The developmental expression of Dlx5 and Msx2 suggests that the exencephaly phenotype associated with the double mutation may be linked to a synergistic interaction between these two genes during a transient period when they are both expressed at the apex of the neural folds during neural fold fusion. Since both Dlx5 and Msx2 are transcriptional regulators that must regulate morphogenetic events by affecting the expression of structural genes, we explored potential downstream target genes that might be linked to the exencephaly phenotype. In a previous report, it has been demonstrated that ephrinA5 and its receptor EphA7 participate in cranial neural tube morphogenesis via cell attraction and cell repulsion, and that embryos that possess a defect in this signaling pathway can display exencephaly (Holmberg et al. 2000). To explore whether Dlx5 and Msx2 have a regulatory role on the expression of ephrinA5, EphA7-FL, and EphA7-T1, we performed gene expression pattern analysis of ephrinA5, EphA7-FL, and EphA7-T1 on WT, Dlx5−/−, Msx2−/−, and Dlx5−/−;Msx2−/− mouse embryos at E9.5 (two embryos were used in each genotype for the gene expression pattern analysis). In wildtype embryos, the expression domain of ephrinA5 and EphA7-FL are almost identical to each other in that expression is restricted to the outer layer of the neural tube (Fig. 4A,E). The expression domain of EphA7-T1 is in the dorsal two-thirds of the neural tube (Fig. 4I, the ventral margin of EphA7-T1 expression domain indicated by arrows), and the domain is broader than ephrinA5 and EphA7-FL. In Dlx5 null mutant embryos, expression of ephrinA5 and EphA7-FL appeared similar to wildtype embryos (Fig. 4B,F). However, EphA7-T1 expression domain was expanded to the ventral region of the neural tube in Dlx5−/− mutants, although the intensity of expression did not appear to be significantly changed (Fig. 4J). In Msx2 null mutant embryos, expression of EphA7-FL was not changed (Fig. 4G), whereas ephrinA5 expression was slightly decreased (Fig. 4C). The expression domain of EphA7-T1 in Msx2 mutant was expanded to the ventral region, but the expression level was not altered (Fig. 4K). In Dlx5 and Msx2 double null mutant embryos displaying exencephaly the expression of ephrinA5 was decreased all over the neural tissue, particularly in the region of Dlx5/Msx2 co-expression at the apex of the neural folds (Fig. 4D). EphA7-FL expression was not modified in the apex of neural folds in Dlx5−/−;Msx2−/− embryos (Fig. 4H), whereas EphA7-T1 transcripts were largely absent throughout the neural tissue including the apex of the neural folds (Fig. 4D,L). These results show that the expression of the ligand, ephrinA5, and one of its receptors, EphA7-T1, is regulated by the combined activity of Dlx5 and Msx2 during anterior neural tube closure.
Figure 4. Expression pattern of ephrinA5, EphA7-FL, and EphA7-T1 in WT, Dlx5−/−, Msx2−/−, and Dlx5−/−;Msx2−/− mice embryos at E9.5. (A–D) Expression of ephrinA5 in wildtype (A), Dlx5−/− (B), Msx2−/− (C), and Dlx5−/−;Msx2−/− (D). Transcripts of ephrinA5 are detected outer layer of the neural tube. This expression is not altered in Dlx5, or slightly decreased in Msx2 mutant. In contrast, the double mutant embryo shows significantly decreased expression of ephrinA5. (E–H) Expression of EphA7-FL in wildtype (E), Dlx5−/− (F), Msx2−/− (G), and Dlx5−/−;Msx2−/− (H). Expression pattern of EphA7-FL is not modified in Dlx5, Msx2, or the double mutant. (I–L) Expression of EphA7-T1 in wildtype (I), Dlx5−/− (J), Msx2−/− (K), and Dlx5−/−;Msx2−/− (L). Expression domain of EphA7-T1 in the neural tube is expanded toward the ventral side in Dlx5 and Msx2 single mutant embryo. In Dlx5/Msx2 double mutant, EphA7-T1 expression is significantly decreased. Scale bars: 100 μm.
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