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Human Molecular Genetics, 2003, Vol. 12, No. 16 2041-2048
DOI: 10.1093/hmg/ddg216
© 2003 Oxford University Press

TBX1 is required for inner ear morphogenesis

Francesca Vitelli1, Antonella Viola1,3, Masae Morishima1, Tiziano Pramparo1, Antonio Baldini1,2 and Elizabeth Lindsay1,*

1Department of Pediatrics (Cardiology) and 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA and 3CEINGE Biotecnologie Avanzate, Via S. Pansini, 5, 80131, Naples, Italy

Received April 21, 2003; Revised June 13, 2003; Accepted June 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TBX1 is thought to be a critical gene in the pathogenesis of del22q11/DiGeorge syndrome (DGS). Morphological abnormalities of the external ear and hearing impairment (conductive or sensorineural) affect the majority of patients. Here we show that homozygous mutation of the mouse homolog Tbx1 is associated with severe inner ear defects that prevent the formation of the cochlea and of the vestibulum. Consistent with phenotypic abnormalities, Tbx1 is expressed early in otocyst development in the otic epithelium and in the periotic mesenchyme. Tbx1 loss-of-function blocks inner ear development at early otocyst stage and after neurogenesis. Analysis of chimeras suggests that Tbx1 function is required in the otic epithelium cell autonomously, but abnormalities of the periotic mesenchyme indicate that the pathogenesis of the inner ear phenotype is complex. We propose a model where Tbx1 is required for expansion of a subpopulation of otic epithelial cells, which is required to form the vestibular and auditory organs. Our data suggest that Tbx1 deletion in del22q11 patients may cause not only external and middle ear defects but also sensorineural and vestibular phenotypes observed in these patients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DiGeorge syndrome (DGS) (Online Merdelian Inheritance in Man, http://www3.ncbi.nlm.nih.gov/Omim/) is a characteristic disorder of pharyngeal development with virtually all the derivatives of the pharyngeal arches and pouches being affected. The clinical phenotype includes craniofacial abnormalities, congenital heart disease, thymic and parathyroid defects (reviewed in 1–3). The great majority of DGS cases results from a heterozygous deletion of chromosome 22q11.2 (del22q11) (4–7). Murine models of del22q11 have shown Tbx1 to be haploinsufficient in the mouse; Tbx1+/- animals have cardiovascular defects that are reminiscent of DGS, while Tbx1-/- animals recapitulate most, if not all, of the pharyngeal arch and pouch-derived abnormalities associated with DGS (8–10). Although Tbx1 mouse mutants strongly suggest that DGS results from Tbx1 haploinsufficiency, no DGS patient has been found yet with a single gene mutation of Tbx1. This suggests that isolated Tbx1 mutations are rare or not associated with typical DGS.

Part of the spectrum of craniofacial anomalies is external ear defects (11), and hearing impairment occurs in 44–77% of DGS/VCFS cases (11–14). The latter is primarily conductive and correlates well with the presence of chronic otitis media, which is found in the majority of patients. However, ~10% of cases of hearing loss is of the sensorineural type (13,14), indicating that at least some patients have inner ear problems. In addition, there are reports of patients with balance problems (reviewed in 15).

Profound abnormalities of the external, middle and inner ear have been reported in mice with homozygous loss-of-function mutations in Tbx1 (9). The external and middle ear abnormalities are most likely to be secondary to the severe developmental defects of the pharyngeal arches and pouches and are not the focus of this report. Inner ear abnormalities in Tbx1-/- embryos are already apparent at E9.5, when the otocyst, from which the inner ear forms, is smaller than normal. As embryonic development proceeds, the otocyst expands minimally, but does not undergo the morphogenic changes required to form the cochlea and the semicircular canals. At term, the otic capsule remains small (~25% of normal) and spherical with very poorly developed or absent semicircular canals (9).

In order to understand the role of Tbx1 in otocyst development and to begin to define a molecular pathway controlling inner ear development, we have analyzed expression of molecular markers that identify precursors of sensory, neuronal and structural components of the otocyst. In addition, we have utilized a Tbx1-LacZ knock-in allele to examine the distribution of Tbx1-expressing cells, which are blue after X-gal staining, in Tbx1 homozygous mutants and we have performed chimera analysis. Results suggest that Tbx1 is required for morphogenesis and growth but not induction of the otocyst in mouse.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tbx1 expression is developmentally regulated during inner ear morphogenesis
The developmental expression pattern of Tbx1 has been extensively studied using RNA in situ hybridization (8–10,16,17) and a Tbx1-LacZ knock-in allele (18,19). However, little attention has been given to its expression in the developing ear.

