofacial development through miR-96-5p and PITX2. Together, these data suggest a new molecular mechanism controlling pathogenesis of dental anomalies in human 22q11.2DS.

Ranbp1, Deleted in DiGeorge/22q11.2 Deletion Syndrome, is a Microcephaly Gene That Selectively Disrupts Layer 2/3 Cortical Projection Neuron Generation.

Ranbp1, a Ran GTPase-binding protein implicated in nuclear/cytoplasmic trafficking, is included within the DiGeorge/22q11.2 Deletion Syndrome (22q11.2 DS) critical region associated with behavioral impairments including autism and schizophrenia. Ranbp1 is highly expressed in the developing forebrain ventricular/subventricular zone but has no known obligate function during brain development. We assessed the role of Ranbp1 in a targeted mouse mutant. Ranbp1(-/-) mice are not recovered live at birth, and over 60% of Ranbp1(-/-) embryos are exencephalic. Non-exencephalic Ranbp1(-/-) embryos are microcephalic, and proliferation of cortical progenitors is altered. At E10.5, radial progenitors divide more slowly in the Ranpb1(-/-) dorsal pallium. At E14.5, basal, but not apical/radial glial progenitors, are compromised in the cortex. In both E10.5 apical and E14.5 basal progenitors, M phase of the cell cycle appears selectively retarded by loss of Ranpb1 function. Ranbp1(-/-)-dependent proliferative deficits substantially diminish the frequency of layer 2/3, but not layer 5/6 cortical projection neurons. Ranbp1(-/-) cortical phenotypes parallel less severe alterations in LgDel mice that carry a deletion parallel to many (but not all) 22q11.2 DS patients. Thus, Ranbp1 emerges as a microcephaly gene within the 22q11.2 deleted region that may contribute to altered cortical precursor proliferation and neurogenesis associated with broader 22q11.2 deletion.

Tbx1 genetically interacts with the transforming growth factor-ß/bone morphogenetic protein inhibitor Smad7 during great vessel remodeling.

RATIONALE: Growth and remodeling of the pharyngeal arch arteries are vital for the development of a mature great vessel system. Dysmorphogenesis of the fourth arch arteries can result in interruption of the aortic arch type B, typically found in DiGeorge syndrome. Tbx1 haploinsufficient embryos, which model DiGeorge syndrome, display fourth arch artery defects during formation of the vessels. Recovery from such defects is a documented yet unexplained phenotype in Tbx1 haploinsufficiency. OBJECTIVE: To understand the nature of fourth arch artery growth recovery in Tbx1 haploinsufficiency and its underlying genetic control. METHODS AND RESULTS: We categorized vessel phenotypes of Tbx1 heterozygotes as hypoplastic or aplastic at the conclusion of pharyngeal artery formation and compared these against the frequency of vessel defects scored at the end of great vessel development. The frequency of hypoplastic vessels decreased during embryogenesis, whereas no reduction of vessel aplasia was seen, implying recovery is attributable to remodeling of hypoplastic vessels. We showed that Smad7, an inhibitory Smad within the transforming growth factor-ß pathway, is regulated by Tbx1, is required for arch artery remodeling, and genetically interacts with Tbx1 in this process. Tbx1 and Tbx1;Smad7 haploinsufficiency affected several remodeling processes; however, concurrent haploinsufficiency particularly impacted on the earliest stage of vascular smooth muscle cell vessel coverage and subsequent fibronectin deposition. Conditional reconstitution of Smad7 with a Tbx1Cre driver indicated that the interaction between the 2 genes is cell autonomous. CONCLUSIONS: Tbx1 acts upstream of Smad7 controlling vascular smooth muscle and extracellular matrix investment of the fourth arch artery.

A Bayesian autoregressive three-state hidden Markov model for identifying switching monotonic regimes in microarray time course data.

When modeling time course microarray data special interest may reside in identifying time frames in which gene expression levels follow a monotonic (increasing or decreasing) trend. A trajectory may change its regime because of the reaction to treatment or of a natural developmental phase, as in our motivating example about identification of genes involved in embryo development of mice with the 22q11 deletion. To this aim we propose a new flexible Bayesian autoregressive hidden Markov model based on three latent states, corresponding to stationarity, to an increasing and to a decreasing trend. In order to select a list of genes, we propose decision criteria based on the posterior distribution of the parameters of interest, taking into account the uncertainty in parameter estimates. We also compare the proposed model with two simpler models based on constrained formulations of the probability transition matrix.

Tbx1 regulates progenitor cell proliferation in the dental epithelium by modulating Pitx2 activation of p21.

