Figure 5

Transcription during early gametocyte development. (A) Flow sorting of putative sexually and asexually committed parasites. Synchronized and stressed schizonts of the transgenic 164/TdTom line were collected and purified at around 45 hours post-invasion, and separated by flow cytometry. Infected RBCs are gated based on nuclear content using the nuclear dye Vybrant Violet, and sexually committed parasites including gametocytes are gated based on the TdTomato signal. Shown is one of two biological replicates containing 5% uninfected RBCs (1), 20% non-fluorescent parasites with a single nucleus (2, trophozoites), 74% non-fluorescent parasites with multiple nuclei (3, schizonts) and 0.6% fluorescent parasites with single and multiple nuclei (4; gametocytes, putative sexually committed schizonts). From populations 3 and 4 a total of 5 × 10⁵ to 10⁶ cells were collected for microarray analysis, including an aliquot for cytospin to confirm stage composition (see inserted picture of representative schizonts and gametocytes). (B) Affymetrix microarray analysis of putative sexually and asexually committed parasites. The scatterplot is based on data from two biological replicates of stressed schizont populations that were subsequently enriched and sorted. Genes with a mean differential expression level of ≥2-fold are marked in green for those up-regulated in the fluorescent population (‘sexually committed’; 305 genes) and in blue for those up-regulated in the non-fluorescent population (‘asexually committed’; 98 genes). (C) Staging of sexually committed cells. The histogram shows the distribution of transcriptional peak times for genes up-regulated in the fluorescent population (‘sexually committed’) in Figure 5B.