EphA3 null mutants do not demonstrate motor axon guidance defects

Abstract

Motor axon projections are topographically ordered. Medial motor column axons project to axial muscles, whereas lateral motor column axons project to limb muscles and, along the rostrocaudal axis of the animal, the more rostral motor neuron pools project to more rostral muscle targets. We have shown that EphA3 is specifically expressed in the developing medial motor column and have postulated that EphA3 might be responsible for directing their axons to axial muscle targets. This hypothesis was supported by our demonstration that EphA3 can direct retinal ganglion cell axon targeting and by studies of ephrin-A5(-/-) mutants that show that EphA receptor signaling controls the topographic innervation of the acromiotrapezius. To test the role of EphA3 in motor axon guidance, we generated an EphA3 null mutant. Retrograde labeling studies in EphA3(-/-) embryos and adults indicate that, contrary to our predictions, EphA3 is not necessary to direct motor axons to axial muscle targets. Our results also demonstrate that ephrin A5's ability to direct topographic innervation of the acromiotrapezius must be mediated through EphA receptors other than, or in addition to, EphA3.

FIG. 1.

Generation and characterization of the EphA3^−/− mutant. (A) The top line represents the wild-type EphA3 genomic locus. The line below is a sketch of the targeting construct used to generate the EphA3 null mutant. The portion of the EphA3 gene removed by homologous recombination includes EphA3's first exon (indicted by a box with an arrow in it) that encodes its signal sequence. (B) Western blots of E13.5 spinal cord protein extracts from wild-type and EphA3^−/− mutants with an anti-EphA3 antibody demonstrate that no EphA3 protein is produced in EphA3^−/− E13.5 spinal cords (n = 6). The same samples were analyzed by Western blotting with an anti-neurofilament antibody (right panel) as a loading control.

FIG. 2.

Motor column organization in the EphA3 mutants. To determine whether the EphA3 null mutant has the normal columnar organization of motor neurons, immunohistochemistry was carried out on cross sections through wild-type (WT) (n = 5) and EphA3^−/− (KO) (n = 10) E13.5 spinal cords. (A and B) Sections probed with an anti-Lim3 antibody to delineate the MMC; (C and D) sections probed with an antibody that recognizes both Islet-1 and Islet-2 and therefore delineates both the MMC (arrowheads) and the LMC (black arrows). Interneurons are also immunoreactive for these markers, and their positions are indicated by white arrows. This analysis demonstrates that the motor column organization in EphA3^−/− mice appears to be unaffected by their mutation. Bar, 0.2 mm.

FIG. 3.

Spinal nerve projection patterns in EphA3^−/− mutants. To analyze the EphA3^−/− mutants for gross abnormalities in the patterns of their spinal nerve projections, E12.5 wild-type (n = 12) and EphA3^−/− (n = 21) embryos were subjected to whole-mount immunohistochemistry by using an anti-neurofilament antibody. The embryos were subsequently sectioned on a Vibratome at 150 μm and viewed under epifluorescence. No significant differences between the mutants and wild-type were detected at this level of analysis. Bar, 0.5 mm.

FIG. 4.

Retrograde labeling studies in EphA3^−/− mutant embryos. Wild-type (A and C) and EphA3^−/− (B and D) E13.5 embryos were retrogradely labeled by injecting a rhodamine-dextran conjugate into their paravertebral muscles (A and B) or limb muscles (C and D). The positions of the MMC and of the LMC (indicated by the white dashed curves) were observed by overexposing the image. In both wild-type and EphA3^−/− mutants, tracer injection of paravertebral muscles consistently retrogradely labeled the MMC, while tracer injection of the limb muscles consistently labeled the LMC. Bar, 0.2 mm.

FIG. 5.

Retrograde labeling studies in EphA3 mutant adults. The acromiotrapezius muscles of wild-type (n = 6) (A) and EphA3^−/− (n = 6) (B) mice were injected with Fluorogold. After we allowed time for the tracer to be transported to the innervating motor neurons, the spinal cords were sectioned and analyzed for the position of labeled motor neurons. Labeled motor neurons fell almost exclusively in spinal segments C1 to C3. Thus, no shift in the motor neuron pool innervating the acromiotrapezius in EphA3^−/− mutants could be detected. Bar, 0.5 mm.

FIG. 6.

The EphA3 and EphA4 genes are coexpressed in motor neurons during development. RNA in situ hybridizations for EphA3 (A), EphA4 (B), and (C) Islet-2 were carried out on 7-μm serial sections of E11.5 wild-type embryos. The sections were counterstained with DAPI (4′,6′-diamidino-2-phenylindole), and the silver grains were viewed with a red filter under dark-field illumination. Islet-2 expression delineates the motor neurons in the field. Note that EphA3 and EphA4 are coexpressed in a subset of MMC motor neurons at this developmental time point. Bar, 0.1 mm.