# GFAP+ FOXF2+ ependymal cells promote blood–brain barrier repair via DLL4–NOTCH signaling after neural injury

## TL;DR

A specific type of ependymal cell helps repair the blood-brain barrier after injury by using DLL4-NOTCH signaling, offering a new therapeutic target for neural injury recovery.

## Contribution

Identifies GFAP+ FOXF2+ ependymal cells as key mediators of blood–brain barrier repair via DLL4–NOTCH signaling after neural injury.

## Key findings

- GFAP+ FOXF2+ ependymal cells selectively expand in the subventricular zone after neural injury.
- Conditional deletion of Foxf2 in these cells leads to impaired blood–brain barrier integrity.
- Exosomal DLL4 secretion by GFAP+ FOXF2+ ependymal cells activates NOTCH signaling to restore barrier function.

## Abstract

Ependymal cells (EP) in the adult ventricular-subventricular zone (V-SVZ) are recognized for diverse functions beyond cerebrospinal fluid (CSF) dynamics, yet their identity, specialization, and roles in niche interactions and postinjury repair remain poorly understood. Our study identifies distinct injury-responsive ependymal subpopulations, highlighting GFAP+ FOXF2+ cells as a pivotal subset following stroke. Conditional deletion of Foxf2 in GFAP+ EP results in markedly aggravated blood–brain barrier (BBB) disruption, demonstrating their indispensable role in BBB repair mediated through DLL4-dependent Notch signaling. These findings underscore GFAP+ FOXF2+ EP as a compelling therapeutic target for enhancing BBB integrity in the context of neural injury.

Ependymal cells in the adult ventricular-subventricular zone are increasingly recognized for functions extending beyond cerebrospinal fluid dynamics; however, their identity and functional specialization remain incompletely understood. While ependymal cells (EP) have been implicated in interactions with the stem cell niche and the vasculature, their role in repair processes following neural injury remains elusive. In this study, we employed region-specific single-cell transcriptomics of the subventricular zone (SVZ) and ipsilesional peri-infarct territory in mice to identify a distinct subpopulation of GFAP+ FOXF2+ EP that selectively expand within the SVZ after neural injury. Notably, these cells were absent from other brain regions. Immunohistochemical validation revealed characteristic ependymal features, including typical pinwheel architecture and expression of Foxj1 and β-catenin. Furthermore, the absence of the proliferation marker Ki67 and the resistance of this subpopulation to Ara-C-mediated ablation indicate that these cells do not possess proliferative properties. Conditional deletion of Foxf2 in GFAP+ cells led to impaired endothelial junction integrity and increased blood–brain barrier (BBB) permeability. In contrast, overexpression of Foxf2 via GFAP promoter-driven adeno-associated virus delivery enhanced vascular repair and facilitated functional recovery. Mechanistically, these GFAP+ FOXF2+ EP secrete exosomal DLL4, which was associated with enhanced NOTCH pathway activity and restoration of BBB function. While the mechanism linking this limited cell population to the broad reparative effect, particularly the complete signaling amplification cascade, remains to be fully elucidated, these findings identify a subset of EP that contributes to BBB repair.