Differences in the Mechanical Properties of the Developing Cerebral Cortical Proliferative Zone between Mice and Ferrets at both the Tissue and Single-Cell Levels

Abstract

Cell-producing events in developing tissues are mechanically dynamic throughout the cell cycle. In many epithelial systems, cells are apicobasally tall, with nuclei and somata that adopt different apicobasal positions because nuclei and somata move in a cell cycle–dependent manner. This movement is apical during G2 phase and basal during G1 phase, whereas mitosis occurs at the apical surface. These movements are collectively referred to as interkinetic nuclear migration, and such epithelia are called “pseudostratified.” The embryonic mammalian cerebral cortical neuroepithelium is a good model for highly pseudostratified epithelia, and we previously found differences between mice and ferrets in both horizontal cellular density (greater in ferrets) and nuclear/somal movements (slower during G2 and faster during G1 in ferrets). These differences suggest that neuroepithelial cells alter their nucleokinetic behavior in response to physical factors that they encounter, which may form the basis for evolutionary transitions toward more abundant brain-cell production from mice to ferrets and primates. To address how mouse and ferret neuroepithelia may differ physically in a quantitative manner, we used atomic force microscopy to determine that the vertical stiffness of their apical surface is greater in ferrets (Young's modulus = 1700 Pa) than in mice (1400 Pa). We systematically analyzed factors underlying the apical-surface stiffness through experiments to pharmacologically inhibit actomyosin or microtubules and to examine recoiling behaviors of the apical surface upon laser ablation and also through electron microscopy to observe adherens junction. We found that although both actomyosin and microtubules are partly responsible for the apical-surface stiffness, the mouse<ferret relationship in the apical-surface stiffness was maintained even in the presence of inhibitors. We also found that the stiffness of single, dissociated neuroepithelial cells is actually greater in mice (720 Pa) than in ferrets (450 Pa). Adherens junction was ultrastructurally comparable between mice and ferrets. These results show that the horizontally denser packing of neuroepithelial cell processes is a major contributor to the increased tissue-level apical stiffness in ferrets, and suggest that tissue-level mechanical properties may be achieved by balancing cellular densification and the physical properties of single cells.

Tissue mechanics regulate mitotic nuclear dynamics during epithelial development

“…We use live imaging of the developing wing disc, in combination with analysis of fixed samples where the cell cycle stage can be unambiguously determined. We confirm through the combinatorial use of live and fixed analysis that in the wing disc, nuclear movement in mitosis, rather than G2 [25,28,29], achieves apical mitotic positioning [26] (Figure 2). Furthermore, our fixed-tissue analysis of mitotic nuclear position recapitulates developmental stage specific differences in mitotic nuclear dynamics ( Figures 1E'-E '', 2 and S1G'-G ''), thereby allowing us to…”

“…We show that mitotic nuclear dynamics change as development progresses and tissue architecture changes (Figures 1 and 2). Previous studies had suggested that tissue density might influence IKNM in the crowded, vertebrate cortex PSE [28,29].…”

“…This idea is supported by evidence for mechanical regulation of cell differentiation in the developing zebrafish neural tube, where gradual apical nuclear crowding alters the positioning of progenitor nuclei and thus their exposure to signalling molecules [30]. To what extent, and how tissue properties influence nuclear dynamics remains unclear, as currently, only epithelia from different species have been directly compared [28,29].…”

“…Even though previous studies have mostly focused on the processes controlling IKNM locally, in individual cells, comparative studies of PSE suggest that IKNM dynamics might also be influenced by tissue architecture. Comparison of IKNM in the mouse and ferret cortex, show a reduced rate of apical motion in denser and stiffer tissue [28,29]. Furthermore, faster apical nuclear movements are observed in the developing zebrafish hindbrain compared to the retina, which differ in tissue shape [25].…”

Two‐photon microscopic observation of cell‐production dynamics in the developing mammalian neocortex in utero

“…In three-dimensional environments, cell production via mitosis depends not only on the progression of the cell cycle but also on the movement of cells' nuclei/somata into place for mitosis, making the overall cell-production events highly temporally and spatially dynamic (Keller, Schmidt, Wittbrodt, & Stelzer., 2008;Kurotaki, Hatta, Nakao, Nabeshima, & Fujimori, 2007;Miyata, 2008;Norden, Young, Link, & Harris, 2009;Taverna & Huttner, 2010). Recent studies using slice culture-based imaging and mechanical assessment of developing mammalian brain walls have shown that such dynamic cytogenetic events occur at a very high density under physiologically crowded cellular conditions (Miyata, Okamoto, Shinoda, & Kawaguchi, 2015;Nagasaka et al, 2016;Okamoto et al, 2013;Okamoto, Shinoda, Kawaue, Nagasaka, & Miyata, 2014;Saito, Kawasoe, Sasaki, Kawaguchi, & Miyata, 2018;Shinoda et al, 2018); the studies also show that delays in the cell cycle or nuclear migration in a subpopulation of progenitors can easily lead, in a cell non-autonomous manner, to secondary (more widespread) disorganization of histogenesis (Okamoto et al, 2013;Watanabe, Kawaue, & Miyata, 2018).…”