Abstract
In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.
Highlights
The ventricular layer (VL) of the embryonic spinal cord is composed of pseudostratified radial glial stem/progenitor cells that line the central lumen
We propose that collapse is initiated by the release of secreted CRB2 (CRB2S) from dmNes+radial glia (RG) that acts on transmembrane CRB2 (CRB2TM)-expressing VL cells, causing down-regulation of polarity and junctional proteins and their decreased cohesion
4E@ and S5D Fig), and continuous labelling at E17 (Fig 4J). These data suggest that apical adhesion/tight junction proteins are retained on dmNes+RG throughout collapse but are reduced on dorsal ventricular layer (dVL) progenitor cells; in particular, they are absent (Zo-1, atypical protein kinase C (aPKC), PAR3) or markedly reduced (CRB2) from the dVL cells that are delaminating
Summary
The ventricular layer (VL) of the embryonic spinal cord is composed of pseudostratified radial glial stem/progenitor cells that line the central lumen. The finding in zebrafish that mutation in pard6yb results in the failure of dorsal collapse [45,46] suggests that apical polarity complex regulation plays a critical role in VL remodelling. DmNes+RG are rich in secretory vesicles, and gain-of-function in vivo studies show that they secrete a CRB2 required for remodelling of ventricular layer to ependymal layer factor that promotes progenitor cell delamination They express a variant of CRB2 that can be secreted (CRB2S) and appears to mediate this activity. We propose that collapse is initiated by the release of CRB2S from dmNes+RG that acts on CRB2TM-expressing VL cells, causing down-regulation of polarity and junctional proteins and their decreased cohesion We suggest that these are a critical step in the exclusion of these cells from the VL and transformation of the VL to the EL. Our findings suggest a model in which CRB2S acts cell non-autonomously to orchestrate progenitor cell delamination from an epithelium through a mechanism that retains epithelial integrity
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