Here we have used the abovementioned Tbx1-LacZ knock-in allele, which recapitulates the expression of the endogenous Tbx1 gene (18,19) (Figs 1A–E and 2G–R), to follow the developmental expression of Tbx1 during the period of intense inner ear morphogenesis that occurs between embryonic day (E9) and E13.5. Tbx1 expression becomes detectable in the otic epithelium at E9, where it localizes to the lateral–ventral wall of the posterior otocyst. This pattern is maintained and extended at E9.5 (Fig. 1A). At E10.5, Tbx1 is widely expressed in the otic epithelium (Figs. 1B and 2A–R), but it is excluded from the most dorsal and medial regions, including the endolymphatic duct. Tbx1 is also expressed in the periotic mesenchyme. This mesenchymal domain is more anterior and ventral than the epithelial expression domain and is, in part, a direct dorsal continuation of the Tbx1 expression domain in the paraxial mesoderm of the second pharyngeal arch (Fig. 1C). From E11.5, the otocyst undergoes dramatic morphogenesis that leads to the development of the acoustic and vestibular apparata that characterize the mature inner ear. Out-pouching of the otic epithelium in the ventral–medial region of the otocyst initiates formation of the cochlear duct. Development of the vestibular apparatus begins more anteriorly with sequential out-pouching of the epithelium. Extension and fusion of these epithelial projections forms the anterior, posterior and lateral semi-circular canals. The morphogenic processes that form the semicircular canals and cochlea are revealed strikingly by the Tbx1-LacZ knock-in allele in E12.5 and E13.5 embryos, as Tbx1 expression defines precisely the entire vestibular apparatus and, less vividly, the developing cochlea (Fig. 1D and E).



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  		Figure 1. Tbx1 expression in the developing inner ear. Saggittal views of X-gal stained Tbx1+/- (A–E) and Tbx1-/- embryos (A', B', D') at E9.5 (A, A'), E10.5 (B, B', C), E12.5 (D, D') and E13.5 (E). (A) Tbx1 is expressed in the ventral–lateral wall of the posterior otocyst (arrowhead) at E9.5. (B) At E10.5, Tbx1 is expressed throughout the lateral otocyst wall but not in the endolymphatic duct (white arrow). (C) Tbx1 is expressed in the periotic mesenchyme where it is continuous with expression in the mesenchyme of the second pharyngeal arch (II). (D) At E12.5, Tbx1 expression delineates the developing semicircular canals and at E13.5 (E), the cochlea. In Tbx1-/- embryos, the otocyst forms (A') but it is smaller than in wild type and Tbx1+/- litter mates (compare B and B') and does not undergo morphogenesis. Arrows indicate the otocyst in C and D.

 


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  		Figure 2. Sagittal sections (A–F and G–L) and coronal sections (M–R) of the otocysts from either a Tbx1 RNA in situ hybridization of a WT embryo at E10.5 or of X-gal stained Tbx1+/- embryos at E10.5.

 
Tbx1 is required for morphogenesis of the otocyst
Inner ear development proceeds apparently normally in Tbx1-/- embryos prior to E9, indicating that Tbx1 is not required for formation of the otic placode or otic cup. At E9.5, the Tbx1-/- otocyst is smaller than in wild type littermates (Fig. 1A' and B') and the cochleo-vestibular ganglion (cvg) is abnormally positioned (Fig. 3A' and F'). Between E10.5 and E11.5, the Tbx1-/- otocyst expands only minimally, while the endolymphatic duct grows apparently normally. The period E12.0–E13.5 is normally one of rapid growth and morphogenesis, characterized by the development of the semicircular canals and cochlea (Fig. 1D and E). However, the Tbx1-/- otocyst shows no morphogenesis and retains the morphology of the earlier unelaborated otocyst (Fig. 1D'). The abnormal otocyst morphology in Tbx1-/- mutants was confirmed on histological sections of X-gal stained embryos. The developmental failure of the otocyst in Tbx1-/- mutants is accompanied by an absence of blue cells in the otic epithelium after E9 (Fig. 1A', B' and D'). Blue cells were present in the periotic mesenchyme and in all other tissues that normally express Tbx1.



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  		Figure 3. Expression of molecular markers in wild type (A–F) and Tbx1-/- (A'–F') embryos at E10.5 (A–D, F, G and A'–D', F', G') and E9.5 (E, E'). (A, A') Fgf10 expression in the lateral otic epithelium is lost in Tbx1-/- embryos but conserved in the cvg (arrowhead) and underlying otic epithelium. The cvg is abnormally positioned (ventrally) in Tbx1-/- embryos (A' and E'). (B, C, B', C') Both Bmp4 domains of expression are lost in Tbx1-/- embryos. (D, D') Gsc expression is absent in Tbx1-/- embryos. (E, E') Expression of Pax2 is conserved medially in Tbx1-/- embryos but the posterior expression domain is lost. (F, F') Expression if Fgf8, which is conserved in Tbx1-/- embryos, shows how malpositioning of the cvg separates the cvg from the ganglion of the VII cranial nerve (arrow). (G, G') Expression of Prx2 in the periotic mesenchyme is greatly reduced or absent in Tbx1-/- embryos.