Tbx1(-/-) mice present with phenotypic effects observed in DiGeorge syndrome patients however, the molecular mechanisms of Tbx1 regulating craniofacial and tooth development are unclear. Analyses of the Tbx1 null mice reveal incisor microdontia, small cervical loops and BrdU labeling reveals a defect in epithelial cell proliferation. Furthermore, Tbx1 null mice molars are lacking normal cusp morphology. Interestingly, p21 (associated with cell cycle arrest) is up regulated in the dental epithelium of Tbx1(-/-) embryos. These data suggest that Tbx1 inhibits p21 expression to allow for cell proliferation in the dental epithelial cervical loop, however Tbx1 does not directly regulate p21 expression. A new molecular mechanism has been identified where Tbx1 inhibits Pitx2 transcriptional activity and decreases the expression of Pitx2 target genes, p21, Lef-1 and Pitx2c. p21 protein is increased in PITX2C transgenic mouse embryo fibroblasts (MEF) and chromatin immunoprecipitation assays demonstrate endogenous Pitx2 binding to the p21 promoter. Tbx1 attenuates PITX2 activation of endogenous p21 expression and Tbx1 null MEFs reveal increased Pitx2a and activation of Pitx2c isoform expression. Tbx1 physically interacts with the PITX2 C-terminus and represses PITX2 transcriptional activation of the p21, LEF-1, and Pitx2c promoters. Tbx1(-/+)/Pitx2(-/+) double heterozygous mice present with an extra premolar-like tooth revealing a genetic interaction between these factors. The ability of Tbx1 to repress PITX2 activation of p21 may promote cell proliferation. In addition, PITX2 regulation of p21 reveals a new role for PITX2 in repressing cell proliferation. These data demonstrate new functional mechanisms for Tbx1 in tooth morphogenesis and provide a molecular basis for craniofacial defects in DiGeorge syndrome patients.

The morphology of the sella turcica in velocardiofacial syndrome suggests involvement of a neural crest developmental field.

We described the morphology of the sella turcica in individuals with velocardiofacial syndrome (VCFS), also known as chromosome 22q11.2 deletion syndrome, and compared the morphology with that of a control group of individuals from the Oslo University Craniofacial Growth Archive. The aim was to measure the cranial base angles in individuals with VCFS and, if possible, to discover the developmental field that may be involved in the condition. The study included 33 patients with VCFS from the Copenhagen Cleft Palate Center, Denmark. The genotype was confirmed by fluorescence in situ hybridization. The morphology of the sella turcica was described and measurements of the cranial base angles were performed on lateral cephalometric radiographs. The VCFS individuals had larger deviations in the morphology of the sella turcica compared to individuals from the Oslo University Craniofacial Growth archive. The deviations were mostly in the posterior part of the dorsum sellae. Individuals with VCFS had increased cranial base angles. The results of this study combined with the information in the literature on the main defects in VCFS (palatal abnormalities, cardiac anomalies, thymic hypoplasia or aplasia, hypothyroidism, and posterior brain abnormality), suggest involvement of a specific developmental field.

22q11 deletion syndrome: a role for TBX1 in pharyngeal and cardiovascular development.

Tbx1 is a member of the Tbox family of binding domain transcription factors. TBX1 maps within the region of 22q11 deleted in humans with DiGeorge or velocardiofacial syndrome. Mice haploinsufficient for Tbx1 have phenotypes that recapitulate major features of the syndrome, notably abnormal growth and remodelling of the pharyngeal arch arteries. The Tbx1 haploinsufficiency phenotype is modified by genetic background and by mutations in putative downstream targets. Homozygous null mutations of Tbx1 have more severe defects including failure of outflow tract septation, and absence of the caudal pharyngeal arches. Tbx1 is a transcriptional activator, and loss of this activity has been linked to alterations in the expression of various genes involved in cardiovascular morphogenesis. In particular, Fgf and retinoic acid signalling are dysregulated in Tbx1 mutants. This article summarises the tissue specific and temporal requirements for Tbx1, and attempts to synthesis what is know about the developmental pathways under its control.

Decreased levels of embryonic retinoic acid synthesis accelerate recovery from arterial growth delay in a mouse model of DiGeorge syndrome.

RATIONALE: Loss of Tbx1 and decrease of retinoic acid (RA) synthesis result in DiGeorge/velocardiofacial syndrome (DGS/VCFS)-like phenotypes in mouse models, including defects in septation of the outflow tract of the heart and anomalies of pharyngeal arch-derived structures including arteries of the head and neck, laryngeal-tracheal cartilage, and thymus/parathyroid. Wild-type levels of T-box transcription factor (Tbx)1 and RA signaling are required for normal pharyngeal arch artery development. Recent studies have shown that reduction of RA or loss of Tbx1 alters the contribution of second heart field (SHF) progenitor cells to the elongating heart tube. OBJECTIVE: Here we tested whether Tbx1 and the RA signaling pathway interact during the deployment of the SHF and formation of the mature aortic arch. METHODS AND RESULTS: Molecular markers of the SHF, neural crest and smooth muscle cells, were analyzed in Raldh2;Tbx1 compound heterozygous mutants. Our results revealed that the SHF and outflow tract develop normally in Raldh2(+/-);Tbx1(+/-) embryos. However, we found that decreased levels of RA accelerate the recovery from arterial growth delay observed in Tbx1(+/-) mutant embryos. This compensation coincides with the differentiation of smooth muscle cells in the 4th pharyngeal arch arteries, and is associated with severity of neural crest cell migration defects observed in these mutants. CONCLUSIONS: Our data suggest that differences in levels of embryonic RA may contribute to the variability in great artery anomalies observed in DGS/VCFS patients.

