Figure 5

Formation of micronuclei from nuclear extrusions in gonocytes. Immunofluorescent staining of frozen tissue sections of ovaries from P. esculentus diploid females at 41 (a,b) and 44 (c–f) Gosner stages: nuclear pore complex proteins, NPC (Nup F/G, green), DNA counterstained with DAPI (blue). (a–c) Single confocal sections of DNA and NPC proteins (0.46 µm). (b,d) 3D reconstructions of natural confocal sections showing the views of germ cells from the angle to visualise chromatin budding (arrowheads). (e,f) 3D models of reconstructed fluorescent signal volumes from cell visible in c,d, (e) NPC proteins and DNA, note that NPC signal is discontinuous probably due to differential distribution of nuclear pore complexes in the nuclear envelope and threshold settings excluding cytoplasmic signal, (f) DNA. (a,b) Early stage of micronucleus formation: gonocyte with small chromatin extrusion possessing a weak NPC protein signal in nuclear envelope and strong DAPI signal (arrowhead), representing emerging micronucleus containing heterochromatin. Second micronucleus with punctate NPC protein signal (a, arrowhead). (c,d,f) Middle stage of micronucleus formation: extruded chromatin has finger-like shape and is connected with main chromatin mass in the cell nucleus (arrowhead). Note the discontinuous NPC signal in the nuclear envelope at budding site. Late stage: micronuclei (double arrowhead, a–e) are separated from the main nucleus by nuclear envelope with strong NPC protein signal (arrow), either protruding towards the micronucleus (a) or flat (c,e). There is no distinct NPC protein signal at the nuclear envelope in micronucleus (c) as well as in finger-like extrusion (arrow, c,e). Scale bars 5 µm.