 
In view of the lack of inner ear morphogenesis in early Tbx1-/- embryos and the complete absence of the auditory and vestibular systems at term, we decided to address the molecular nature of these abnormalities at the otocyst stage. The inner ear, has six major sensory organs, which though morphologically characteristic, conform to a basic pattern comprising sensory hair cells and supporting cells. The vestibular apparatus comprises five sensory organs: the three semicircular canals, the utricle and the saccule. At the base of each semicircular canal is a swelling (ampulla) within which is located a sensory patch (crista). The maculae are the sensory patches of the saccule and utricle. The auditory apparatus comprises a single sensory organ, the cochlea. We have analyzed molecular markers that identify the various compartments (known or presumed) of the otocyst. These include markers of the sensory patches of the presumptive semicircular canals, cochlea, the cvg and the periotic mesenchyme.

To assess the impact of Tbx1 loss of function on early development of the sensory organs, we first analyzed the expression of Fgf10, which is a marker of presumptive vestibular sensory epithelium (20). Fgf10 has a domain of expression in the lateral otic epithelium and it is also expressed in the cvg and underlying epithelium (20). Fgf10 expression in the lateral otic epithelium was lost in Tbx1-/- embryos, but its expression in the cvg and underlying epithelium was preserved (Fig. 3A and A'). Conservation of the cvg domain was expected because the cvg grows apparently normally in Tbx1-/- mutants. Bmp4 is a marker of the presumptive vestibular and cochlear sensory patches and is expressed in two discrete domains in the otocyst: an anterior–lateral domain encompassing the presumptive anterior and lateral cristae (21) and a posterior domain encompassing the presumptive maculae and cochlea (Fig. 3B and C). Both Bmp4 expression domains were lost in Tbx1-/- embryos (Fig. 3B' and 3C'). Otx2, a gene that is required for cochlea development (22), and Goosecoid (Gsc) are both expressed in the ventral–posterior otocyst. Otx2, like Bmp4 identifies the presumptive maculae and cochlea (22). Hence, the Otx2 and Gsc expression domains are encompassed within the Tbx1 expression domain. Expression of Otx2 (data not shown) and Gsc (Fig. 3D and D') were both lost in Tbx1-/- embryos. Pax2, is also required for cochlear development (23,24). The ventral–posterior part of the Pax2 expression domain overlaps with Otx2 (25), and identifies the presumptive cochlear region, while the ventral–medial expression domain encompasses the endolymphatic duct. In Tbx1-/- embryos, the presumptive cochlea expression domain was lost, while the ventral–medial domain was conserved (Fig. 3E and E'), consistent with the preservation of the endolymphatic duct, which expresses Pax2, in Tbx1-/- mutants. In addition, expression of lunatic fringe (Lfng), which overlaps with the ventral–medial expression domain of Pax2, but not with the Pax2 cochlear domain (25) was preserved in Tbx1-/- embryos (data not shown). Overall, these results reveal that the expression of multiple molecular markers that identify vestibular and cochlear sensory epithelium, and which overlap completely or partially with Tbx1 expression is lost or reduced in Tbx1-/- embryos. Although it is formally possible that all these genes are regulated by Tbx1, we think it is more likely that it reflects the loss of a population of otic epithelial cells in Tbx1-/- mutants. This is supported by the fact that all these markers, with the exception of Fgf10 (19), are expressed normally in the rest of the Tbx1-/- embryo.

The cvg, which is the proximal ganglion of the VIII cranial nerve, derives from neuronal precursors that delaminate from the anterior–ventral region of the otic epithelium from E9.5. Later in development, the cvg partitions into the acoustic and vestibular ganglia. Tbx1 is not required for formation or growth of the cvg as assessed by histology and by conservation of Fgf10 (Fig. 3A') and Fgf8 (Fig. 3F') expression, which is consistent with the largely distinct expression domain of these genes compared to Tbx1. Two other markers of the cvg, Six1 and Eya1, were expressed normally in Tbx1-/- embryos (data not shown). However, although the cvg expresses specific markers normally in Tbx1-/- embryos, it is anomalously positioned from its inception, occupying a more posterior and ventral position than the normal anterior position, and thereby losing its proximity to the VII cranial nerve ganglion (Fig. 3F'). This suggests that the otic epithelium that gives rise to the cvg neuronal precursors is abnormally specified or abnormally positioned.

Epithelial–mesenchymal interactions have a key role in otic induction and inner ear development (26,27) and loss-of-function mutations in mice have implicated several genes in these interactions (27–30). As Tbx1 is expressed in both otic epithelium and periotic mesenchyme, it is possible that Tbx1 may be required in epithelium and mesenchyme during inner ear morphogenesis. Expression of Prx2, which is detected in the lateral periotic mesenchyme, in addition to other tissues, was considerably reduced or absent in the lateral periotic mesenchyme of Tbx1-/- embryos (Fig. 3G') but not elsewhere (data not shown). Col2aI, which is expressed throughout the head mesenchyme was normally expressed in Tbx1-/- embryos (data not shown). Loss of Prx2 expression is not likely to contribute to the mutant phenotype because Prx2-/- mice are normal (29), but it suggests that the periotic mesenchyme is abnormal in Tbx1-/- embryos. Indeed, the lateral periotic mesenchyme is considerably reduced in Tbx1-/- mutants from E9.75 because the mutant otocyst remains small and superficially positioned beneath the surface ectoderm (Fig. 3G').

