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William J Brunken, Dmitri Serjanov, Galina Bachay, Dale D Hunter; Laminin β2 Chain Regulates Retinal Progenitor Cell Mitotic Spindle Orientation Via Dystroglycan. Invest. Ophthalmol. Vis. Sci. 2018;59(9):586.
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© ARVO (1962-2015); The Authors (2016-present)
The orientation of cytokinesis plays an important role in controlling timely cell cycle exit and reentry of the retinal progenitor cells (RPCs) during development. Here, we demonstrate that extracellular matrix (ECM) laminins provide orientation cues to the RPC regulating basal process (BPs) formation and concomitantly mitotic spindle orientation and proliferation. We have identified the laminin receptor, dystroglycan (DG), as the key mediator of this signaling.
IHC was performed on WT and laminin β2-/- retinas using markers for centrosome, mitosis, DG, integrin β1 (intβ1), and phospho-Vimentin. RPC mitotic spindles were visualized via 3D-reconstructions and spindle angles were calculated. DG and intβ1expression patterns were assayed and quantified. Organotypic cultures from P0 mice were used for ex vivo experiments; addition of recombinant β2-containing laminins was used to rescue Lamb2 deletion while DG or intβ1 function-blocking antibodies were used to block DG-mediated signaling in WT cultures.
Lamb2 deletion results in a significant shift of the RPC mitotic spindle pole orientation towards asymmetric (neurogenic) cell divisions (p≤0.01). This is accompanied by reduction (56%; p≤0.01) in the incidence of RPCs with BPs. We show that RPCs with BPs divide symmetrically, while those without BPs divide stochastically. The shift to fewer BP-bearing-RPC is accompanied by a reduction in proliferation and premature RPC pool depletion (-70%; at P5, p≤0.01) with a concomitant reduction in bipolar cells (-38%; p≤0.002) and Müller cells (-39%; p≤0.003). In vivo and ex vivo the expression of DG and intβ1 was decreased at the vitreal surface in Lamb2-/- retinas. Exogenous laminin 521 restored the normal receptor distribution, spindle orientation, BPs, and increased RPC proliferation ex vivo while treating WT retina with DG-blocking antibodies phenocopied laminin β2 deletion.
These data demonstrate that b2-containing laminins at the ILM regulate critical developmental patterning by regulating ECM-RPC contact, matrix receptor expression, and RPC cytokinesis dynamics and proliferation. The data identify DG as mediating Lamb2 signaling events. This is the first report of dystroglycan signaling affecting mitotic spindle orientation. Our data suggest a mechanism by which contact with the BM modulates RPC proliferation and fate choice.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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