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Hmx2 homeobox gene control of murine vestibular morphogenesis

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

View larger version (22K): [in a new window]   Fig. 1. Disruption of Hmx2 by homologous recombination, mRNA analysis and genotyping of embryos carrying an Hmx2lacZ mutant allele. (A) Hmx2 wild-type locus, targeting construct and Hmx2 mutant alleles. Homologous recombination of the targeting construct into the Hmx2 locus results in the insertion of ires.lacZ.neo into the Hmx2 homeobox. Owing to the presence of translation stop codons in all three open reading frames in the ires.lacZ.neo cassette, this mutant allele produces ß-galactosidase and a nonfunctional truncated Hmx2 protein. The black boxes show the positions of the two Hmx2 exons. The homeobox is located in the second exon. The transcriptional orientation of Hmx2 is shown by an arrow. The positions and lengths of the probes used in Southern blot analysis and RNAse protection are also indicated by small black rectangles. (B) Southern blot assay from yolk sac DNA of wild-type, Hmx2lacZ+/– and Hmx2lacZ–/– embryos. Probe1 and Probe 2 are located outside of the targeting construct. Probe 1 detects a 17.0 kb wild-type NotI+ClaI fragment, as well as a 12.5 kb mutant NotI+ClaI band. Probe 2 hybridizes with a 5.7 kb mutant XbaI fragment instead of a 6.7 kb wild-type XbaI band because of the introduction of an additional XbaI by the ires.lacZ.neo cassette. Note: the XbaI site 5' to the Hmx2 gene is methylated in dam+ bacterial hosts. (C) RNAse protection analysis of Hmx2 RNA expression in wild-type, Hmx2lacZ+/– and Hmx2lacZ–/– embryos. A 242 genomic fragment spanning the Hmx2 homeobox was amplified by PCR using primers TL245 and TL246, and used as a template to prepare an antisense RNA riboprobe. RNA transcripts produced by the wild-type allele protect a fragment of 242 bp, whereas the Hmx2 mutant transcripts protect two fragments of 175 bp and 67 bp.

View larger version (83K): [in a new window]   Fig. 2. Early embryonic expression pattern of Hmx2 revealed by staining Hmx2lacZ+/– whole mounts for ß-galactosidase. Embryonic stages are indicated at the lower-right corner of each panel. ß-galactosidase expression patterns are identical to that obtained by RNA in situ hybridization. (A) No ß-galactosidase activity can be detected at E8.5. Hmx2 is first turned on at E9.0. (B) At E9.5 and (C) E10.5, Hmx2 expression becomes more prominent in the anterior portion of otic vesicle and otocyst, respectively and the cleft between the first and second branchial arches. (D) At E12.0, expression of Hmx2 can also be seen in the developing neural tube and hypothalamus. Arrows indicate the positions of the otic anlagen. The arrowhead in B indicates the Hmx2 expression in the cleft between the first and second branchial arches. Arrowheads in D indicate the Hmx2 expression in the central nervous system. Letters AB, CD and EF indicate the level and orientation of corresponding sections shown in Fig. 6.

View larger version (59K): [in a new window]   Fig. 3. Structural alteration of the Hmx2lacZnull inner ears as shown by ß-galactosidase activity in whole-mount embryos and dissected inner ears. The embryonic stages are indicated on the left. The genotype of each embryo is shown at the top. E and F are high power views of dissected Hmx2lacZ+/– and Hmx2lacZ–/– inner ears. Arrows in A-D indicate the positions of the inner ear. Arrowheads in panel A and B indicate Hmx2lacZ expression in the developing neural tube and hypothalamus. In total, more than 20 embryos of each genotype from different embryonic stages were examined. CD, cochlear duct; ES, endolymphatic sac; LD, lateral semicircular duct; PD, posterior semicircular duct; S, saccule; SD, superior semicircular duct; SV, stria vascularis; VD, vestibular diverticulum.

View larger version (138K): [in a new window]   Fig. 4. Absence of semicircular duct formation and formation of a common macula in a fused utriculosaccular chamber in Hmx2lacZ null mutants. (A) Section illustrating normal development of the semicircular ducts in the control embryo at E13.5. (B) Hmx2lacZ–/– inner ear at E13.5 illustrating the formation of two vestibular diverticulae that are dorsal and lateral to a fused utriculosaccular chamber. (C) Control inner ear at E16.5 illustrating normally developed utricle and saccule chambers with associated maculae and horizontal ampulla. (D) Hmx2lacZ–/– inner ear at E16.5 illustrating a common macula within a fused utriculosaccular chamber, lacking any distinction between the utricle and saccule other than the location of the maculae, as both appear to be combined ventrally. (E) Section illustrating normal development of the horizontal crista and ampulla coming off the utricular chamber in a control embryo at E18.5. (F) Hmx2lacZ–/– inner ear at E18.5 demonstrating a more severe dysgenic vestibular system and increased fusion and enlargement of the common utriculosaccular chamber relative to earlier stages. D, dorsal; HC, horizontal crista; L, lateral; M, medial; MS, macula of saccule; MU, macula of utricle; S, footplate of the stapes; SD, semicircular duct; V, ventral; VD, vestibular diverticum.

