Chihiro Yamagishi

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

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 is Regulated by Forkhead Proteins in the Secondary Heart Field

Transcriptional regulation in a tissue‐specific and quantitative manner is essential for developmental events, including those involved in cardiovascular morphogenesis. Tbx1 is a T‐box–containing transcription factor that is responsible for many of the defects observed in 22q11 deletion syndrome in humans. Tbx1 is expressed in the secondary heart field (SHF) and is essential for cardiac outflow tract (OFT) development. We previously reported that Tbx1 is regulated by sonic hedgehog by means of forkhead (Fox) transcription factors in the head mesenchyme and pharyngeal endoderm, but how it is regulated in the SHF is unknown. Here, we show that Tbx1 expression in the SHF is regulated by Fox proteins through a combination of two evolutionarily conserved Fox binding sites in a dose‐dependent manner. Cell fate analysis using the Tbx1 enhancer suggests that SHF‐derived Tbx1‐expressing cells contribute extensively to the right ventricular myocardium as well as the OFT during early development and ultimately give rise to the right ventricular infundibulum, pulmonary trunk, and pulmonary valves. These results suggest that Fox proteins are involved in most, if not all, Tbx1 expression domains and that Tbx1 marks a subset of SHF‐derived cells, particularly those that uniquely contribute to the right‐sided outflow tract and proximal pulmonary artery. Developmental Dynamics 235:701–710, 2006.

Sonic Hedgehog Is Essential for First Pharyngeal Arch Development

The secreted protein sonic hedgehog (Shh) is essential for normal development of many organs. Targeted disruption of Shh in mouse leads to near complete absence of craniofacial skeletal elements at birth, and mutation of SHH in human causes holoprosencephaly (HPE), frequently associated with defects of derivatives of pharyngeal arches. To investigate the role of Shh signaling in early pharyngeal arch development, we analyzed Shh mutant embryos using molecular markers and found that the first pharyngeal arch (PA1) was specifically hypoplastic and fused in the midline, and remaining arches were well formed at embryonic day (E) 9.5. Molecular analyses using specific markers suggested that the growth of the maxillary arch and proximal mandibular arch was severely defective in Shh-null PA1, whereas the distal mandibular arch was less affected. TUNEL assay revealed an increase in the number of apoptotic signals in PA1 of Shh mutant embryos. Ectodermal expression of fibroblast growth factor (Fgf)-8, a cell survival factor for pharyngeal arch mesenchyme, was down-regulated in the PA1 of Shh mutants. Consistent with this observation, downstream transcriptional targets of Fgf8 signaling in neural crest–derived mesenchyme, including Barx1, goosecoid, and Dlx2, were also down-regulated in Shh-null PA1. These results demonstrate that epithelial-mesenchymal signaling and transcriptional events coordinated by Shh, partly via Fgf8, is essential for cell survival and tissue outgrowth of the developing PA1.

Functional attenuation of UFD1l, a 22q11.2 deletion syndrome candidate gene, leads to cardiac outflow septation defects in chicken embryos.

Microdeletion of chromosome 22q11.2 is commonly associated with congenital cardiovascular defects that involve development of cranial neural crest cells (NCC) that emigrate through the pharyngeal arches. UFD1l is one of several candidate genes for 22q11.2 deletion syndrome (22q11DS). UFD1l encodes a protein whose yeast counterpart is involved in a ubiquitin-dependent proteolytic degradation pathway; however, the role of UFD1L in NCC development remains unknown. Mouse embryos that lack Ufd1l die before organogenesis. We have therefore studied the function of Ufd1l in the chick system. Chick Ufd1l encoded a 307–amino acid protein that was highly conserved with mouse and human UFD1L. Chick Ufd1l was expressed in the developing neural tube, NCC, and mesenchyme of the head and pharyngeal arch structures, as well as in the conotruncal region (cardiac outflow tract), consistent with the clinical features of 22q11DS. To determine loss-of-function effects of chick Ufd1l in NCC, we infected cardiac NCC with a retrovirus expressing antisense Ufd1l transcripts in chick embryos before their migration. Morphologic analysis of infected embryos at a later developmental stage demonstrated that functional attenuation of chick Ufd1l in cardiac NCC resulted in an increased incidence of conotruncal septation defects. These data suggest that Ufd1l may play a role in cardiac NCC during conotruncal septation.

A common cis-acting sequence in the DiGeorge critical region regulates bi-directional transcription of UFD1L and CDC45L

Two to three megabase deletions on chromosome 22q11 are the cytogenetic findings most commonly associated with cardiac and craniofacial defects in humans. The constellation of clinical findings associated with these deletions is termed the 22q11 deletion syndrome. We had earlier described a patient with the 22q11 deletion phenotype who was hemizygous for an atypical 20 kb microdeletion in this region. The deletion included coding regions of two genes organized head-to-head, UFD1L and CDC45L, along with an 884 bp CpG-rich intervening region. Based on this genomic organization, we hypothesized that both genes may be co-expressed and co-regulated by sequences within this region. We demonstrate that expression of both genes is enhanced in a similar pattern in precursors of structures affected by the deletion. The intergenic region is sufficient to direct transcription most strongly in the developing pharyngeal arches and limb buds of transgenic mice and can also direct bi-directional transcriptional activation in a neural crest-derived cell line. Deletion analyses revealed that a 404 bp fragment closest to UFD1L is necessary and sufficient to direct this bi-directional transcriptional activity. These results reveal the presence of a conserved regulatory region in the 22q11 deletion locus that can direct simultaneous transcription of genes involved in ubiquitin mediated protein processing (UFD1L) and cell cycle control (CDC45L).