Overall, the results of the marker analysis reveal that expression of multiple markers that overlap with the Tbx1 expression domain is lost in Tbx1-/- embryos (Otx2, Gsc, Bmp4, Pax2 posterior domain, Fg f10 lateral epithelial domain), while expression of non-overlapping markers is conserved (Lfng, Pax2 ventral–medial domain). This suggests that there is a problem with the otic epithelium that normally expresses Tbx1. Although it is formally possible that all the genes that are lost are regulated by Tbx1, we think it is more likely that it reflects the loss of a population of otic epithelial cells in Tbx1-/- mutants. This is supported by the fact that all these markers, with the exception of Fgf10, are expressed normally in the rest of the Tbx1-/- embryo.

An extensive population of otic epithelial cells is absent in the otocyst of Tbx1-/- mutants
The Tbx1-LacZ knock-in allele is particularly useful for correlating phenotype and gene expression, because it visualizes cells with a transcriptionally active Tbx1 gene in the absence of a functional gene product. This allele revealed that a small population of LacZ-positive epithelial cells is present the posterior otocyst of Tbx1-/- animals at E9 (Fig. 4A' and B'), but by E9.5, this population is no longer detectable (Fig. 1A', B' and D'). This result could be interpreted in at least three ways. (i) Tbx1 is required cell autonomously for expansion of a subpopulation of otic epithelial cells. In general, Tbx1 is not required for survival of Tbx1-expressing cells (18) but the ear may be an exception to this general rule. (ii) Maintenance of Tbx1 expression is dependent upon signalling from the periotic mesenchyme. If the periotic mesenchyme is abnormal in Tbx1-/- embryos, as Prx2 expression suggests, this signalling may fail. (iii) Epithelial cells destined to express Tbx1 take on a different fate. Hypotheses (i) and (ii) are not mutually exclusive and a combination of the two is likely. This is supported by the loss of expression of apparently unrelated genes that partially overlap with the Tbx1 expression domain. Hypothesis (iii) is less likely because we did not detect expansion of any marker into abnormal domains.



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  		Figure 4. X-gal staining of wholemount embryos at E9 reveals the presence of LacZ-positive epithelial cells in the otocyst of Tbx1+/- (A) and Tbx1-/- (A') mutants. Sections of the same embryos (B, B') show that the LacZ-positive cells are localized to the lateral wall of the posterior otocyst. (C, C') anti-BrdU staining of proliferating cells in the otic epithelium of Tbx1+/- and Tbx1-/- mutants at E9.5. LysoTracker red identifies cells in apoptosis in the endolymphatic duct (D, D') and in the ventral otic epithelium (E, E') of Tbx1+/- and Tbx1-/- mutants at E10.5. (F) Gene targeting strategy to generate Tbx1-/- ES cells. An ES cell line carrying a LacZ knock-in allele on one chromosome was retargeted to modify the remaining wild type allele. Chimeric embryos at E9.5 (G) and E10.5 (H) show that Tbx1-/- cells (blue) do not populate the otocyst (arrow) but they are present in other tissues that normally express Tbx1.

 
The Tbx1-/- inner ear phenotype is not explained by wide-spread cell proliferation or survival defects
The much reduced size of the mutant otocyst could be due to reduced cell proliferation and/or to increased apoptosis. Also, we observe that the small population of blue cells present in the otocyst of Tbx1-/- embryos at E9 is rapidly lost. We have therefore analyzed the Tbx1 mutant and WT embryos for differences in cell proliferation and apoptosis. We tested cell proliferation at E9.5 by BrdU incorporation. At this stage, we found that ~50% of the otic epithelial cells were proliferating, but after counting ~500 cells/genotype we did not detect any difference between Tbx1+/- (276/565, 48.8%) and Tbx1-/- (236/493, 47.8%) otocysts (Fig. 4C and C'). We tested apoptosis using LysoTracker (Molecular Probes) staining (31) at E9, E9.5 and E10.5. Apoptosis is normally found in two domains; in the endolymphatic duct and in a ventral–medial region of the otocyst (32,33). At E9 and E9.5, we did not detect any difference in LysoTracker staining between Tbx1+/- and Tbx1-/- otocysts (data not shown). At E10.5, the endolymphatic duct domain was essentially identical in Tbx1+/- and Tbx1-/- mutants (Fig. 4D and D'), but the ventral–medial domain, which was well defined in Tbx1+/- embryos, was more diffuse, though still well delimited, in Tbx1-/- embryos (Fig. 4E and E'). However, these techniques may not provide definitive data if cell death and/or reduced proliferation affect a limited number of cells in a narrow time window, as the LacZ staining suggests. Nevertheless, the LysoTracker data suggest that increased apoptosis may play a role from E10.5, but one should also consider that this ‘late’ apoptosis may be a secondary event in an already compromised otocyst.

