TAG-1–assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding

Abstract

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Ferret–mouse differences in interkinetic nuclear migration and cellular densification in the neocortical ventricular zone

“…Cross-sectional and tangential immunohistochemistry were performed as described previously (Okamoto et al, 2013). Brains were fixed with periodate-lysine-paraformaldehyde (PLP) fixative (McLean and Nakane, 1974), immersed in 20% sucrose, embedded in OCT compound (Miles), frozen, and sectioned coronally and tangentially (16 m).…”

“…For live observation, sporadic visualization was achieved by electroporation with a mixture of conditional expression plasmids, EFX-LPL-LynN-EGFP (0.5 g/l) and BA-LPL-H2B-mRFP1 (0.5 g/l), and a Cre-recombinase expression plasmid, EFX-cre (0.001 g/l), as described previously (Okamoto et al, 2013).…”

“…We recently obtained experimental evidence that acute alterations of density and/or traffic of progenitor cell nuclei within the VZ can lead to abnormal separation of a certain fraction of undifferentiated progenitors from the VZ (Okamoto et al, 2013). When we induced over-proliferation of undifferentiated (Pax6 + ) progenitors via artificial expression of Wnt3a in early embryonic mouse neocortical walls, nuclear packing density in the VZ increased, concomitant with a slight thickening of the VZ.…”

“…Similar Pax6 + heterotopia also occurred when we imposed another acute physical load on VZ cells by inhibiting their interkinetic nuclear migration (INM): progenitors that were overcrowded periventricularly as a result of insufficient basalward nucleokinesis (due to removal of their basal processes, induced by knockdown of the cell-surface molecule TAG-1) left the VZ and invaded the neuronal territory. Based on further examinations carried out using physical techniques (such as laser ablation), as well as in silico mechanical simulations, we proposed that progenitors evacuate (or are forced to exit) from the VZ in response to excessive acute mechanical stress (Okamoto et al, 2013 Those results, obtained in mice, and previous suggestions on the expansion of progenitors' proliferative zones under the influence of spatial limitations (Smart, 1965;Charvet and Striedter, 2011;Poluch and Juliano, 2013) together prompted us to wonder whether the evolutionary emergence of OSVZ progenitors might have been influenced by mechanical factors within the VZ. Vertical mitotic spindle orientation (and resultant cytokinesis perpendicular to the apical surface) can contribute to the supply of non-apically-connected VZ cells that move basally and eventually adopt an oRG-like morphology (Konno et al, 2008;Postiglione et al, 2011;Shitamukai et al, 2011;LaMonica et al, 2013); therefore, regulation of the cleavage orientation of stem-like cells at the apical surface may underlie the evolutionary changes that have generated OSVZ.…”

“…(4) The average area of individual endfeet comprising the apical surface was smaller in ferrets. Furthermore, we performed mean-squared displacement (MSD) analysis (Norden et al, 2009;Leung et al, 2011;Okamoto et al, 2013) on nuclear movements, and found that (5) both apicalward and basalward INM phases in ferret VZ are quite different from those observed in mice.…”