Pial collaterals form between E14.5 and P21, have a continuous lumen with arteries and recruit mural cells postnatally.

Immunofluorescence staining against ICAM2 (green), aSMA (magenta) and CD93 (blue) was performed throughout the pial arterial development. Scale bar: 100μm. A. At E12.5, the MCA trunk and main branches are lumenized and show mural cell progenitor recruitment. B. First collateral vessels are observed on the pial surface at E14.5, appear lumenized (ICAM2) arching over a dense venous plexus (CD93) and are devoid of aSMA coverage. C. At E16.5, pial collateral network is more complex, still intimately connected to the underlying plexus (now also ICAM2 positive) and still aSMA-negative. D. At P1, pial collateral network is clearly a part of the superficial arterial circulation (ICAM2), shows a more complete aSMA-coverage and lies on top of a very dense venous plexus. E. At P21, mature pial collaterals display an even more complete aSMA-coverage and are now surrounded by well-defined veins, venules and the residues of the pial plexus (CD93).

Mature collaterals are mosaic in origin: composed of mostly Bmx-(arterial) and, to a lesser degree, Vegfr3-(microvascular) endothelial lineage.

Pregnant dams were injected with tamoxifen at E13.5: samples were harvested at E18.5 (A,B) or P21 (C,D), as indicated in the experimental scheme (top left) and immunolabeled against ICAM2, GFP and endothelial nuclear marker, ERG. Scale bars: 100um. GFP (lineages) are shown separately under each merged image. A. E18.5 BmxCreERT;R26mTmG pial collaterals. Bmx-lineage cells (GFP) label the majority of the nascent collateral. B. E18.5 Vegfr3CreERT;R26mTmG pial collaterals. Some of the Vegfr3-lineage ECs (GFP) are embedded in the forming collaterals and in contact with the lumen (indicated by white arrows), while most of them belong to the plexus. C. P21 BmxCreERT;R26mTmG pial collateral. Embryonic (E13.5) Bmx-lineage labeling of the mature collateral. D. P21 Vegfr3CreERT;R26mTmG pial collateral. Embryonic Vegfr3-lineage labels fewer cells in mature collaterals. E. Percentage of Bmx- or Vegfr3-lineage, quantified as the number of double positive GFP+ERG+ cells over all ERG+ cells within embryonic collaterals (E18.5) F. Percentage of Bmx- or Vegfr3-lineage, quantified as the number of double positive GFP+ERG+ cells over all ERG+ cells within mature collaterals (P21). N≥3 animals. Mean and SD are calculated using average data points per animal. For transparency reasons, all data points are represented as scatter dot plots and individual animals are color-coded.

Postnatal growth of collaterals involves mostly Bmx-lineage.

Pups were injected with tamoxifen at P1 and samples were collected at P21 and immunostained for GFP (green), ICAM2 (magenta) and ERG (blue), as indicated in the experimental scheme (top left). White dotted lines represent individual collaterals (A,B). Postnatal Bmx-lineage (GFP) labels most of mature collateral vessels and arteries (A, C) whereas postnatal Vegfr3-lineage contributes much less to collaterals (B,D) and is mostly located around veins and venules at P21. Contribution of each lineage was quantified as the number of GFP+ERG+ within all ERG+ cells in mature collaterals and depicted in E. Percentage of Bmx- or Vegfr3-lineage, quantified as the number of double positive GFP+ERG+ cells over all ERG+ cells within mature collaterals. N=3 animals, Data are mean ±SD. Mean and SD are calculated using average data points per animal. For transparency reasons, all data points are represented as scatter dot plots and individual animals are color-coded.

Bmx- and Vegfr3-lineage cells appear to be actively migrating within already existing capillaries.

Bmx- and Vegfr3-lineage ECs were traced from E13.5 - E16.5 and E13.5 - E18.5, as shown in the experimental plan (upper left). GFP (green), ICAM2/CD93/Tomato (magenta). GFP-channel is shown separately A and B, respectively. White dotted lines represent each individual pre-collateral A. E16.5 BmxCreERT;R26mTmG pial pre-collaterals. B. E16.5 Vegfr3CreERT;R26mTmG pial pre-collaterals. C. E18.5 BmxCreERT;R26mTmG pial pre-collaterals. D. E18.5 Vegfr3CreERT;R26mTmG pial pre-collaterals. Blue dotted lines indicate vascular segments that are enlarged in the lower panel of C, D.

Bmx- and Vegfr3-lineage ECs mark pre-collateral vessels which are undergoing arterialization.

Bmx- and Vegfr3-lineage ECs were traced from E13.5 - E16.5. GFP (green), Cx40 (magenta), ICAM2/CD93/Tomato (blue). ECs derived from Bmx-lineage were found in wider arterial segments which showed Cx40-positive staining and in narrower pre-collateral segments (faintly Cx40-positive) - indicated by white arrowheads (A,C,E). ECs derived from Vegfr3-lineage were found both in vessel segments with weak Cx40 staining (white arrowhead) and Cx40-negative vessel segments (yellow arrowhead) (B,D,E).

Arterial cells proliferate during collateral formation.

EdU injections were performed at E16.5 and E18.5. Upper panel shows ICAM/Tomato (gray) and EdU (yellow) staining, middle panel shows ICAM/Tomato (gray) and ERG (magenta) staining and lower panel shows ERG (magenta) and EdU (yellow) labeling. At E16.5 there were double positive cells (EdU+ERG+) in pial arteries and the surrounding plexus (A). At E18.5, we also found proliferating endothelial cells (EdU+ ERG+) in both arteries and plexus. C. Quantification of proliferating ECs at E16.5. D. Quantification of proliferating ECs at E18.5. N=3 animals per stage. Mean and SD are calculated using average data points per animal. For transparency reasons, all data points are represented as scatter dot plots and individual animals are color-coded.

After stroke (dMCAO), collateral outward remodeling happens through proliferation of collateral (local) and nearby arterial endothelial cells and not through recruitment of microvascular cells.

To combine lineage tracing and dMCAO, adult (8 - 12 weeks old) mice lineage-labeled at P1 underwent a dMCAO/sham procedure. Following operations, animals were injected with EdU daily for the next 7 days and then sacrificed. GFP (green), ICAM/Tomato (magenta) and EdU (gray). White dotted lines represent each individual collateral. Collaterals in animals with dMCAO underwent a massive remodeling, marked by their bulging morphology and many proliferating ECs in both lines (A,D). Collaterals from the sham BmxCreERT;R26mTmG animals or contralateral hemisphere of dMCAO animal group displayed normal morphology, GFP and few proliferating ECs (B,C). MCA branches directly upstream of the remodeling collateral showed marked EC proliferation in the dMCAO BmxCreERT;R26mTmG animals (F) and no proliferation in the sham BmxCreERT;R26mTmG animals (E). G. Quantification of EdU+ endothelial cells in ipsilateral collateral vessels (collaterals stroke) and sham control collateral vessels (collaterals sham). H. Quantification of EdU+ endothelial cells in ipsilateral upstream MCA branches (arteries stroke) and sham upstream MCA branches (arteries sham). N=2 animals for sham (technical control) and N=5 for stroke animals. Mean and SD are calculated using average data points per animal. For transparency reasons, all data points are represented as scatter dot plots and individual animals are color-coded.