Abstract
Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.
Highlights
Mutations in the occludin (OCLN) gene cause a recessively inherited severe human disorder of microcephaly and band-like calcifications with polymicrogyria (BLC-PMG) characterized by loss of cortical convolutions, shallow or absent sulci, and multiple small gyri giving the cortex surface a roughened irregular appearance (Abdel-Hamid et al, 2017; O’Driscoll et al, 2010; Jenkinson et al, 2018; Aggarwal et al, 2016; Elsaid et al, 2014)
OCLN localizes to interphase and mitotic centrosomes in embryonic mouse cortex It is widely held that OCLN functions in tight junctions and its expression in the embryonic cortex is limited to neuroepithelial (NE) junctions (Gotz and Huttner, 2005) prior to the NE-to-radial glial cell (RGC) transition at the onset of neurogenesis, at which point OCLN expression is believed to be turned off (AakuSaraste et al, 1996; Sahara and O’Leary, 2009; Gotz and Huttner, 2005)
By E14.5, mouse OCLN (mOCLN) was absent at the cell membrane, in accordance with previous studies in which tight junction (TJ) are replaced by adherens junctions as neurogenesis begins (Aaku-Saraste et al, 1996)
Summary
Mutations in the occludin (OCLN) gene cause a recessively inherited severe human disorder of microcephaly and band-like calcifications with polymicrogyria (BLC-PMG) characterized by loss of cortical convolutions, shallow or absent sulci, and multiple small gyri giving the cortex surface a roughened irregular appearance (Abdel-Hamid et al, 2017; O’Driscoll et al, 2010; Jenkinson et al, 2018; Aggarwal et al, 2016; Elsaid et al, 2014). We use human and mouse models of corticogenesis to explore the role of OCLN in the developing cortex, to investigate its potential interaction with the centrosome and elucidate mechanisms through which its loss-offunction produces microcephaly. We use mouse and human models to show that OCLN functions in cortical development, playing a previously unappreciated role in neural progenitor proliferation through promoting centrosomal and mitotic spindle integrity. Specific loss of the full-length OCLN isoform results in altered spindle and astral microtubules, prolonged M-phase, premature cell cycle exit and early neuronal differentiation. These defects are consistent with observed microcephaly and PMG associated with human OCLN mutations
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