Fig. 5.

Endothelial cells forming the additional network in BLEC-depleted fish represent a distinct endothelial cell population with lymphatic vessel features. (A) Confocal images of a control brain with BLECs (mrc1a:mCitrine) and meningeal blood vessels (kdrl:mCherry), and a brain from a T-DM1-injected fish with the endothelial network (mrc1a:mCitrine) and meningeal blood vessels (kdrl:mCherry). Meninges of control and T-DM1-treated fish were FACS sorted into two different plates based on their mCitrine and mCherry signals (four groups: negative cells, mCit⁺ cells, mCherry⁺ cells and double positive mCit⁺;mCherry⁺ cells). Control plate: 203,627 live cells; T-DM1 plate: 286,098 live cells. (B,C) The scRNA sequencing data were analyzed with the Seurat pipeline, revealing eight clusters. The BLECs cluster is exclusively composed of cells from the control plate, whereas the network/BEC cluster is mainly composed of cells from the T-DM1-treated plate. (D,E) Distribution of the FACS-sorted cells with respect to their transgene expression and origin: control (D) and T-DM1-treated (E). (F) Cluster 1 subanalysis. Location of cells from pagoda2 algorithm sub-clustering in the original UMAP space. Analysis with Seurat pipeline (20 dim). Differentially expressed (DE) gene analysis of the sub-clusters 1 and 3 (network) versus sub-cluster 2 (BECs) (log2FC>1). (G) DE gene analysis of sub-clusters 1 and 3 (network) versus cluster 7 (BLECs) (log2FC>1). (H) Dot plot indicating expression levels of selected BLEC, LEC and BEC markers (y-axis) in BECs, network cells and BLECs. Color scale represents average expression; point size is relative to the percentage of cells expressing the gene. (I) Analysis of network cells (sub-clusters 1 and 3) in terms of their BEC and LEC identity shows that cells in the network cluster exhibit significantly higher expression of genes typically found in LECs. A list of marker genes indicative of either the BEC or LEC phenotype was generated based on a dataset from Grimm et al. (2023). The network cells were profiled by applying these gene lists, and the per-cell index is shown (see Materials and Methods for details). A value of 1 indicates expression of all analyzed BEC or LEC marker genes in a given cell. (J) Differentially expressed genes in network cells versus BLECs. (K) Differentially expressed genes in network cells versus meningeal BECs.