Fig. 5. Pax6 can induce ectopic expression of Necab. (A) Schematized view of the electroporation procedure. Mouse embryos are microinjected with a needle containing the DNA solution (in blue) and electroporated. (B,C) Double whole-mount in situ hybridization (8.5 dpc), 6 hours after electroporation with a Pax6 expression vector. (B) Ectopic expression of Necab is detected in a subset of cells (blue arrowhead). Endogenous expression is detected in the pretectum and optic vesicle (white arrowheads). (C) The expression domain of the Pax6 transgene (red arrowhead) is broader but overlaps ectopic Necab-positive cells. (D-F) Double in situ hybridization (8.5 dpc) on midbrain explants. (D,E) Twenty four hours after electroporation with a Pax6 expression vector, cells expressing the transgene (in red) are also expressing Necab (in blue). (E) Control explants (no electroporation) cultured for 24 hours and hybridized with both probes. (G,H) Double in situ hybridization (8.5 dpc), 6 hours after electroporation with a Six3 expression vector. Transgene expression is present (red arrowhead) but ectopic Necab-expressing cells are not detected. (I) Cross section at the hindbrain level of the embryo in G, showing that the transgenic cells do not expressed Necab. (J,K) Six hours after the electroporation of a Necab expression vector, ectopic expression of Chx10 is detected in the midbrain region (J) where transgenic cells are present (K). Red arrowhead, transgene expression; blue arrowhead, ectopic expression of the target gene; white arrowhead, endogenous expression.
