ARTICLE |
Correspondence to: Lynne M. Bianchi, Neuroscience Program, Oberlin College, Oberlin, OH 44074. E-mail: lynne.bianchi@oberlin.edu
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Summary |
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Immunostaining in transgenic mice carrying the lac z gene can be used to map gene and protein distribution in a single tissue. In this study, we examined inner ears from ephrin-B3 homozygous and ephrin-B2 heterozygous mice. Ephrin-B3 lac z expression was limited in these mice. However, immunostaining revealed ephrin-B3 throughout cochlear and vestibular regions. Immunoreactivity was absent in ephrin-B3-homozygous null mutants, demonstrating the specificity of the antibody. Ephrin-B2 lac z reactivity was detected in a limited number of cells in cochlear and vestibular regions. Different immunostaining patterns were found with different antibodies. Comparison with lac z expression indicated which antibody was specific for the transmembrane-bound ephrin-B2 ligand.
(J Histochem Cytochem 50:16411645, 2002)
Key Words: cochlea, vestibular, ephrin-B2, ephrin-B3, transgenic mice
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Introduction |
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IN RECENT YEARS, methods such as in situ hybridization (ISH) and immunohistochemistry (IHC) have been useful for examining the distribution of genes and proteins in tissues. The development of transgenic mice provides additional ways to evaluate gene expression and to test the specificity of reagents and methods. For example, transgenic mice are now often generated with the lac z reporter gene substituted into the coding region of the gene of interest, permitting endogenous gene expression to be mapped in animals carrying one (+/-) or two (-/-) copies of the mutated genes. IHC can also be performed for comparison of gene and protein expression in a single tissue. Furthermore, in mice homozygous for a gene mutation (knockout mice), IHC analysis can be used to test the specificity of an antibody by comparing immunoreactivity in wild-type and knockout mice. Any immunoreactivity detected in wild-type mice should be absent from the knockout mice, because any remaining reactivity would be indicative of nonspecific binding of that antibody. Therefore, transgenic mice can be used not only to examine morphological changes but also to map gene and protein distribution.
A number of transgenic mice have been generated in which the lac z gene is substituted for the gene of interest. For example, mice lacking members of the Eph family have been used to examine Eph distribution and function. The Eph family is comprised of ephrin ligands and Eph receptors. The ephrins are membrane-associated ligands. Ephrin-A ligands are attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage, whereas ephrin-B ligands are transmembrane-bound. These ligands bind to the Eph receptor tyrosine kinases. The EphA subclass of receptors binds primarily A ligands, whereas EphB receptors bind primarily B ligands (
This study examined inner ear tissues of mice homozygous for an ephrin-B3 or heterozygous for an ephrin-B2 null mutation. Specifically, this study evaluated (a) gene expression patterns using lac z histochemical methods, (b) protein expression using IHC analysis, and (c) morphological changes in the inner ear using light microscopy.
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Materials and Methods |
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The lac z gene was used to replace the ligand-coding region of the ephrin-B2 gene or the first coding exon of the ephrin-B3 gene (
For IHC, tissue sections were rehydrated through a graded series of alcohols, incubated in 3% H2O2, then blocked in 1% normal serum. Sections were incubated in a primary polyclonal antibody overnight at 4C (anti-ephrin-B3 1:501:100 (SC-7281; Santa Cruz Biotechnology, Santa Cruz, CA); anti-ephrin-B2 1:501:100 (SC-1010, Santa Cruz Biotechnology); anti-ephrin-B2 1:501:100 (AF496; R&D Systems, Minneapolis, MN). The following day, sections were rinsed in PBS, then incubated in biotinylated secondary antibody (1:200; Vector Labs, Burlingame, CA). Sections were then rinsed and treated with the avidinbiotin complex (ABC; Vector Labs). Sites of antibody binding were visualized with diaminobenzidine (DAB; Sigma, St Louis, MO). Ephrin-B2 inner ears were also processed for IHC following lac Z reactivity. Prior processing for lac z histochemistry did not interfere with subsequent IHC results other than reducing the visibility of the DAB signal.
Estimates of neuron numbers in cochlear and vestibular ganglia were made from hematoxylin- and eosin-stained serial sections. The number of neurons with a clearly identifiable nucleus was counted in every fourth section (16-µm intervals) as described previously (
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Results |
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In the adult inner ear, ephrin-B3 lac z reactivity was detected in the cochlear (spiral ganglion) neurons (Fig 1A) and weakly in the stria vascularis (not shown) in four of the six inner ears analyzed. Other regions of the inner ear did not exhibit lac z reactivity. Wild-type mice did not show any reactivity, even after 96 hr in developing solution.
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Because the lac z expression was so limited in the inner ear, it was not clear whether the lac z reactivity accurately reflected ephrin-B3 distribution. IHC analysis was therefore completed to examine ephrin-B3 protein production in the adult inner ear. Ephrin-B3 immunoreactivity was widely distributed throughout the inner ear in areas including the cochlear spiral limbus, the organ of Corti, and the lateral wall (Fig 1B). In the vestibular regions, the sensory epithelia and connective tissue areas below the sensory epithelia were reactive (not shown). Both cochlear (Fig 1B) and vestibular neurons (not shown) were also immunoreactive. In contrast to the widespread distribution of ephrin-B3 in the adult inner ear, ephrin-B3 immunoreactivity was absent in the inner ear and brain at E18 (not shown).
