Fig. 1: Pia mater and arachnoid mater cells can be identified by VE-cadherin-GFP expression.

A Schematic representation of the meningeal layers on the surface of the spinal cord. B–L Representative images of the cervical spinal cord and brain as observed by 2P-IVM imaging of VE-cadherin GFP reporter mice via a cervical spinal cord window and skull thinning preparation, respectively. The dura mater is visible by the second-harmonic generation (blue) due to its richness in collagen type 1. VE-cadherin-GFP is shown in green. The subarachnoid space (SAS) is bordered by the VE-cadherin-GFP⁺ arachnoid and pia mater. VE-cadherin-GFP also marks blood vessels. The lumen of the spinal cord blood vessels is visible in red (B, D–G) due to systemic injection of TRITC-BSA. In the skull thinning preparation (I–L) the SAS is visible in red due to a cisterna magna injection of 10 kDa-TRITC dextran. Data are representative of three different mice. B YZ MIP of the 2P-IVM of the cervical spinal cord. Below the dura mater, a GFP signal is visible at the expected level of the arachnoid mater. The SAS is breached by VE-cadherin-GFP⁺ trabeculae (white arrowhead). A GFP signal can be seen below the SAS at the expected level of the pia mater. The lumen of the dorsal vein (DV) and subarachnoid blood vessels (red) ensheathed by VE-cadherin-GFP⁺ endothelial cells are visible. C YZ maximum intensity projection (MIP) of 2P-IVM of the meningeal layers on the surface of the brain of a VE-cadherin-GFP mouse. Within the dura mater, VE-cadherin-GFP⁺ blood vessels are visible. Below the dura mater, a GFP signal highlights the expected level of the arachnoid mater (AM). The VE-cadherin GFP signal can also be seen at the expected level of the pia mater. D Cervical spinal cord window preparation. YZ MIP of 2P-IVM of the meningeal layers on the surface of the spinal cord of a VE-cadherin-GFP mouse highlighting the arachnoid (A.M.) and pia mater (P.M). E XY MIP of the meningeal layers of the spinal cord. VE-Cadherin GFP⁺ AJs on the endothelial cells are visible on the walls of the dorsal vein and branching vessels. The blood vessel lumen is visible in red. Additional VE-cadherin GFP signal is visible outside the blood vessels. F XY MIP of the arachnoid mater level of the spinal cord from the region highlighted as A.M. from D. VE-cadherin GFP⁺ signal with junctional properties and no distinct cellular morphology that is not associated with blood vessels (red) is seen. G XY MIP of the pia mater from the region highlighted as P.M. from D. Large cells with VE-cadherin GFP⁺ AJ not associated with blood vessels (in red) are visible. H The skull thinning preparation and a schematic representation of the imaged anatomical location of the brain surface is shown. YZ MIP of 2P-IVM of the meningeal layers on the surface of the brain of a VE-cadherin-GFP mouse highlighting the dura, arachnoid (A.M.), and pia mater (P.M). I XY MIP of the meningeal layers of the surface of the brain is shown. VE-cadherin-GFP⁺ blood vessels are visible. Additional VE-cadherin GFP signal is visible outside the blood vessels. Perivascular red tracer is visible in the SAS. J XY MIP of the dura mater of the brain from the region. Remnants of the skull bone and the dura mater are visualized by the second-harmonic generation (blue). The VE-cadherin GFP⁺ AJs in the blood vessels of the dura mater are visible. White arrowheads point to non-endothelial VE-cadherin-GFP⁺ cells directly below the second-harmonic generation, possibly representing individual dural border cells. K XY MIP of the arachnoid and pia mater of the brain from the region highlighted as A.M. + P.M. VE-cadherin-GFP⁺ AJs in the blood vessels is also clearly visible. VE-cadherin-GFP⁺ signal with no association with blood vessels is also visible. Cisterna magna infusion of a 10 kDa TRITC-Dextran allows for visualization of the SAS along a meningeal artery. L 2P-IVM imaging after skull thinning in a VE-cadherin-GFP mouse cisterna magna infused with a 10kDa-TRITC dextran (red) and systemically injected with a 10kDa-AF647 dextran (white). The VE-cadherin-GFP⁺ signal is shown in green. Second-harmonic generation is shown in blue. DV dorsal vein, A.M. arachnoid mater, P.M. pia mater, SAS subarachnoid space, BV blood vessel.