Tbx1-/- cells are unable to populate the otocyst in chimeric embryos
To understand the developmental potential of Tbx1-/- cells we generated embryonic stem (ES) cell lines that have a LacZ reporter knocked into both Tbx1 alleles (Fig. 4F). These ES cells express the LacZ reporter when the Tbx1 gene is activated. The ES cell lines were injected into wild type blastocysts and chimeric embryos were harvested at E9.5 and E10.5 and stained with X-gal. If Tbx1 is required cell autonomously in the otic epithelium we expect two scenarios: (i) mutant cells, which may have a proliferation/survival disadvantage, are rapidly replaced by wild type cells and the otocyst develops normally; or (ii) mutant cells contribute to the otocyst and cause developmental abnormalities. Experimental results were consistent with the first scenario, as we did not observe any LacZ positive cells in the otic epithelium (Fig. 4G and H) in 19 embryos that had good levels of chimerism. The otocysts of 11 of these chimeras were sectioned for histological analysis and all were apparently normal, although we cannot exclude subtle abnormalities due to the presence of Tbx1-/- cells in the surrounding periotic mesenchyme. Blue cells were observed in all other tissues that normally express Tbx1 at those stages and eight of these chimeras presented with developmental abnormalities of the pharyngeal apparatus (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Hearing impairment affects the majority of patients with del22q11 and although it is mainly attributable to the effects of chronic otits media, ~10% of cases involve sensorineural hearing loss. In addition, there are reports in the literature of patients with balance problems (15), although it is not known whether these are due to vestibular defects or to hypotonia. Here we show that Tbx1, which is a candidate gene for del22q11/DiGeorge syndrome, has a primary role in early morphogenesis of the inner ear, suggesting that some del22q11-associated hearing and balance impairments may result from Tbx1 haploinsufficiency.

In the broader context of inner ear develoment, our data establish an early temporal requirement for Tbx1 in the mouse from the time the otic cup closes to form the otocyst (E9–E9.5). Tbx1 is not required in mouse for formation of the endolymphatic duct or the cochlear–vestibular ganglia, but by E9.5, the otic vesicle in Tbx1-/- mutants is visibly abnormal, being smaller than the wild type otocyst with a thickened epithelial wall. No appreciable morphogenesis occurs between E9.5 and E13.5, although the otocyst does enlarge minimally. The lack of visible development of the semicircular canals and cochlea, and the loss of epithelial expression of several markers of the precursors of these structures (e.g., Bmp4, Pax2) suggest that Tbx1 may be required for expansion of a population of epithelial cells that is destined to form the vestibular apparatus and the cochlea. As Tbx1 is expressed in the periotic mesenchyme as well as in the otic epithelium, potentially, Tbx1 could function cell autonomously and/or cell non-autonomously. What the relative contribution of epithelial versus mesenchymal expression of Tbx1 is to inner ear development will require tissue-specific deletion of Tbx1.

A clue as to why the otocyst develops abnormally in Tbx1-/- mutants is provided by the observed distribution of mutant cells in Tbx1-/- embryos and in Tbx1 chimeras. The rapid disappearance of cells with transcriptionally activated Tbx1 in the otocyst of Tbx1-/- embryos and the lack of LacZ positive cells in the otocyst of chimeras suggests that mutant cells may have a proliferation/survival disadvantage that prevents them from expanding and populating the otocyst. In germline mutants this results in the failure of a cell population, while in chimeras mutant cells are replaced by wild type cells. This concept is supported by the analysis of region-specific molecular markers, which shows loss of expression of multiple markers that have expression patterns that are overlapping with each other and with Tbx1. We propose the following model to explain our findings. Tbx1 is required after neurogenesis but before the complex morphogenic changes occur. There is a subpopulation of otic epithelial cells that depends upon Tbx1 function for expansion and this population is required for development of the cochlea and vestibulum. The chimera analysis data support the hypothesis that Tbx1 functions cell autonomously in the epithelium. However, it is clear that the periotic mesenchyme in Tbx1-/- mutants is abnormal, as shown by Prx2 expression and morphological observation.

The ear phenotype caused by Tbx1 loss-of-function is, to our knowledge, one of the most severe that has been reported to date, because it results in complete developmental failure of the inner, middle and outer ear. Eya1 loss-of-function also results in severe abnormalities in all three ear compartments (34) and indeed Eya1-/- mutants have more extensive inner ear defects than Tbx1-/- mutants, in that the endolymphatic duct and cvg fail to develop as well as the semicircular canals and cochlea, whereas in Tbx1-/- mutants the endolymphatic duct and cvg form. However, Eya1-/- mice do have rudimentary development of the middle and outer ears (34). Sonic hedgehog (Shh) loss-of-function also results in severe malformation or absence of the vestibular and auditory systems (30) and several studies have shown that Tbx1 is a target of Shh signalling in the pharyngeal arches (17,35) and periotic mesenchyme (30). However, the earlier manifestation of inner ear abnormalities in Tbx1-/- embryos compared to Shh-/- embryos (36), together with the conservation of robust Tbx1 expression in the otic epithelium of Shh-/- embryos (30), suggest separate roles for these genes in inner ear development. Furthermore, a Shh-responsive enhancer element that binds forkhead box (Fox)-containing transcription factors has been recently identified upstream of the Tbx1 start codon (35). This enhancer was shown to regulate Tbx1 transcription in the embryonic head mesenchyme and in the pharyngeal endoderm, but not in the otic epithelium, as judged by LacZ expression. These data suggest that Fox proteins may function as intermediaries in Tbx1 regulation by Shh in some but not all Tbx1 expressing tissues.