Diminished dosage of 22q11 genes disrupts neurogenesis and cortical development in a mouse model of 22q11 deletion/DiGeorge syndrome.

The 22q11 deletion (or DiGeorge) syndrome (22q11DS), the result of a 1.5- to 3-megabase hemizygous deletion on human chromosome 22, results in dramatically increased susceptibility for "diseases of cortical connectivity" thought to arise during development, including schizophrenia and autism. We show that diminished dosage of the genes deleted in the 1.5-megabase 22q11 minimal critical deleted region in a mouse model of 22q11DS specifically compromises neurogenesis and subsequent differentiation in the cerebral cortex. Proliferation of basal, but not apical, progenitors is disrupted, and subsequently, the frequency of layer 2/3, but not layer 5/6, projection neurons is altered. This change is paralleled by aberrant distribution of parvalbumin-labeled interneurons in upper and lower cortical layers. Deletion of Tbx1 or Prodh (22q11 genes independently associated with 22q11DS phenotypes) does not similarly disrupt basal progenitors. However, expression analysis implicates additional 22q11 genes that are selectively expressed in cortical precursors. Thus, diminished 22q11 gene dosage disrupts cortical neurogenesis and interneuron migration. Such developmental disruption may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.

Craniofacial malformations: intrinsic vs extrinsic neural crest cell defects in Treacher Collins and 22q11 deletion syndromes.

The craniofacial complex is anatomically the most sophisticated part of the body. It houses all the major sensory organ systems and its origins are synonymous with vertebrate evolution. Of fundamental importance to craniofacial development is a specialized population of stem and progenitor cells, known as the neural crest, which generate the majority of the bone, cartilage, connective and peripheral nerve tissue in the head. Approximately one third of all congenital abnormalities exhibit craniofacial malformations and consequently, most craniofacial anomalies are considered to arise through primary defects in neural crest cell development. Recent advances however, have challenged this classical dogma, underscoring the influence of tissues with which the neural crest cells interact as the primary origin of patterning defects in craniofacial morphogenesis. In this review we discuss these neural crest cell interactions with mesoderm, endoderm and ectoderm in the head in the context of a better understanding of craniofacial malformations such as in Treacher Collins and 22q11 deletion syndromes.

Tbx1 is expressed at multiple sites of epithelial-mesenchymal interaction during early development of the facial complex.

TBX1 encodes a T-box-containing transcription factor, which is thought to be a key player in the aetiology of the DiGeorge and Velocardiofacial syndromes (DGS/VCFS). In addition to defects affecting structures derived from the pharyngeal pouches, these patients exhibit varying degrees of facial dysmorphology and cleft palate. We have analysed the expression of murine Tbx1 during early facial development and found transcripts at sites of known epithelial-mesenchymal interaction. In particular, Tbx1 was expressed in epithelium of the early facial processes, including the fronto-nasal, medial and lateral nasal and palatine. Transcripts were also localised to the epithelium of developing tooth germs and hair follicles at several stages during their early development. Together, these expression domains suggest a role for Tbx1 in mediating epithelial-mesenchymal signalling in regions of the developing face, a finding which is consistent with the spectrum of facial deformity encountered amongst subjects affected by DGS/VCFS.

Timed mutation and cell-fate mapping reveal reiterated roles of Tbx1 during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion.

The definition of time-specific requirements for a developmental gene can pinpoint the processes within which the gene is involved and can reveal potential late functions in structures and organs that fail to develop in germline mutants. Here, we show the first systematic time-course deletion, in parallel with timed cell fate mapping, of a developmentally crucial gene, Tbx1, during mouse embryogenesis. Tbx1 mouse mutants model DiGeorge syndrome, a disorder of pharyngeal and cardiovascular development. Results revealed different time requirements for the development of individual structures, as well as multiple and time-distinct roles during the development of the same organ or system. We also show that Tbx1 is required throughout pharyngeal segmentation for the regulation of endoderm expansion, thus this is the first gene implicated directly in this process. A genetic-based blueprint of crucial developmental times for organs and systems should be a valuable asset for our understanding of birth defect pathogenesis.