Figure 3

Expression of Amvasa in Apis mellifera embryos. Scale bars indicate 100 micrometers. A, C, E, G, and K, Brightfield images of embryos hybridised with probes against Amvasa, Lateral view. B, D, F, H, J, Fluorescent micrographs of the same embryos stained using the nuclear stain DAPI.A and B)) A stage 1 embryo stained (blue) for Amvasa RNA. Expression is present in the cytoplasm of all energids. The dark band running across the embryo (arrows) is due to mitosis occurring in that region (see B and magnified in M). C-F) embryos of stage 3 (C and D) and stage 8 (E and F) showing no expression of Amvasa RNA. G-J) embryos of stage 9 stained for Amvasa RNA. Expression can be seen in a line of cells starting in the 3^rd abdominal segment (arrow in G and I) and stretching back to the 6^th abdominal segment. The line of cells expressing Amvasa (magnified in N) lies under the epidermis and is just ventral to the boundary between the germband and the extra-embryonic membranes. A small group of cells expressing Amvasa can also be seen in the mandibular segment (asterisks in G and I). K) View of the dorsal surface of a stage 9 embryo stained for both Amvasa RNA (blue) and engrailed-like proteins (brown). Arrow marks the third abdominal segment. L) View of the dorsal surface of a just-hatched honeybee larva stained for Amvasa RNA. Expression can be seen in two crescents of cells near the midline of the larva (arrows) in the approximate position of the future gonads. The background in this larva is due to the forming cuticle. M) Magnification of the stage 1 embryo shown in A and B stained with the nuclear dye DAPI showing a region of mitoses in the embryo. N) Magnification of the Amvasa expressing cells of a stage 9 embryo, dorsal view, segments marked. O) Magnification of the anterior region of a stage 9 embryo stained for Amvasa RNA. A group of cells in the mandibular segment are weakly expressing Amvasa RNA (arrow). P) RT-PCR analysis of Amvasa expression. RT-PCR was carried out on RNA from just-laid eggs (JL), eggs between stages 3 and 7 (3–7) and eggs older than stage 10 (10-L). Each RT-PCR experimental lane lies next to a control reaction where amplification was from RNA treated as for the experimental lanes but without the addition of reverse transcriptase in the first stand synthesis. This controls for genomic DNA contamination of the cDNA. These lanes are labelled with -RT. RT-PCR was also attempted from ten times more cDNA from stages 3–7 in an attempt to detect low level expression (10 × 3–7) Successful amplification of Amvasa gives a 916 bp fragment. Amplification is seen only in cDNA from just laid embryos and stages 10 and above.