Additional evidence for the importance of Tbx1 dosage for inner ear development comes from transgenic mice that overexpress four genes, Tbx1, Pnutl1, Gp1bB and Wdr14. These mice have abnormal behaviour indicative of vestibular defects and sensorineural hearing loss (37). The inner ear defects reported included hypoplastic semicircular canals, shortened or malformed cochlear duct and abnormal endolymphatic sac and duct. They also had middle ear defects. The authors presented data supporting overexpression of Tbx1 as the cause of the ear defects which, although not conclusive because a Tbx1 only transgenic was not available, when considered together with Tbx1-/- phenotype, indicate that the inner ear, like the embryonic cardiovascular system (8–10) is highly sensitive to Tbx1 gene dosage. If similar dosage sensitivity prevails in humans, it is possible that some of the cases of hearing loss in del22q11 patients may be due to Tbx1 haploinsufficiency.

Many outstanding questions remain about the role of Tbx1 in inner ear development. We do not know which genes regulate Tbx1 expression in the otocyst. A strong case can be made for Shh as a regulator of Tbx1 expression in several tissues, but diverse experimental data do not support this relationship in the ear. The downstream targets of Tbx1 are also unknown. Fgf10 and Fgf8 have been shown to have Tbx1-dependent domains of expression in the heart and pharynx (19) and here we have shown loss of the epithelial domain of Fgf10 expression in Tbx1-/- mutants. However, if our hypothesis is correct, this may be a consequence of a failure of a subpopulation of otic epithelial cells to proliferate and expand in the absence of Tbx1, rather than evidence of a genetic relationship between the two genes.

Currently, we do not know whether Tbx1 has a later role in inner ear development because most of the structures that comprise the mature inner ear do not form in the absence of Tbx1. The answer to this question may come from drug-inducible conditional alleles that will permit time-controlled elimination of Tbx1 expression at later stages of development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
B-galactosidase detection, histology and immunohistochemistry
B-gal activity was detected using an X-gal substrate on 4% paraformaldehyde fixed embryos, according to standard protocols. Embryos were photographed as wholemount specimens prior to embedding in paraffin and cutting in 10 µm sections. To visualize X-gal staining, histological sections were counterstained with Nuclear Fast Red. For cell proliferation assays, pregnant female mice were injected intraperitoneally with bromodeoxyuridine (BrdU) at 5 mg/100 g body weight and sacrificed 1 h after injection and embryos harvested. Embryos were ethanol fixed prior to embedding in paraffin and were cut into 7 µm sections. BrdU was detected using an anti-BrdU monoclonal antibody (Novacastra, Colne no. 85-2C8). Apoptosis was assayed with LysoTracker Red-DND-99 (Molecular Probes) using a protocol modified form (31). Briefly, embryos were incubated for 30 min at 37°C with slow rotation in 5 µm LysoTracker Red in HBSS, followed by 3x washes in HBSS and 3x washes in PBS, and fixed overnight at 4°C in 4% paraformaldhyde/PBS. After fixation, embryos were photographed as wholemout specimens or were ethanol fixed prior to embedding in paraffin and cut into 10 µm sections. LysoTracker Red staining was visualized by fluorescence microscopy.

In situ hybridization
Embryos were staged by somite count. Embryos were harvested between E9 and E13.5, fixed overnight in 4% paraformaldehyde/PBS, dehydrated in ethanol, embedded in paraffin and cut into 10 µm sections. RNA in situ hybridization was performed in accordance with a published protocol (38). Sense and antisense riboprobes were prepared by reverse transcription of DNA and labelled by incorporation of 35S-UTP (ICN).

Chimeras
To generate Tbx1-/- mouse ES cells, we retargeted the original Tbx1 mutant ES cell line so that the LacZ reporter would be knocked into both Tbx1 alleles. The targeting construct used was identical to that used previously (8), except that the neomycin resistance gene was replaced by a puromycin resistance gene to permit selection of double targeted clones. Tbx1-/- ES cells were injected into wild type C57BL/6 blastocysts and transferred into pseudopregnant CD1 females. Chimeric embryos were harvested at E9.5 and E10.5 and were stained with X-gal prior to ethanol fixation and embedding in paraffin. Histological sections (10 µm) were counterstained with Nuclear Fast Red.


