Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors

Abstract

SUMMARYMitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10°/minute) and migrate (3 m/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

Ci-Pem-1 localizes to the nucleus and represses somatic gene transcription in the germline of Ciona intestinalis embryos

“…S6C,D in the supplementary material) RNAs were readily detected as a subcortical crescent in the posterior-most region of the germline blastomere pair in 16-cell stage embryos, indicating that Ci-Pem-1 is dispensable for the asymmetric inheritance of postplasm by the germline blastomeres during the cleavage stages. These results are consistent with the effects of pem-1 KD in other ascidian species (Negishi et al, 2007;Prodon et al, 2010), and suggest that roles of Pem-1 during the cleavage stages are conserved in ascidians.…”

“…The postplasmic/PEM RNAs are packed into a specialized structure in the postplasm, called the centrosome-attracting body (CAB), which is connected to the centrosome through microtubules. The CAB was initially identified as an electron-dense cortical structure in detergent-treated cleavage-stage embryos (Iseto and Nishida, 1999;Hibino et al, 1998;Nishikata et al, 1999), and it is now known to be a multilayered structure, consisting of an outer layer enriched in the polarity protein complex (Patalano et al, 2006), and an inner compacted cortical endoplasmic reticulum (cER) layer, to which many postplasmic/PEM RNAs are anchored (Sardet et al, 2003;and asymmetric cell division of the posterior blastomeres during the cleavage stages (Hibino et al, 1998;Nishikata et al, 1999;Patalano et al, 2006;Prodon et al, 2010). It also plays a role in the fate determination of the posterior-vegetal blastomeres, by regulating the accumulation and translation of specific maternal RNAs (Nishida, 2005;Prodon et al, 2007), such as macho-1, which encodes a determinant for muscle and posterior-vegetal cells (Nishida and Sawada, 2001).…”

“…Consistent with this hypothesis, Pem-1 plays several roles in ascidian embryogenesis during the cleavage stages. The knockdown (KD) of pem-1 causes irregular cleavages of the posterior blastomeres because the centrosome-CAB linkage is interrupted, owing to collapse of the microtubule bundles that connect them (Negishi et al, 2007;Prodon et al, 2010). Furthermore, although -catenin normally accumulates in the nucleus of only the vegetal somatic blastomeres, where it regulates the expression of -catenin/TCF-dependent genes during the cleavage stages (Imai et al, 2000;Kawai et al, 2007), pem-1 KD in H. roretzi embryos causes the nuclear accumulation of -catenin in the germline blastomeres, resulting in the ectopic expression of -catenin/TCF-dependent genes, such as FoxA-a, FoxD and FGF9/16/20 (Kumano and Nishida, 2009).…”

Asymmetric division of cyst stem cells in Drosophila testis is ensured by anaphase spindle repositioning

“…We hypothesize that the slight rounding up of cells during mitosis gives enough room for the spindle to move within CySCs, and anaphase spindle repositioning ensures appropriate asymmetric divisions. This may be analogous to asymmetric cleavage in ascidian blastomeres, where spindle pole migration is coupled with spindle rotation (Prodon et al, 2010), suggesting this mechanism is conserved across species. Although the molecular mechanisms that allow cells to round up during mitosis have been investigated intensively, the reasons why cells round up in mitosis (or why cells are flattened during interphase) are less clear.…”

Cortical localization of Maternal Embryonic Leucine zipper Kinase (MELK) implicated in cytokinesis in early Xenopus embryos

“…1). The role of asymmetric furrowing is unknown, but it is encountered in divers organism including worm and ascidian embryos [21][22][23] as well as MDCKII cells cultured in vitro. 24 In the latter case, the asymmetric furrowing proceeds from the basolateral side towards the apical domain.…”