1 Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574, USA
2 Departments of Otolaryngology and Neuroscience, Albert Einstein College of Medicine, 1410 Morris Park Avenue, Bronx, NY 10461, USA
*Author for correspondence (e-mail: thomas.lufkin{at}mssm.edu)
Accepted September 18, 2001
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SUMMARY |
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Key words: Homeobox gene, Hmx, Inner ear development, Semicircular ducts, Gene knockout, Mouse, Cell proliferation
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INTRODUCTION |
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During development, the otic placode invaginates and pinches off from the surface ectoderm to form an ellipsoid-shaped vesicle termed the otic vesicle (Hilfer et al., 1989). During the closure of the otic vesicle, positional information is gradually acquired by different regions of the otic epithelium, as indicated by the regionally restricted expression of different combinations of inner ear marker genes. Axial specification in the inner ear for sensory organs happens earlier than that for nonsensory organs. Transplantation experiments performed in chick demonstrated that patterning of sensory organs along the anterior-posterior (AP) axis is fixed first, followed by specification of the dorsal-ventral (DV) axis. At the same time, nonsensory epithelial cells maintain a certain degree of plasticity, since they can be reprogrammed when placed in a new environment (Wu et al., 1998). Different regions of the otocyst possess distinct positional information and express unique combinations of inner ear marker genes, which in turn ultimately determines their specific identities (Fekete, 1996). Regions with different identities display distinct capabilities in cell differentiation, proliferation and programmed cell death, a prerequisite for the transformation from an otocyst to an elaborate three-dimensional labyrinth. Precise outgrowth and fusion of the otic epithelium are morphogenetic milestones in the maturation of the inner ear. In the mouse, around E9.5 the anlagen of the endolymphatic duct bulges from the dorsal portion of the otic vesicle and elongates dorsally to form a hollow tube, which enlarges at its distal end to form a sac. Meanwhile, the otic epithelium destined to give rise to the cochlea enlarges ventrally and finally develops into a coiled duct. Morphogenesis of the otocyst pars superior into a vestibule requires very sophisticated shape changes. Formation of the semicircular ducts initiates from paired outpocketings of otic epithelium. In the central regions of these pockets, the two epithelial layers on the opposing walls first become thinner and then detach from the underlying mesenchyme. Afterwards, these two walls approach one another to form an extensive region termed the fusion plate. Epithelial cells meeting at the fusion plate will meld into a single layer, and subsequently disappear via a possible mechanism involving either programmed cell death or epithelium cell retraction, or both. The semicircular ducts and associated canals are formed by the interactions between the remaining otic epithelium and its adjacent periotic mesenchyme (Fekete et al., 1997; Frenz and Van De Water, 1991; Lang et al., 2000; Martin and Swanson, 1993). Undoubtedly, a balance between cell proliferation and programmed cell death plays a critical role in this process. Previous work has confirmed the existence of several regions of programmed cell death in the developing inner ear, including the fusion plates of the semicircular canals, the ventromedial otic vesicle and the base of the endolymphatic sac. In the chick, the overexpression of inhibitors of normal programmed cell death, such as bcl2, block semicircular canal fusion (Fekete et al., 1997). It has been proposed that the driving force pushing the apposing walls inward to form the fusion plate is generated by the interaction between the otic epithelium and the surrounding mesenchyme. The analysis of the inner ears of netrin 1 null mice support this mechanism (Salminen et al., 2000). netrin 1 appears to be involved in a signaling pathway regulating cell proliferation in the periotic mesenchyme. When netrin 1 production by the presumptive fusion plate was disrupted, reduced cell proliferation in the underlying mesenchyme resulted in severe defects in fusion plate formation and therefore semicircular duct morphogenesis.