The immunoreactivity noted throughout adult inner ear tissues suggested that the antibody was binding nonspecifically. However, immunostaining of ephrin-B3-homozygous mutants revealed that ephrin-B3 immunoreactivity was lost in the null mutants (Fig 1C). Furthermore, in the E18 mouse ephrin-B3 immunoreactivity was detected in whisker follicles of wild-type mice (Fig 1D) but was absent in the whisker follicles of null mutant mice (Fig 1E). Therefore, the IHC studies demonstrated that the anti-ephrin-B3 antibody was specific for ephrin-B3 protein, confirmed that ephrin-B3 protein was not produced in the mutant mice, and allowed more detailed analysis of expression than was possible with lac z histochemistry in these mice.
The gross morphology of the inner ear was normal in the ephrin-B3-null mutants. The hair cells and supporting cells of the cochlear and vestibular epithelia, were present and nerve fibers were seen entering both regions of sensory epithelia, as observed by light microscopy (not shown). Neither cochlear nor vestibular neurons showed a significant decrease in number in the ephrin-B3 null mutant mice (mean number of cochlear neurons in wild-type 1911 ± 169 SE, n=2; homozygous null mutants 2060 ± 57 SE, n=4; mean vestibular neurons in wild-type 1047 ± 54 SE, n=6; homozygous null mutants 934 ± 53 SE, n=6). Although a slight decrease in vestibular neurons was noted in the ephrin-B3-null mutants compared to wild-type mice, no significant difference was noted (p>0.05).
Mice heterozygous for the ephrin-B2 mutation were used to compare lac z and IHC expression in the adult inner ear. Mice homozygous for the ephrin-B2 mutation are embryonic lethal; therefore, they could not be evaluated (
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IHC studies of ephrin-2-heterozygous mice revealed different results with two different anti-ephrin-B2 antibodies. One antibody (R&D Systems; Fig 2B and Fig C) showed immunoreactivity in areas that overlapped with lac z expression, as would be expected because ephrin-B2 is a transmembrane-anchored protein (Fig 2B and Fig 2C). In contrast, a second anti-ephrin-B2 antibody (Santa Cruz Biotechnology) showed some regions of expression that overlapped with lac z staining, such as cochlear and vestibular neurons, as well as several immunopositive regions adjacent to structures positive for lac z (Fig 2D). The areas of adjacent immunoreactivity were limited to specific sites in the adult inner ear, giving the appearance of immunoreactivity "specific" to certain cell regions such as the spiral limbus, lateral wall, and basilar membrane of the cochlea and the fibrocyte-rich area below the vestibular epithelia. A second lot of this anti-ephrin-B2 antibody (Santa Cruz Biotechnology) failed to stain any region of the inner ear, further suggesting that the areas of adjacent reactivity observed with the first lot of the antibody were largely nonspecific. In summary, lac z reactivity detected ephrin-B2 expression in specific inner ear regions and was useful in determining which commercially available antibody specifically recognized ephrin-B2 in paraffin-embedded tissues. Because ephrin-B2-homozygous mice are not available for study, it was not possible to confirm whether immunoreactivity is lost in homozygous ephrin-B2 mice.
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Discussion |
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The present study demonstrated the need for both lac z histochemistry and IHC to accurately identify the sites of ephrin-B3 and ephrin-B2 expression in the inner ear. Lac z reactivity revealed ephrin-B3 to be only weakly expressed in cochlear and vestibular neurons and in the stria vascularis. Increasing the time in developing solution did not increase the intensity or distribution of the lac z reaction product in the inner ear of the null mutant mice. Further, wild-type mice never showed any reaction product. In contrast, previous studies using the same ephrin-B3-null mutant mice reported that the lac z reactivity was below the level of detection in embryonic brain (
Because lac z reactivity alone could not be used to map ephrin-B3 expression, IHC analysis was completed. These experiments revealed that ephrin-B3 was widely distributed throughout the adult inner ear of wild-type mice. The observation that this immunoreactivity was absent in the adult inner ear and embryonic whisker follicles of homozygous null mutants indicated that the antibody specifically detected ephrin-B3. These studies also confirmed that the ephrin-B3 protein was not produced in the null mutants.
No gross morphological deficits were noted in ephrin-B3-homozygous mice, suggesting that ephrin-B3 is not required for cell formation and maintenance in the inner ear, or that other ephrins can compensate for the loss of ephrin-B3.
Experiments using heterozygous ephrin-B2 mice revealed lac z expression in vestibular neurons and supporting cells, similar to the distribution reported with another ephrin-B2-null mutant mouse (
When the lac z expression patterns were compared to the immunoreactivity patterns in the heterozygous ephrin-B2 mice, the specificity of a commercially available antibody was questioned. Whereas one antibody (R&D Systems) overlapped lac z expression, the second antibody (Santa Cruz Biotechnology) showed several areas of immunoreactivity that were adjacent to lac z-positive areas. Although the immunoreactivity with this second antibody looked "specific" in these areas, several of the regions, such as the basilar membrane, have previously been found to nonspecifically bind a variety of antibodies, including antibodies that recognize their appropriate protein in other inner ear regions (B. Schulte, personal communication). Therefore, it appeared that the majority of the immunoreactivity detected with this antibody was nonspecific. The reason for the nonspecific binding to regions adjacent to lac z reactivity is unclear. The immunostaining patterns were not identical to any of the ephrin staining patterns previously observed in paraffin-embedded tissues using antibodies from the same company (
In summary, transgenic mice are useful tools for assessing antibody specificity in mice that are homozygous for a null mutation or heterozygous for a mutation in a protein that is not secreted.
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Acknowledgments |
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Supported by NSF IBN 9904566.
We wish to thank S. Anne Hastings for technical assistance, Dr Bradley Schulte for helpful discussion on this project, and Nicole Falk and Annegret Falkner for assisting with analysis of neuron counts.
Received for publication June 4, 2002; accepted July 3, 2002.
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Literature Cited |
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