View larger version (27K): [in a new window]   Fig. 5. Histograms showing a progressive loss of hair cells from most of the vestibular sensory epithelia and of vestibular ganglion neurons in the Hmx2lacZ null inner ears. (A-E) Cell counts of the cristae of all three semicircular canals, maculae of the utricle and saccule, as well as ganglion neurons in the vestibular and spiral ganglia, were performed on the Hmx2lacZ+/– and Hmx2lacZ–/– inner ears. Black bars represent the number of hair cells and ganglion neurons of the Hmx2lacZ+/– inner ear; gray, those of the Hmx2lacZ–/– inner ear. Statistical analysis was performed using an unpaired Student’s t-test. Error bars indicate standard deviations.

View larger version (91K): [in a new window]   Fig. 6. Cell proliferation in the developing inner ear is affected by the loss of Hmx2. A-F show the morphological alteration of the Hmx2lacZ–/– inner ear at E11.5 and E12.0 by examining ß-galactosidase activity. Arrows indicate the presumptive fusion plate that is undergoing thinning and invagination. E and F are sections dorsal to C and D, respectively. The approximate level of sectioning is also indicated in Fig. 2. G-L are the comparable anti-BrdU-labeled sections showing the reduced rate of cell proliferation in the developing Hmx2lacZ–/– inner ears. Genotypes and embryonic stages are indicated at the top of each column. Control corresponds to either Hmx2lacZ+/+ or Hmx2lacZ+/– genotypes. Overt morphological differences can be seen at E11.5 when the invagination of epithelial cells around the posterolateral boundary of the otic vesicle is delayed in the homozygotes (A and B). At E12.0, the close apposition of epithelial walls to form the fusion plates was not present in the otocyst of the Hmx2lacZ–/– embryo (C,D,E,F). Both the epithelial cells and underlying periotic mesenchymal cells of the corresponding regions are undergoing reduced cell proliferation (J and L). M-P show the apoptotic activities of the otic epithelial cells in the control and Hmx2lacZ–/– inner ears at E11.5 and E12.0. Arrows indicate the regions of the fusion plates. A higher percentage of cells in the fusion plates are undergoing programmed cell death relative to other regions in both control and Hmx2lacZ–/– embryos. However, the Hmx2lacZ–/– otic vesicles do not show an altered rate of cell death relative to the corresponding regions in the control embryos. L, lateral; R, rostral.

View larger version (115K): [in a new window]   Fig. 7. Expression profile of developmental control genes in the otocysts of wild-type and Hmx2lacZnull embryos. The Hmx2 genotype is indicated on the top of each column. The molecular markers examined are listed on the lower right corner of each panel. The embryonic stages are indicated on the upper right corner of each panel. Arrows in K,L indicate the position of the developing sensory patches in the otic vesicle. Arrowheads indicate the presumptive fusion plate. (A,B) No discernible difference in Hmx3 expression can be seen between wild-type and Hmx2lacZnull backgrounds. (C,D) Altered Dlx5 expression in the Hmx2lacZ–/– inner ear is demonstrated by the loss of its expression in the discrete epithelial patches and ectopic Dlx5 expression in the presumptive fusion plate. (E,F) Down regulation of Pax2 expression in the lateral aspect of the Hmx2lacZnull otocyst can be clearly seen at this stage. (G-J) Although both genes play a critical role in inner ear development, no discernible alteration of either BF1 or netrin 1 expression was observed in the developing inner ears in the Hmx2lacZnull embryos. At early embryonic stages, Bmp4 mRNA is restricted to the sensory epithelial cells in the developing otic vesicle. K-N show the Bmp4 expression in the otic vesicles in the wild-type and Hmx2lacZ–/– embryos by whole mount in situ hybridization. (K,L) At E10.5, Bmp4 is expressed in a correct spatial fashion in the Hmx2lacZ–/– otocyst. No differences in Bmp4 expression are seen at this stage of development. However, from E11.5, the sensory patches fail to express Bmp4 (M,N). Instead, preferential expression of Bmp4 in discrete regions of the lateral portion of the wild-type otic vesicle is replaced by a homogenous expression in the corresponding region in the Hmx2lacZnull mutants (O,V). P, R, T and V show the expression pattern of Bmp4 in the wild-type and Hmx2lacZ–/– otic vesicles examined by in situ hybridization on paraffin sections of embryos of E11.5 and E12.0. O, Q, S and U are the corresponding bright-field views of P, R, T and V. C, caudal; L, lateral; M, medial; R, rostral.

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