In the mouse, by E14.5, the basic architecture of the membranous labyrinth of the inner ear has been fully established. From E14.5 through adult stages, maturation occurs and a functional inner ear emerges, with the vestibule and cochlea being responsible for the senses of balance and hearing, respectively. The identification of developmentally important genes expressed in the inner ear has been steadily increasing, and the function of certain of these genes has been determined (Represa et al., 2000). Antagonizing the normal Bmp4 signaling pathway with noggin disrupts inner ear development and demonstrates that local signals in the otic epithelium are essential for the correct formation of the inner ear (Chang et al., 1999; Gerlach et al., 2000). Different members of the homeobox gene superfamily are involved in all steps of inner ear development ranging from otic placode induction to maturation of a fully functional inner ear. Recent progress in mouse molecular genetics has allowed us to acquire specific information regarding the developmental contribution of individual genes to inner ear development. Certain homeobox genes are involved in regional specification of the inner ear, and some of these genes determine the fate of cells in their restricted expression domains. For example, Pax2 is predominately expressed in the ventral otic epithelium, and Pax2 null inner ears lack an identifiable cochlea (Represa et al., 2000; Torres et al., 1996). In addition, expression of a functional Dlx5 gene in the dorsal portion of the otic vesicle is required for proper morphogenesis of the semicircular canals (Acampora et al., 1999; Depew et al., 1999). Murine Otx1, an orthologue of the Drosophila orthodenticle gene, is expressed in the ventrolateral wall of the otocyst with a dorsal limit in the presumptive lateral semicircular canal. Otx1null inner ears show an absence of the lateral semicircular canals and a malformed cochlea (Acampora et al., 1996; Morsli et al., 1999). However, it is common for a homeobox gene family that has several members to exhibit similar or even identical expression profiles and to be functionally interchangeable in certain aspects (Greer et al., 2000). The Hmx homeobox genes belong to a distinct family, which is of ancient origin, and their existence has been reported in many species (Bober et al., 1994; Stadler et al., 1995; Stadler et al., 1992; Stadler and Solursh, 1994; Wang et al., 1990; Wang et al., 2000). Three members of the Hmx homeobox gene family, designated Hmx1, Hmx2 and Hmx3 have been identified in mouse (Wang et al., 2000; Yoshiura et al., 1998). Regions where Hmx1 likely exerts its developmental function are neural crest derivatives including the dorsal root ganglion, cranial ganglia, branchial arches and in the developing neural retina. Unlike Hmx1, Hmx2 and Hmx3 are clustered together on the same chromosome and have nearly identical expression domains that include the inner ear, CNS and uterus. Defects caused by the inactivation of Hmx3 have been reported for the inner ear and in female fertility (Hadrys et al., 1998; Wang et al., 1998). An absence of horizontal cristae, a fused utriculosaccular chamber, a significant loss of vestibular hair cells and perimplantation infertility have been observed, indicating unique functions for Hmx3. However, analysis of these defects also suggests that some aspects of Hmx3 function might have been compensated for by its sibling Hmx2, since not all cells expressing Hmx3 are affected in the Hmx3 null mice. In this paper, we will present the defects exhibited by Hmx2 null mice to reveal its unique developmental function.
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MATERIALS AND METHODS |
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For the cell proliferation assay, pregnant mice were injected intraperitoneally with BrdU (50 µg/g body weight) at E10.5, E11.5 and E12.0. Embryos were collected 1 hour after injection and embryos processed as previously described (Tribioli and Lufkin, 1999; Wang and Lufkin, 2000). TUNEL was performed on paraffin sections as previously described (Tribioli and Lufkin, 1999; Wang and Lufkin, 2000; Wang et al., 1998). Embryos from E10.5 to E13.5 were collected. At least three control embryos (Hmx2lacZ+/+ or Hmx2lacZ+/) and three Hmx2lacZ/ embryos at each embryonic stage were examined for both BrdU-labeling and TUNEL. Light micrographs were taken and the color was inverted using Adobe Photoshop 5.5.
