Figures & data
Figure 1 Innexin7 (inx7) antibody generation and Innexin7 protein expression pattern in Drosophila embryos. (A) Predicted structure of the Drosophila Innexin7 protein. Striking features, such as the putative PDZ domain, and phosphorylation sites are depicted using different symbols. The peptide derived from the C-terminal region, which was used for antibody generation is depicted in green. (B) Peptide competition experiment (Western blot). The specific Innexin7 signal (51.62 kDa, arrow) was competed with excess of the C-terminally derived Innexin7 peptide. (C) Ectopic expression of UASinx7myc in seven stripes using the paired-Gal4 driver. The anti-Innexin7 antibody (red) detects ectopic Innexin7 myc expression in the seven stripes of the paired pattern (see Methods) and thereby colocalizes with the anti-Myc antibody (green). (D–F) Lateral view of stage 5 (D, E) and stage 6 (F) embryos. Innexin7 (red) is localized within the nucleus and does not colocalize with armadillo/β-catenin (green), an apicolateral located core component of adherens junctions. (G) Dorsal view of a stage 7 embryo. Innexin7 staining is detected in the invaginating embryonic midline and in the developing neuroblasts, which delaminate from the epidermis. (H, I) Innexin7 is identified in the midgut (mg), hindgut (hg), and within a subset of cells within the ventral nerve cord (vnc) along the anterior-posterior axis. Innexin7 localization is found in a punctate pattern in the cytoplasm and at the membrane of epithelial cells (I, arrow). In contrast, nuclear staining is kept only in a few cell types including in the developing central nervous system (I, arrowhead; see also ). (J) Peptide competition experiment in Drosophila embryos. Dorsal view of a stage 15 embryo. Innexin7 staining is completely abolished upon competition with excess of Innexin7 peptide (compare to H and I).
Figure 2 Innexin7 is dynamically expressed in the Drosophila central nervous system (CNS) during embryonic nervous system development. Innexin7 expression in a stage 7 (A) and a stage 9 (B) embryo. Innexin7 (red) is localized to the developing neuroectoderm and enriched within the embryonic midline (ml) (B). (C–G) During progression of the embryonic development, two different expression domains of Innexin7 can be detected: the midline (C) and neuronal cells next to the midline (E, F, G). During embryogenesis, Innexin7-expressing cells in the embryonic midline are restricted to only two types of midline glia (mlg), here indicated by colocalization with the marker slit (C, green), which is made by midline glia cells. Innexin7 is not coexpressed with the homeodomain protein repo (green), which is expressed in all CNS glia except the midline glia (D). (E, F, G) Double staining of Innexin7 with neuronal markers prospero (pros, green) and FasciclinII (FasII, green). The Innexin7-expressing cells next to the midline colocalize with the nuclear marker prospero (F) and are ensheathed by FasII-positive fascicles (G), which led to their identification as vMP2 and pCC cells. Additionally, three other Innexin7-positive neuronal cells could be detected (G, arrow), which are not yet identified.
Figure 3 RNAi-mediated knockdown of innexin7 in the nervous system causes severe defects in the Drosophila embryonic nervous system. (A) Scheme of the UASwizinx7 construct used for knockdown of innexin7. (B) UASwizinx7 can reduce the level of innexin7 mRNA expression. RT-PCR shows that the UASwizinx7 knockdown decreases innexin7 mRNA expression level down to 7% as compared to wild type. (C, D, F–H) Wild-type pattern and 31-1-Gal4: UASwizinx7 knockdown phenotypes stained with 22C10 (green). (C, D) Lateral view of wild-type embryos stained with 22C10. (E) 31-1-Gal4–mediated expression pattern monitored using UAS-GFP. (F–H) Lateral view of innexin7 RNAi knockdown embryos. By overexpressing the RNAi construct in the nervous system, a severe disorganisation of the vnc along the anterior posterior axis can be observed (arrows in G and H). (H) Magnification of the embryo depicted in (G). (I–M) Wild-type pattern and sim-Gal4: UASwizinx7 knockdown phenotypes stained with 22C10 (green) and BP102 (green). (I, K) Dorsal view of wild-type embryos stained with 22C10 (I) or BP102 (K). (J, L, M) Expression of UASwizinx7 in midline cells only results in intrasegmental fusion of the ventral nerve cord and disruption of longitudinal, posterior, and anterior commissures in the CNS.