   ACKNOWLEDGEMENTS
 
We thank Hedda Sobotka and Chelsey Sparks for technical assistance. This work was funded in part by the National Heart, Lung and Blood Institute, NIH (to A.B.) and by the American Heart Association Texas Affiliate (to E.A.L.). F.V. is the recipient of a fellowship from Italian Telethon.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Pediatrics (Cardiology), Baylor College of Medicine-Feigin Center, 1102 Bates, Houston, TX, 77030, USA. Tel: +1 8328244162; Fax: +1 8328254153; Email: elindsay{at}bcm.tmc.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Scambler, P.J. (2000) The 22q11 deletion syndromes. Hum. Mol. Genet., 9, 2421–2426.[Abstract/Free Full Text]

  2. Lindsay, E. (2001) Chromosomal microdeletions: dissecting Del22q11 syndrome Nat. Rev. Genet., 2, 858–868.[CrossRef][ISI][Medline]

  3. McDermid, H.E. and Morrow, B.E. (2002) Genomic disorders on 22q11. Am J Hum. Genet., 70, 1077–1088.[CrossRef][ISI][Medline]

  4. Greenberg, F., Crowder, W.E., Paschall, V., Colon-Linares, J., Lubianski, B. and Ledbetter, D.H. (1984) Familial DiGeorge syndrome and associated partial monosomy of chromosome 22. Hum. Genet., 65, 317–319.[CrossRef][ISI][Medline]

  5. Scambler, P.J., Kelly, D., Lindsay, E., Williamson, R., Goldberg, R., Shprintzen, R., Wilson, D.I., Goodship, J.A., Cross, I.E. and Burn, J. (1992) Velo-cardio-facial syndrome associated with chromosome 22 deletions encompassing the DiGeorge locus. Lancet, 339, 1138–1139.[CrossRef][ISI][Medline]

  6. Driscoll, D.A., Budarf, M.L. and Emanuel, B.S. (1992) A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11. Am. J. Hum. Genet., 50, 924–933.[ISI][Medline]

  7. Burn, J., Takao, A., Wilson, D., Cross, I., Momma, K., Wadey, R., Scambler, P. and Goodship, J. (1993) Conotruncal anomaly face syndrome is associated with a deletion within chromosome 22q11. J. Med. Genet., 30, 822–824.[Abstract]

  8. Lindsay, E.A., Vitelli, F., Su, H., Morishima, M., Huynh, T., Pramparo, T., Jurecic, V., Ogunrinu, G., Sutherland, H.S., Scambler, P.J. et al. (2001) Tbx1 haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice. Nature, 410, 97–101.[CrossRef][Medline]

  9. Jerome, L.A. and Papaioannou, V.E. (2001) DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet., 27, 286–291.[CrossRef][ISI][Medline]

  10. Merscher, S., Funke, B., Epstein, J.A., Heyer, J., Puech, A., Min Lu, M.M., Xavier, R.J., Demay, M.B., Russell, R.G., Factor, S. et al. (2001) TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell, 104, 619–629.[CrossRef][ISI][Medline]

  11. Ford, L.C., Sulprizio, S.L. and Rasgon, B.M. (2000) Otolaryngological manifestations of velocardiofacial syndrome: a retrospective review of 35 patients. Laryngoscope, 110, 362–367.[CrossRef][ISI][Medline]

  12. Shprintzen, R.J., Goldberg, R.B., Young, D. and Wolford, L. (1981) The velo-cardio-facial syndrome: a clinical and genetic analysis. Pediatrics, 67, 167–172.[Abstract/Free Full Text]

  13. Digilio, M.C., Pacifico, C., Tieri, L., Marino, B., Giannotti, A. and Dallapiccola, B. (1999) Audiological findings in patients with microdeletion 22q11 (Di George/velocardiofacial syndrome). Br. J. Audiol., 33, 329–333.[ISI][Medline]

  14. Reyes, M.R., LeBlanc, E.M. and Bassila, M.K. (1999) Hearing loss and otitis media in velo-cardio-facial syndrome. Int. J. Pediatr. Otorhinolaryngol., 47, 227–233.[CrossRef][ISI][Medline]

  15. Swillen, A., Devriendt, K., Legius, E., Prinzie, P., Vogels, A., Ghesquiere, P. and Fryns, J.P. (1999) The behavioural phenotype in velo-cardio-facial syndrome (VCFS): from infancy to adolescence. Genet. Couns., 10, 79–88.[ISI][Medline]

  16. Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M. and Papaioannou, V.E. (1996) Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn., 206, 379–390.[CrossRef][ISI][Medline]

  17. Garg, V., Yamagishi, C., Hu, T., Kathiriya, I.S., Yamagishi, H. and Srivastava, D. (2001) Tbx1, a digeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol., 235, 62–73.[CrossRef][ISI][Medline]

  18. Vitelli, F., Morishima, M., Taddei, I., Lindsay, E.A. and Baldini, A. (2002) Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum. Mol. Genet., 11, 915–922.[Abstract/Free Full Text]

  19. Vitelli, F., Taddei, I., Morishima, M., Meyers, E.N., Lindsay, E.A. and Baldini, A. (2002) A genetic link between Tbx1 and fibroblast growth factor signaling. Development, 129, 4605–4611.[Abstract/Free Full Text]