35S-labeled RNA in situ hybridization was performed as previously described (Tribioli and Lufkin, 1999; Wang and Lufkin, 2000; Wang et al., 1998) and embryos from E10.5 to E18.5 were collected. Comparable sections from at least three wild-type or Hmx2lacZ/ embryos of different stages were examined for each in situ probe. The in situ probe for Hmx3 has been described previously (Wang et al., 1998). Other antisense probes used in this experiment were 1.2 kb Bmp4; 1.0 kb Brain Factor 1(BF1; now known as Foxg1); 400 bp Dlx5; 0.6 kb Otx1; 600 bp netrin 1; 500 bp Pax2; 1.0 kb Sek1 (also known as Epha4). Wild-type and Hmx2lacZ/ embryos from E10.5 to E12.5 were isolated for whole-mount in situ hybridization. Preparation of antisense digoxiginin-labeled Bmp4 RNA probe, hybridization and visualization of signals were performed essentially as previously described (Tribioli et al., 1997). Five embryos of the same genotype (+/+ or /) at different stages were pooled and probed with a dig-labeled Bmp4 probe.
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RESULTS |
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Since no Hmx2-transcription control elements were deleted in the targeting strategy, integration of the ires.lacZ.neo reporter gene into Hmx2 exonic sequences enables us to follow Hmx2 gene expression by examining ß-galactosidase activity. ß-galactosidase expression of the Hmx2lacZ heterozygotes faithfully reproduced Hmx2 expression patterns as revealed by in situ hybridization on paraffin sections and in whole-mount embryos (Wang et al., 2000). Unlike Hmx3, Hmx2 is still negative in the otic placode at E8.5 (Fig. 2A). Hmx2 expression is first detectable at E9.0 and its expression becomes prominent from E9.5 onward in the anteriodorsal portion of the otic vesicle, as well as the cleft between the first and second branchial arches (Fig. 2B,C). In addition to its expression in the vestibular portion of the otic vesicle, from E12.0, Hmx2 transcripts are strongly present in the central nervous system, including the developing neural tube, pons and hypothalamus. Its expression in the CNS is maintained at later stages (Fig. 2D and Fig. 3A,C). After E13.5, ß-galactosidase activity from the Hmx2lacZ+/ allele is detected in both the sensory and nonsensory epithelia of all three well-formed semicircular canals, endolymphatic sac, utricle and saccule (Fig. 3A,C,E). From E14.5, the stria vascularis of the cochlea begins to show ß-galactosidase activity (Fig. 3C,E). Hmx2lacZ+/ and wild-type mice are indistinguishable in their viability, behavior and fertility. Approximately 65% of the Hmx2lacZ/ mice in the outbred genetic background C57BL/6Jx129X1/SvJ (previously 129/SvJ; Jackson Laboratory #000691) show classic vestibular defects as indicated by hyperactivity, head tilting and circling activity. The remaining Hmx2lacZ/ mice do not display any abnormalities in their behavior or fertility. Despite its strong expression in the central nervous system, loss of Hmx2 was insufficient to cause any observable defect in the CNS as determined by the behavior, ß-galactosidase activity and histological analysis of Hmx2lacZ/ mice and embryos (data not shown). Furthermore, RNA in situ hybridization with numerous probes corresponding to neurogenic developmental regulators from the paired, forkhead and homeodomain families (e.g. Pax, Fox, Dlx), as well as cell-cell signaling molecules of the TGFß superfamily, failed to reveal any alterations in expression between wild-type and Hmx2 lacZ/ null embryos in the CNS (data not shown).
