Abstract
ABSTRACTCellular generation of mechanical forces required to close the presumptive spinal neural tube, the ‘posterior neuropore’ (PNP), involves interkinetic nuclear migration (INM) and apical constriction. Both processes change the apical surface area of neuroepithelial cells, but how they are biomechanically integrated is unknown. Rho kinase (Rock; herein referring to both ROCK1 and ROCK2) inhibition in mouse whole embryo culture progressively widens the PNP. PNP widening is not caused by increased mechanical tension opposing closure, as evidenced by diminished recoil following laser ablation. Rather, Rock inhibition diminishes neuroepithelial apical constriction, producing increased apical areas in neuroepithelial cells despite diminished tension. Neuroepithelial apices are also dynamically related to INM progression, with the smallest dimensions achieved in cells positive for the pan-M phase marker Rb phosphorylated at S780 (pRB-S780). A brief (2 h) Rock inhibition selectively increases the apical area of pRB-S780-positive cells, but not pre-anaphase cells positive for phosphorylated histone 3 (pHH3+). Longer inhibition (8 h, more than one cell cycle) increases apical areas in pHH3+ cells, suggesting cell cycle-dependent accumulation of cells with larger apical surfaces during PNP widening. Consequently, arresting cell cycle progression with hydroxyurea prevents PNP widening following Rock inhibition. Thus, Rock-dependent apical constriction compensates for the PNP-widening effects of INM to enable progression of closure.This article has an associated First Person interview with the first authors of the paper.
Highlights
Abnormalities in embryonic cellular biomechanics are increasingly recognised as underlying congenital structural malformations in organ systems, including the heart (Hoog et al, 2018), eye (Hosseini et al, 2014; Oltean et al, 2016), joints (Singh et al, 2018) and central nervous system (Galea et al, 2017, 2018)
To minimise the potential for secondary changes owing to prolonged culture, we first characterised the morphological changes caused by 8 h of Rho-associated kinase (Rock) inhibition with the extensively used compound Y27632 in embryonic day (E)9–9.5 CD1 mouse embryos
These morphometric studies reveal tissue shape changes caused by 8 h of Rock inhibition, of which posterior neuropore (PNP) widening is the most marked, for which biomechanical mechanisms were further investigated
Summary
Abnormalities in embryonic cellular biomechanics are increasingly recognised as underlying congenital structural malformations in organ systems, including the heart (Hoog et al, 2018), eye (Hosseini et al, 2014; Oltean et al, 2016), joints (Singh et al, 2018) and central nervous system (Galea et al, 2017, 2018). Mechanical forces must be generated to change the shape of embryonic structures into the. Received January 2019; Accepted May 2019 presumptive organs. These forces may be generated non-cellautonomously, such as during osmotic swelling of the lumen of the closed neural tube, the embryonic precursor of the brain and spinal cord (Desmond and Jacobson, 1977). The best studied force-generating mechanism is apical constriction of epithelial cells, which requires recruitment of non-muscle myosin motor proteins, such as myosin-II, onto the apical F-actin cytoskeleton. When coordinated across an epithelium, this causes tissue bending (Nishimura et al, 2012)
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