  20. Pirvola, U., Spencer-Dene, B., Xing-Qun, L., Kettunen, P., Thesleff, I., Fritzsch, B., Dickson, C. and Ylikoski, J. (2000) FGF/FGFR-2(IIIb) signaling is essential for inner ear morphogenesis. J. Neurosci., 20, 6125–6134.[Abstract/Free Full Text]

  21. Morsli, H., Choo, D., Ryan, A., Johnson, R. and Wu, D.K. (1998) Development of the mouse inner ear and origin of its sensory organs. J. Neurosci., 18, 3327–3335.[Abstract/Free Full Text]

  22. Morsli, H., Tuorto, F., Choo, D., Postiglione, M.P., Simeone, A. and Wu, D.K. (1999) Otx1 and Otx2 activities are required for the normal development of the mouse inner ear. Development, 126, 2335–2343.[Abstract]

  23. Torres, M., Gomez-Pardo, E. and Gruss, P. (1996) Pax2 contributes to inner ear patterning and optic nerve trajectory. Development, 122, 3381–3391.[Abstract]

  24. Favor, J., Sandulache, R., Neuhauser-Klaus, A., Pretsch, W., Chatterjee, B., Senft, E., Wurst, W., Blanquet, V., Grimes, P., Sporle, R. et al. (1996) The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl Acad. Sci. USA, 93, 13870–13875.[Abstract/Free Full Text]

  25. Fekete, D.M. and Wu, D.K. (2002) Revisiting cell fate specification in the inner ear. Curr. Opin. Neurobiol., 12, 35–42.[CrossRef][ISI][Medline]

  26. Noramly, S. and Grainger, R.M. (2002) Determination of the embryonic inner ear. J. Neurobiol., 53, 100–128.[CrossRef][ISI][Medline]

  27. Liu, W., Oh, S.H., Kang Yk, Y., Li, G., Doan, T.M., Little, M., Li, L., Ahn, K., Crenshaw, E.B., III and Frenz, D.A. (2003) Bone morphogenetic protein 4 (BMP4): A regulator of capsule chondrogenesis in the developing mouse inner ear. Dev. Dyn., 226, 427–438.[CrossRef][ISI][Medline]

  28. Paradies, N.E., Sanford, L.P., Doetschman, T. and Friedman, R.A. (1998) Developmental expression of the TGF beta s in the mouse cochlea. Mech. Dev., 79, 165–168.[CrossRef][ISI][Medline]

  29. Ten Berge, D., Brouwer, A., Korving, J., Martin, J.F. and Meijlink, F. (1998) Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs. Development, 125, 3831–3842.[Abstract]

  30. Riccomagno, M.M., Martinu, L., Mulheisen, M., Wu, D.K. and Epstein, D.J. (2002) Specification of the mammalian cochlea is dependent on Sonic hedgehog. Genes Dev., 16, 2365–2378.[Abstract/Free Full Text]

  31. Zucker, R.M., Hunter, E.S., III and Rogers, J.M. (1999) Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. Methods, 18, 473–480.[CrossRef][ISI][Medline]

  32. Fekete, D.M., Homburger, S.A., Waring, M.T., Riedl, A.E. and Garcia, L.F. (1997) Involvement of programmed cell death in morphogenesis of the vertebrate inner ear. Development, 124, 2451–2461.[Abstract]

  33. Nishikori, T., Hatta, T., Kawauchi, H. and Otani, H. (1999) Apoptosis during inner ear development in human and mouse embryos: an analysis by computer-assisted three-dimensional reconstruction. Anat. Embryol. (Berl), 200, 19–26.[CrossRef][Medline]

  34. Xu, P.X., Adams, J., Peters, H., Brown, M.C., Heaney, S. and Maas, R. (1999) Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordial. Nat. Genet., 23, 113–117.[CrossRef][ISI][Medline]

  35. Yamagishi, H., Maeda, J., Hu, T., McAnally, J., Conway, S.J., Kume, T., Meyers, E.N., Yamagishi, C. and Srivastava, D. (2003) Tbx1 is regulated by tissue-specific forkhead proteins through a common Sonic hedgehog-responsive enhancer. Genes Dev., 17, 269–281.[Abstract/Free Full Text]

  36. Liu, W., Li, G., Chien, J.S., Raft, S., Zhang, H., Chiang, C. and Frenz, D.A. (2002) Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo. Dev. Biol., 248, 240–250.[CrossRef][ISI][Medline]

  37. Funke, B., Epstein, J.A., Kochilas, L.K., Lu, M.M., Pandita, R.K., Liao, J., Bauerndistel, R., Schuler, T., Schorle, H., Brown, M.C. et al. (2001) Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects. Hum. Mol. Genet., 10, 2549–2556.[Abstract/Free Full Text]

  38. Albrecht, U., Eichele, G., Helms, J.A. and Lu, H.C. (1997) Visualization of gene expression patterns by in situ hybridization. In Daston, G.P., (ed.), Molecular and Cellular Methods in Developmental Toxicology. CRC Press, Inc., New York, pp. 23–48.


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