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. 2019 Mar 4;20(4):e46762. doi: 10.15252/embr.201846762

Figure 6. Zygotic replacement of H3.3K36 and H3.2K36 with H3.2R36 does not cause ectopic repression of Abd‐B in the central nervous systems.

Figure 6

  1. The Abd‐B expression in the Central Nervous System (CNS) of stage 16 embryos was assayed by immunostaining with antibodies against Abd‐B (red). In the ash1 22/TM3, Sb, e Kr::GFP or ash1 9011/TM3, Sb, e Kr::GFP embryos (heterozygous control), Abd‐B is expressed in parasegments 14–10 in a gradient that recedes from the posterior to anterior parasegment. In the ash1 22/ash1 9011 mutants, the Abd‐B gradient is much steeper, with reduced staining of parasegments 13 and 12 and at the edge of the detection in parasegments 11–10. Heterozygous control and ash1 22/ash1 9011 mutant embryos were stained together and separated by strong GFP expression (green) in the Bolwig's organs (white arrows). Here and in (B) the embryos are oriented with anterior pole facing the top and scaling bars correspond to 50 μm.
  2. Unlike ash1 mutants, embryos homozygous for His3.3A, His3.3B, ΔHisC deletions and supplemented with 12x H3K36R transgene (His3.2 − ,His3.3 − ,H3K36R) display the wild‐type Abd‐B immunostaining pattern (red), the same as the control embryos heterozygous for His3.3A, and ΔHisC deletions. In this experiment, immunostaining with antibodies against acetylated H3K18 (green) served as positive control.
  3. The schematic of the Abd‐B expression in the Drosophila embryo. Although homeotic transformations are often described according to the adult segments affected, the expression of the bithorax complex genes is regulated at the level of embryonic parasegments (PS), which are directly related to adult segments but slightly shifted relative to one another 5, 64.
  4. Reverse transcription and quantitative PCR (RT–qPCR) measurement of Abd‐B expression in His3.2 − ,His3.3 − ,H3K36R embryos. Expression of Abd‐B in stage 16 embryos, which are homozygous for His3.3A, His3.3B and ∆HisC deletions and carry 12x H3K36R transgene (mutant), is not reduced compared to their wild‐type counterparts (control 1) or embryos heterozygous for His3.3A, and ΔHisC deletions (control 2). Histograms show the mean of the two independent experiments (n = 2) with dots indicating individual experimental results.