Tuong Huynh

Tbx1 haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice

DiGeorge syndrome is characterized by cardiovascular, thymus and parathyroid defects and craniofacial anomalies, and is usually caused by a heterozygous deletion of chromosomal region 22q11.2 (del22q11) (ref. 1). A targeted, heterozygous deletion, named Df(16)1, encompassing around 1 megabase of the homologous region in mouse causes cardiovascular abnormalities characteristic of the human disease. Here we have used a combination of chromosome engineering and P1 artificial chromosome transgenesis to localize the haploinsufficient gene in the region, Tbx1. We show that Tbx1, a member of the T-box transcription factor family, is required for normal development of the pharyngeal arch arteries in a gene dosage-dependent manner. Deletion of one copy of Tbx1 affects the development of the fourth pharyngeal arch arteries, whereas homozygous mutation severely disrupts the pharyngeal arch artery system. Our data show that haploinsufficiency of Tbx1 is sufficient to generate at least one important component of the DiGeorge syndrome phenotype in mice, and demonstrate the suitability of the mouse for the genetic dissection of microdeletion syndromes.

Mesodermal expression of Tbx1 is necessary and sufficient for pharyngeal arch and cardiac outflow tract development

The development of the segmented pharyngeal apparatus involves complex interaction of tissues derived from all three germ layers. The role of mesoderm is the least studied, perhaps because of its apparent lack of anatomical boundaries and positionally restricted gene expression. Here, we report that the mesoderm-specific deletion of Tbx1, a T-box transcription factor, caused severe pharyngeal patterning and cardiovascular defects, while mesoderm-specific restoration of Tbx1 expression in a mutant background corrected most of those defects in the mouse. We show that some organs, e.g. the thymus, require Tbx1 expression in the mesoderm and in the epithelia. In addition, these experiments revealed that different pharyngeal arches require Tbx1 in different tissues. Finally, we show that Tbx1 in the mesoderm is required to sustain cell proliferation. Thus, the mesodermal transcription program is not only crucial for cardiovascular development, but is also key in the development and patterning of pharyngeal endoderm.

Genetic factors are major determinants of phenotypic variability in a mouse model of the DiGeorge/del22q11 syndromes

The del22q11 syndrome is associated with a highly variable phenotype despite the uniformity of the chromosomal deletion that causes the disease in most patients. Df1/+ mice, which model del22q11, present with reduced penetrance of cardiovascular defects similar to those seen in deleted patients but not with other del22q11-like findings. The reduced penetrance of cardiovascular defects is caused by the ability of mutant embryos to recover from a fourth pharyngeal arch artery growth abnormality that is fully penetrant in early embryos. Here we show that genetic background has a major effect on penetrance of cardiovascular defects by affecting this embryonic recovery process. This effect could not be explained by allelic variation at the haploid locus, and it is likely to be caused by genetic modifiers elsewhere in the genome. We also show that genetic factors control extension of the Df1/+ phenotype to include thymic and parathyroid anomalies, establishing the Df1 mouse as a model for the genetic analysis of three major features of human del22q11 syndrome. We found that in Df1/+ mice, as in human patients, expression of the heart and thymic phenotypes are essentially independent from each other, suggesting that they may be controlled by different genetic modifiers. These data provide a framework for our understanding of phenotypic variability in patients with del22q11 syndrome and the tools for its genetic dissection.

Tbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner

Tbx1 is a T-box transcription factor implicated in DiGeorge syndrome. The molecular function of Tbx1 is unclear although it can transactivate reporters with T-box binding elements. We discovered that Tbx1 binds Smad1 and suppresses the Bmp4/Smad1 signaling. Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity. In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction. Expression of Tbx1 in transgenic mice generates phenotypes similar to those associated with loss of a Bmp receptor. One phenotype could be rescued by transgenic Smad1 expression. Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.

Early thyroid development requires a Tbx1-Fgf8 pathway

The thyroid develops within the pharyngeal apparatus from endodermally-derived cells. The many derivatives of the pharyngeal apparatus develop at similar times and sometimes from common cell types, explaining why many syndromic disorders express multiple birth defects affecting different structures that share a common pharyngeal origin. Thus, different derivatives may share common genetic networks during their development. Tbx1, the major gene associated with DiGeorge syndrome, is a key player in the global development of the pharyngeal apparatus, being required for virtually all its derivatives, including the thyroid. Here we show that Tbx1 regulates the size of the early thyroid primordium through its expression in the adjacent mesoderm. Because Tbx1 regulates the expression of Fgf8 in the mesoderm, we postulated that Fgf8 mediates critical Tbx1-dependent interactions between mesodermal cells and endodermal thyrocyte progenitors. Indeed, conditional ablation of Fgf8 in Tbx1-expressing cells caused an early thyroid phenotype similar to that of Tbx1 mutant mice. In addition, expression of an Fgf8 cDNA in the Tbx1 domain rescued the early size defect of the thyroid primordium in Tbx1 mutants. Thus, we have established that a Tbx1->Fgf8 pathway in the pharyngeal mesoderm is a key size regulator of mammalian thyroid.

Gain of function of Tbx1 affects pharyngeal and heart development in the mouse

Mammalian development is highly sensitive to Tbx1 gene dosage reduction. Gene function insights can also be learned from increased or ectopic expression. The authors generated a novel mouse transgenic line, named COET, which expresses Tbx1 upon Cre‐mediated recombination. The authors crossed this transgenic line with Tbx1Cre animals to activate expression in the Tbx1‐expression domain. Compound mutant COET;Tbx1Cre/+ animals died after birth and showed heart enlargement. At E18.5, compound mutants showed ventricular septal defects and thymic abnormalities. The authors crossed compound mutants into a Tbx1 null background to understand whether this phenotype is caused by gene overdosage. Results showed that gene dosage reduction at the endogenous locus could not rescue heart and thymic defects, although the transgene rescued the loss of function phenotype. Thus, the transgenic phenotype appears to be due to gain of function. Resultant data demonstrate that Tbx1 expression must be tightly regulated to be compatible with normal embryonic development. genesis 47:188–195, 2009.

Partial rescue of the Tbx1 mutant heart phenotype by Fgf8: genetic evidence of impaired tissue response to Fgf8.

Tbx1 is the candidate gene of DiGeorge syndrome and is required in humans and mice for the development of the cardiac outflow tract (OFT) and aortic arch arteries. Loss of function mutants present with reduced cell proliferation and premature differentiation of cardiac progenitor cells of the second heart field (SHF). Tbx1 regulates Fgf8 expression hence the hypothesis that the proliferation impairment may contribute to the heart phenotype of mutants. Here we show that forced Fgf8 expression modifies and partially rescues the OFT septation defects of Tbx1 mutants but only if there is some residual expression of Tbx1. This genetic experiment suggests that Tbx1, directly or indirectly, affects tissue response to Fgf8. Indeed, Tbx1(-/-) mouse embryonic fibroblasts were unable to respond to Fgf8 added to the culture media and showed defective response of Erk1/2 and Rsk1. Our data suggest a coordinated pathway modulating Fgf8 ligand expression and tissue response to it in the SHF.