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In addition to a fused utriculosaccular chamber in the Hmx2lacZ/ embryos, a portion of this chamber had an area of a common macula, which was identifiable as early as E14.5 in the vestibule of the Hmx2lacZ/ embryos (data not shown). In other areas of the common cavity, sensory epithelium corresponding to the macula of the utricle was located on the lateral wall of the caudal utriculosaccular chamber where the utricle normally would be located (see Fig. 4). Accordingly, sensory epithelium corresponding to the macula of the saccule was also present in the Hmx2lacZ/ embryo, located on the medial wall of the rostral utriculosaccular chamber, which is where the saccule would normally be located (Fig. 4B,D,F). In contrast, the thickened sensory epithelia of the maculae fuse on the caudal aspect of the utriculosaccular chamber in the Hmx2lacZ/ embryos at E16.5 and persist until later stages (Fig. 4C,D). The posterior ampulla is present in Hmx2lacZ/ embryos along with the posterior crista ampullaris. A distinctly formed superior ampulla, however, is not present, although a patch of sensory epithelium that corresponds to the superior crista ampullaris can be identified in the area where the superior ampulla would normally be located (data not shown). Furthermore, the absence of the lateral crista and lack of a distinct lateral ampulla become evident as early as E14.5. In the Hmx2lacZ/ inner ear, the lateral crista ampullaris never forms and only a rudimentary attempt at formation of a lateral ampulla is evident off the vestibular diverticula, with a lack of any transected loops to provide evidence of developed semicircular ducts. The attempt at formation of a lateral ampulla may also simply be a transection through the lateral vestibular diverticulum, which is melding with the superior/posterior diverticulum as the Hmx2lacZ/ embryo develops in utero, further illustrating the increasingly severe lack of distinction in the vestibular diverticula as the mutants age.
Quantitative analysis of histological variables between control and Hmx2lacZ/ embryos
Progressive impairment of the sensory system of the inner ear was carefully examined during development. Statistically significant deficits in the total number of ampullary hair cells were found in the Hmx2lacZ/ embryos, with an 86% loss at E14.5, 64% at E16.5, and 68% at E18.5 (P-value=0.02, 0.0017 and 0.00056 respectively; Fig. 5A). Hair cell counts revealed statistically significant deficits in the number of utricular hair cells in the Hmx2lacZ/ embryos of 63% at E16.5 and 69% at E18.5 of 63% (P-value=0.006 and 0.0005 respectively; Fig. 5B). In contrast, saccular hair cell counts revealed no statistically significant differences between the controls and mutants at all embryonic ages examined (P-value=0.14, 0.25 and 0.58 at E14.5, E16.5 and E18.5 respectively; Fig. 5C).
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Reduced cell proliferation in the otic epithelium and the periotic mesenchymal cells in the Hmx2lacZnull inner ears
Since gross morphogenetic abnormalities in the developing vestibular system can be observed as early as E13.5 (Fig. 3), molecular events mediating these defects must have occurred before this stage. Even though no gross morphological alterations can be detected between control and Hmx2lacZ/ whole-mount inner ears at E11.5, transverse sections through inner ears stained for ß-galactosidase revealed clear differences in the epithelial invaginations between the Hmx2lacZ+/ and Hmx2lacZ/ embryos (Fig. 6A and 6B). At this stage, Hmx2 expression visualized by ß-galactosidase activity is present in the entire dorsal otic vesicle excluding the otic epithelium cells destined to form the endolymphatic duct and sac (Fig. 6A). A small region of the lateral epithelium becomes thinner and the entire lateral aspect of the otic epithelium begins moving medially (arrow in Fig. 6A). In the Hmx2lacZ/ otic vesicle, neither the thinning nor the invagination of the lateral epithelium takes place (Fig. 6B) but instead, the thickness of the entire Hmx2lacZ/ otic epithelium remains uniform (Fig. 6B). In wild-type embryos at E12.0, a thinned otic epithelial layer delaminates from the underlying mesenchyme and further moves toward the medial face, forming a structure termed the fusion plate (arrows in Fig. 6C,E). Fig. 6E shows the fusion plate that has been pushed inward within close proximity of the medial face and is about to fuse into a single epithelial layer (which will eventually disperse) leaving behind an intact semicircular duct. However, in the Hmx2lacZ/ embryos, regional thinning and invagination of the lateral face of the otic epithelium towards the medial layer is not observed at this stage (Fig. 6D,F). Interestingly, at E13.5 when the 3-dimensional structure of the otic labyrinth has been well formed in the wild-type inner ear (Fig. 3A), the lateral epithelial layer of the Hmx2lacZ/ vestibular diverticulum begins to become thin and eventually detaches from the underlying mesenchyme (data not shown). However, the subsequent fusion plate process never occurs in the Hmx2lacZ/ inner ear, and this appears to be a principal reason for the failed vestibular morphogenesis in mice lacking Hmx2.
