Identification of putative retinoic acid target genes downstream of mesenchymal Tbx1 during inner ear development.

BACKGROUND: The T-box transcription factor Tbx1 is expressed in the otic vesicle and surrounding mesoderm of the periotic mesenchyme (POM) during inner ear development. Mesenchymal Tbx1 is essential for inner ear development, with conditional mutants displaying defects in both the auditory and vestibular systems. We have previously reported that mesodermal Tbx1 loss of function mutants (Mest-KO) have reduced expression of retinoic acid (RA) metabolic genes, Cyp26a1 and Cyp26c1, in the POM, consistent with other studies showing an increase in mesodermal RA reporter expression in Tbx1-/- embryos. However, putative RA effector genes whose expression is altered downstream of increased otic mesenchymal-epithelial RA signaling have remained elusive. RESULTS: Here we report the identification of 18 retinoic acid responsive genes altered in Mest-KO conditional mutants by microarray gene profiling. Nine were chosen for biological validation including quantitative RT-PCR and in situ hybridization (Otor, Mia, Col2a1, Clu, Adm, Myt1, Dlx3, Itgb3, and Itga2b). CONCLUSION: Here study provides a series of newly identified RA effector genes for inner ear development downstream of mesenchymal Tbx1 that may contribute to the inner ear phenotype observed in Tbx1 loss of function mouse models.

Mutational analysis of HOXA2 and SIX2 in a Bronx population with isolated microtia.

OBJECTIVE: Microtia is a developmental malformation of the external ear with genetic and environmental causes. The prevalence of microtia varies but several studies suggest increased incidence in Hispanic and African American populations. No causal genetic mutations have been identified in these populations. Mutations in the homeobox gene HOXA2 caused microtia in a single Iranian family. Another homeobox gene, SIX2, acts downstream of HOXA2 during development and provides another possible candidate for mutational analysis. METHODS: To determine whether mutations in HOXA2 or SIX2 cause sporadic microtia, DNA sequencing analysis was performed on exons in both genes in 8 patients of Hispanic and African descent in the Bronx. Identified variants were assayed in an additional 4 patients and 100 Hispanic control samples using Sequenom MassArray to rule out causality in heterozygous patients. RESULTS: No mutations were identified in the coding sequence of HOXA2 or SIX2. Four novel single nucleotide variants were identified among the patient samples. These variants lie in the intron and 3' UTR of HOXA2 and the 5' and 3' UTRs of SIX2. One variant in the intron of HOXA2 lies in a conserved predicted transcription factor binding site for SMARCA3. All four variants are also present at >5% frequency in Hispanic control samples, ruling out these novel variations as causal. CONCLUSIONS: Lack of mutations in the coding regions of HOXA2 or SIX2 among the sporadic microtia patients studied indicate different etiologies. Identification of four novel single nucleotide polymorphisms in patients and controls of Hispanic descent, but not of Caucasian populations, points to genetic diversity in an understudied population.

Tbx1 and Brn4 regulate retinoic acid metabolic genes during cochlear morphogenesis.

BACKGROUND: In vertebrates, the inner ear is comprised of the cochlea and vestibular system, which develop from the otic vesicle. This process is regulated via inductive interactions from surrounding tissues. Tbx1, the gene responsible for velo-cardio-facial syndrome/DiGeorge syndrome in humans, is required for ear development in mice. Tbx1 is expressed in the otic epithelium and adjacent periotic mesenchyme (POM), and both of these domains are required for inner ear formation. To study the function of Tbx1 in the POM, we have conditionally inactivated Tbx1 in the mesoderm while keeping expression in the otic vesicle intact. RESULTS: Conditional mutants (TCre-KO) displayed malformed inner ears, including a hypoplastic otic vesicle and a severely shortened cochlear duct, indicating that Tbx1 expression in the POM is necessary for proper inner ear formation. Expression of the mesenchyme marker Brn4 was also lost in the TCre-KO. Brn4-;Tbx1+/-embryos displayed defects in growth of the distal cochlea. To identify a potential signal from the POM to the otic epithelium, expression of retinoic acid (RA) catabolizing genes was examined in both mutants. Cyp26a1 expression was altered in the TCre-KO, while Cyp26c1 showed reduced expression in both TCre-KO and Brn4-;Tbx1+/- embryos. CONCLUSION: These results indicate that Tbx1 expression in the POM regulates cochlear outgrowth potentially via control of local retinoic acid activity.