Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors

Abstract

Birth defects, which occur in one out of 20 live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription factor TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. Here, we have generated an allelic series of Tbx1 deficiency that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription factor Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. We identified a Fox-binding site upstream of Tbx1 that interacted with Foxa2 and was necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, we found a fibroblast growth factor 8 (Fgf8) enhancer that was dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function resulted in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgf10. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11.

Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm

“…Furthermore, we have shown that the Tbx1 requirement is intrinsic to the mesoderm. The loss and hypoplasia of the second and first branchial arch muscles in the Tbx1 mutant mouse may be due to intrinsic defects in the mesoderm or secondary to endodermal defects such as the absence of Fgf8 (Vitelli et al, 2002b;Hu et al, 2004;Kelly et al, 2004;Zhang et al, 2005). The endoderm is known to have a key role in patterning the facial structures signaling to the underlying mesoderm and ectomesenchyme (Couly et al, 2002;Ladher et al, 2005).…”

“…In addition to the endodermal signal Fgf8, these include Pax9, Gbx2 in the endoderm, and Fgf10 in the mesoderm (Hu et al, 2004;Ivins et al, 2005). The loss of Fgf10 expression has been proposed to result in the decrease in proliferation in the secondary heart field (Hu et al, 2004).…”

“…In addition to the endodermal signal Fgf8, these include Pax9, Gbx2 in the endoderm, and Fgf10 in the mesoderm (Hu et al, 2004;Ivins et al, 2005). The loss of Fgf10 expression has been proposed to result in the decrease in proliferation in the secondary heart field (Hu et al, 2004). Similarly, Fgf10 expression is lost in the developing myogenic cells of the first and second arch, although as development of the craniofacial muscles is normal in the Fgf10 null mice, the myogenic defects in the Tbx1 null mouse cannot simply be attributed to loss of Fgf10 signaling (Vitelli et al, 2002b;Hu et al, 2004;Kelly et al, 2004).…”

“…In its absence, the second arch muscles fail to develop while the mandibular or first arch muscles are hypoplastic and sporadic (Kelly et al, 2004). In the developing head, Tbx1 is expressed in both the endoderm and mesoderm and, therefore, myogenic defects may be a consequence of defective endoderm/ectoderm signaling (Vitelli et al, 2002b;Hu et al, 2004;Ataliotis et al, 2005, Zhang et al, 2005. To analyze whether Tbx1 is intrinsically required in the mesoderm, we performed micromass assays, cell cultures generated by plating single cell suspensions made from the mesoderm and neural crest-derived ectomesenchyme of branchial arch 1, at high density.…”

“…Deletion of Tbx1 in either the endoderm or mesoderm results in a phenotype very reminiscent of the Tbx1 null, showing that Tbx1 function is required in both tissues for normal branchial arch development (Arnold et al, 2006;. The pharyngeal arch and cardiac abnormalities are associated with a down-regulation of Fgf8 and Fgf10 expression in the endoderm and mesoderm, respectively (Kochilas et al, 2002;Vitelli et al, 2002bVitelli et al, , 2006Hu et al, 2004). There is also an increase in retinoic acid signaling anteriorly within the branchial arch mesoderm and endoderm Guris et al, 2006).…”