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However, when the BrdU incorporation rate was examined, reduced cell proliferation was observed in the Hmx2lacZ/ inner ears in both the otic epithelium and the periotic mesenchymal cells (Fig. 6G-L). In the wild-type otic vesicle at E11.5, epithelial cells, including the sensory and nonsensory epithelial cells show active cell proliferation (Fig. 6G and 6H), which was dramatically reduced in the Hmx2lacZ/ otic vesicle (Fig. 6G and 6H). In wild-type embryos at E12.0, when the fusion plate is being pushed toward the medial epithelial face, mesenchymal cells underlying the presumptive fusion plate as well as the recipient face of the medial epithelial layer display elevated cell proliferation activity as indicated by the many BrdU-labeled cells (Fig. 6I,K). But in the Hmx2lacZ/ otic vesicle, less BrdU-positive cells were present in the corresponding regions and the distribution of proliferating cells was quite uniform throughout the periotic mesenchyme (Fig. 6J,L). Previous work has demonstrated that the periotic mesenchyme may provide the major driving force pushing the neighboring epithelial cells to undergo morphological changes (Salminen et al., 2000; Van De Water, 1983). Therefore, slowed cell proliferation exhibited by the otic epithelium and the surrounding periotic mesenchyme in the Hmx2lacZ/ inner ear may account, in part, for disruption of the morphogenesis of the vestibular system.
Inactivation of Hmx2 affects the expression profile of developmental regulators that control inner ear ontogeny
Expression of genes with a demonstrated role in inner ear morphogenesis and differentiation was examined on paraffin sections and whole mounts of wild-type and Hmx2lacZ/ inner ears at embryonic stages preceding and coincident with the disruption in vestibular morphogenesis. At E10.5, all genes examined, including BF1, Bmp4, Dlx5, Hmx3, netrin 1, Pax2, Otx1and Sek1, showed no overt alteration in their expression in the Hmx2lacZ/ inner ear (Fig. 7K and 7L; data not shown). Homeobox genes Hmx3 and Otx1, as well as the receptor tyrosine kinase gene Sek1, which all play a critical role in inner ear morphogenesis, do not show any altered expression patterns at any of the embryonic stages examined (Fig. 7A,B; and data not shown). Either the lack of a gene regulatory relationship between Hmx2 and the above mentioned genes or the inability of Hmx2 alone to alter the regulatory cascade may account for these findings. Owing to the severe morphological discrepancies between wild-type and Hmx2lacZ/ inner ears older than E13.5, altered expression of certain markers at later stages may be caused by indirect structural changes rather than direct regulatory effects. Thus in situ hybridization results on sections of embryos immediately preceding and coinciding with Hmx2lacZ/ inner ear dysmorphology (i.e. E11.5) are presented.
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In summary, loss of functional Hmx2 leads to an alteration of the molecular identities of the cells in the dorsolateral aspect of the otic vesicle including the sensory cells and the prospective fusion plate. Taken together with the results from the cell proliferation assay, Hmx2lacZ/ fate-altered lateral epithelium acquired a reduced cell proliferation capability, which in turn impacted on the adjacent periotic mesenchyme, which normally does not express Hmx2. Hence changes in cell fate in restricted regions of the otic epithelium subsequently affects global morphogenesis of the entire vestibular system. In addition, the above RNA in situ data also indicate the existence of a genetic regulatory interaction between Hmx2 and other inner ear developmental regulatory genes such as Bmp4, Dlx5 and Pax2. Other factors showing restricted expression pattern in the inner ear were also investigated in the Hmx2lacZ/ embryos. Brain factor 1 (BF1), a winged helix transcription factor is confined to the epithelium on the medial aspect of the otic vesicle (Hebert and McConnell, 2000), however its absence in the dorsolateral face was maintained (Fig. 7G and 7H) in the Hmx2lacZ/ embryos. Likewise netrin 1, a member of the laminin-related secreted proteins, is expressed in the nonsensory epithelium known to form the fusion plate. Expression of netrin 1 remains unchanged in the Hmx2lacZ/ embryos (Fig. 7I and 7J), indicating that inactivation of Hmx2 alone is insufficient to affect netrin 1 expression even though loss-of-function alleles for these two genes present a similar phenotypic mechanism affecting inner ear morphogenesis.
