Figure 4

Expression of Amnos in Apis mellifera embryos. Scale bars denote 100 micrometers. A, C, E, G and I, Brightfield images of embryos hybridised with probes against Amnos, Lateral view. B, D, F, H, J, Fluorescent micrographs of the same embryos stained using the nuclear stain DAPI.A and B) A late stage 1 embryo stained for Amnos RNA. Amnos RNA is present in a gradient with highest concentrations at the posterior pole. C and D) a stage 2 embryo stained for Amnos RNA. Faint staining is present in a similar pattern to that in A. Amnos RNA expression fades very fast in stage 2 embryos. E-H) Stage 5 (E and F) and stage 8 (G and H) embryos stained for Amnos. No expression is seen. I and J) Stage 9 embryo stained for Amnos RNA, expression is seen in a line of cells starting in the third abdominal segment (arrow in I) and stretching to the 6^th abdominal segment. The Amnos expressing cells underlie the ectoderm of the embryo and lie just ventral to the border between the germband and the extraembryonic membranes. K) RT-PCR analysis of Amnos 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 Amnos gives a 257 bp fragment. Amplification is seen in cDNA from just laid embryos (JL), and cDNA stages 10 and above.