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DISCUSSION |
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Molecules from different gene families have been shown to play a critical role in epithelial fusion. One well-characterized molecule is the laminin-like protein, netrin 1. In addition to its function in axon guidance and cell migration in the central nervous system, netrin 1 is also required for fusion plate formation during inner ear morphogenesis (Salminen et al., 2000). Laminins are a major component of the ECM and previous work has shown that remodeling of the extracellular matrix (ECM) in the basement membrane is a key event in axonal guidance, cell migration, angiogenesis and morphogenesis of complex organs. It had been proposed that the robust presence of netrin 1 in the fusion plate might either compete with other laminin molecules to disrupt the laminin network in the basement membrane, or upregulate the production of matrix metalloproteinases to remodel the ECM network. In the netrin 1 null otocyst, the epithelial wall of the presumptive fusion plate became thinner. However, these thinned layers failed to change their epithelial morphology and subsequently detach from the underlying basement membrane. The strong expression of netrin 1 in the Hmx2lacZ/ inner ears at all stages suggests that netrin 1 alone is insufficient for the formation of the fusion plate. It is possible that the prospective epithelium has to first become competent so that it can be responsive to netrin 1. In this respect, Hmx2 may be needed to determine the fate of epithelial cells in specific regions of the otocyst. The reduced cell proliferation rate observed in the Hmx2lacZ/ periotic mesenchymal cells further suggests that Hmx2 and netrin 1 may work cooperatively to control fusion plate formation by affecting cell proliferation of the neighboring mesenchyme. Also, the entry points when the two genes exert their functions are different since Hmx2 clearly functions at an earlier stage than netrin 1. Based upon our in situ hybridization data, although these two genes share many overlapping expression domains, a clear regulatory interaction cannot be established between Hmx2 and netrin 1. There are two possibile mechanisms to account for this. First, both genes may use independent pathways to regulate cell proliferation. Second, there is a regulatory interaction existing between these two genes, however, inactivation of Hmx2 alone is insufficient to alter netrin 1 expression, as other genes might compensate for the function of Hmx2. One such candidate is Hmx3, however, the persistence of netrin 1 in the developing Hmx3 null inner ear suggests either a possible functional redundancy between Hmx2 and Hmx3 in regulating netrin 1 (Salminen et al., 2000), or no regulatory interaction. The assessment of an overlapping role of Hmx2 and Hmx3 in controlling netrin 1 (and other inner ear-specific genes) will be obtained from the analysis of embryos carrying combined loss-of-function alleles for Hmx2 and Hmx3. Interestingly, certain phenotypic aspects of a previously generated Hmx3-PGKneo null allele share some overlap with the Hmx2 phenotype described here (Hadrys et al., 1998). However, this overlap was not observed in mice carrying either of two independent Hmx3 null alleles that lacked PGKneo and which were additionally shown to have no affect on the expression of the neighboring Hmx2 gene (Wang et al., 1998). As PGKneo is notorious for affecting adjacent gene expression within tens of kilobases (Olson et al., 1996), a plausible explanation for the partial phenotypic overlap is that the previously described Hmx3-PGKneo allele (Hadrys et al., 1998) is also affecting, in a negative manner, the expression of the closely linked Hmx2 gene.
Differential expression of varied combinations of developmental patterning genes gives distinct molecular identities to different cell populations, which consequently display unique capabilities in cell proliferation, apoptotic activity and responsiveness to the environment (Fekete, 1996). In the Hmx2lacZ/ otocyst, cells in a subset of the Hmx2-expressing domains have changed their fate as indicated by the altered expression of specific inner ear markers. The loss of expression in the presumptive sensory epithelium of Bmp4, Dlx5 and Pax2, together with ectopic activation of Bmp4 and Dlx5 in the prospective fusion plate, indicate a substantial alteration in cell fate in the otic epithelium of the pars superior. As a result of this cell fate alteration, the otic epithelium fails to communicate properly with the periotic mesenchyme, and in return, proper inner ear morphogenesis is severely impaired (Van De Water, 1983). Here we show that Hmx2, a homeodomain transcription factor affects the rate of cell proliferation of the otic epithelium, as well as the neighboring mesenchymal tissue. The identification of cell-cell signaling factors bridging the gap between the different tissues will be important. In this study, it is interesting that BMP4, a putative crista marker was present at E10.5, but disappeared by E11.5, suggesting that the cristae might be specified initially but fail to develop properly in the absence of Hmx2.
In summary, a normal function of Hmx2 is to govern the specification and commitment of epithelial cells in the pars superior portion of the otocyst to undergo the proliferative growth and fusion processes to form a mature and functional vestibular system.
Functional relationship between Hmx2 and Hmx3 in mouse development
Hmx2 and Hmx3 are an ideal pair of homeobox genes to investigate functional redundancy existing between members of the same gene family. The highly similar expression patterns and close linkage on chromosome 7 suggest that these two genes may share certain of the same transcriptional regulatory elements during mouse development and the similarity in the DNA-binding homeodomain suggest that these two genes may share many downstream regulatory targets. In the central nervous system and uterus, Hmx2 and Hmx3 show identical expression profiles (Wang et al., 2000; Wang et al., 1998), but in the Hmx2lacZ/ animals, no obvious defect was detected in either of these tissues. By comparing the defects in the individual Hmx2 and Hmx3 null inner ears, the unique developmental function of each of these genes can be identified. In the Hmx3 null inner ear, the gross structure of the three semicircular canals forms except the horizontal ampulla and its associated crista are absent. In the ventral part of the vestibule, the utricle and saccule are fused into one chamber in which a significant cell loss occurs in both sensory maculae. This indicates that Hmx3 alone uses a different mechanism to effect cell fate determination than by facilitating fusion plate formation and subsequent closure of the semicircular ducts. Disruption of Hmx2, despite its later onset of expression, results in a more severe inner ear phenotype than loss of Hmx3. In the Hmx2lacZ/ inner ear, anterior and dorsal regions along the corresponding AP and DV axes of the developing vestibule are more severely affected than in Hmx3 null mice. Even though Hmx2 and Hmx3 are both involved in cell fate determination in specific regions of the otic epithelium, the consequences are different. Loss of Hmx3 primarily influences the development of a subset of sensory receptor cells, but not the overall morphogenesis of the inner ear. In contrast, Hmx2 is not only involved in cell fate determination of vestibular sensory areas, but also the morphological transformation mediated by the nonsensory epithelial cells. Even though Hmx3 is transcriptionally activated about 8 hours earlier in the otic placode, the time point when Hmx3 exerts its function seems to be later than that of Hmx2. Moreover, inactivation of Hmx3 generates a milder phenotype, as fewer structures and tissue types are affected. It is also notable that some regions coexpressing Hmx2 and Hmx3, such as the saccule, posterior ampulla and endolymphatic duct are not severely affected in either mutant. One explanation is that Hmx2 and Hmx3 may share redundant functions during inner ear and CNS development or they may be expressed in tissues where they exert no developmental function, possibly owing to a lack of a necessary cofactor(s). The unique and overlapping functions of each gene will be more clearly understood by comparing the inner ear phenotypes of mice carrying individual as well as a combined mutation in Hmx2 and Hmx3.
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ACKNOWLEDGMENTS |
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