Fig. S1. Notch4* repression occurs within 24hrs of doxycycline treatment
Frozen sagittal brain sections with fluorescent immunostaining for Notch4 intracellular domain, with labeling of perfused vessels by tomato lectin staining, and nuclear labeling by DAPI. Images show overlap of Notch4 stain with DAPI in cells lining lectin-perfused vessels. Isolation and amplification of the Notch4 staining shows light Notch4 signal in the control vessel nuclei, strong expression in the vessels of mutants before Notch4* suppression (0hrs Notch4*-Off), and light expression after 24hrs doxycycline treatment (24hrs Notch4*-Off). Graph shows quantification of fluorescent intensity in endothelial cell nuclei of brain vasculature in Notch4* mutants before and after Dox treatment, and their littermate controls. Error bars represent s.d. between individual animals (n=6 at 0hrs Notch4*-Off, n=6 at 24hrs Notch4*-Off, and n=6 controls). In each animal, three nuclei in each of three vessels were quantified to yield an average intensity. *P=0.0007.
Fig. S2. Placement of the chronic imaging window
Perfusion of the whole brain with fluorescein-labeled agarose shows pial arteries (arrowheads) and the typical placement of the window (boxed area).
Fig. S3. Normal blood velocities in artery, capillary and vein in wild-type mice
Two-photon timelapse imaging of cortical brain vessels through cranial window in wild-type mouse. Plasma labeling was provided by intravenous FITC-dextran. (A) Line depicts the path of blood from artery through arteriole, capillary, venule and vein. (B) Images of the artery, capillary and vein in which blood velocity was measured by line scan along the axis depicted. Diameters of the vessels were measured transaxially. (C) Velocity tracing, as calculated from line scans. Note that both velocity and the pulse (the range in velocity) were reduced from artery to capillary to vein.
Fig. S4. Specific regression of AV shunts after repression of Notch4*
Two-photon timelapse imaging of cortical brain vessels through cranial window in Notch4* mutant mice. Plasma labeling was provided by intravenous FITC-dextran. (A) Regression of existing AV shunts following 48hrs of Notch4* repression (B) Regression did not occur without Notch4* repression. Note the reduction in the size of the distal vessels to the enlarging AV shunt in B. Scale bars = 100μm.
Fig. S5. Repression of Notch4* induces regression of well-established large AV malformation
Two-photon timelapse imaging of cortical brain vessels in Notch4* mutant mice after well-established AV malformation. Vessel topology was visualized by intravenous FITC-dextran. Windows were placed on mice demonstrating ataxic symptoms, corresponding with advanced-stage disease. The diameters of large AV shunts were reduced following repression of Notch4* (n = 20 AV shunts in 3 mice). The majority of regression occurred rapidly within the first week and continued to two weeks. Detection of FITC-dextran-perfused capillaries adjacent to AV malformations increases over time. Scale bars = 100μm.
Fig. S6. AV shunts are stable until repression of Notch4*
(A–C) Color-coded depiction of the AV shunt imaged by two-photon timelapse imaging in (D–F), arteries are in red and veins are in blue. AV shunt remained for 24 hours without repression of Notch4* (Compare D and E), but regressed with 48hrs of Notch4* repression (Compare E and F). Bottom panels show velocity traces obtained by line scan in the vessels indicated by yellow or green arrows (A–C). Velocity in AV shunt feeding artery (green) decreased, while velocity in the non-AV shunt artery increased (yellow), during regression of the AV shunt. Figure is representative of results in 10 AV shunts in 8 mice. Scale bars = 100μm.
Fig. S7. Regression of AV shunts to capillary diameter vessels in mice with and without cranial window
(A) Two-photon timelapse imaging of cortical brain vessels through cranial window in Notch4* mutant mouse. Plasma labeling was provided by intravenous FITC-dextran. Large AV shunt regressed to capillary diameter within 9 days of Notch4* repression. (B) Ex vivo imaging of whole mount cortex from mice perfused with FITC-lectin. Prior to Notch4* repression, large AV shunts were observed between artery and vein. In a similarly affected littermate mutant, following 28 days of Notch4* repression, AV connections were reduced to the diameter of capillaries in littermate control mice. n=3 mutants before Notch4*-Off, n=5 littermate mutants after Notch4*-Off, n=5 controls. Scale bars = 100μm
Fig. S8. Smooth muscle coverage is normalized by suppression of Notch4*.
Whole mount immuno-staining of cerebral cortex. VE-cadherin staining shows vasculature and outline of individual ECs. α-Smooth muscle actin staining shows smooth muscle cell coverage and alignment. Note the reduction in smooth muscle cell coverage in the vein after repression of Notch4*, relative to the littermate mutant before Notch4* repression. n=3 Notch4*-On mutants, n=5 Notch4*-off mutants, n=5 controls. Scale bars = 200μm.
Fig. S9. Velocity changes coincide with narrowing of AV shunts and distal vein beginning by 12–24hrs after Notch4* repression
In vivo timelapse imaging of Tie2-tTA; TRE-Notch4*; Tie2-cre; mT/mG mice (green endothelial fluorescence), with TRITC-dextran labeling of plasma (red). (A) With repression of Notch4*, AV shunt regression began between 12 and 24hrs, coinciding with a reduction in blood velocity through the AV shunt. (B) Without repression of Notch4*, AV shunt diameter and velocity were maintained. Note the reduction of distal vessel size. Blood flow was measured in this distal vessel, and was reduced over time.
Fig. S10. Narrowing of ephrinB2-GFP+ AV shunt occurs specifically after Notch4* repression
(A–C) Two-photon time-lapse imaging through a cranial window following repression of Notch4* in Tie2-tTA; TRE-Notch4*; ephrinB2+/H2B-eGFPmutant with labeling of plasma by Texas-Red dextran. An AV shunt showed little change in diameter or centerline velocity (bottom left corner) after 24hrs without repression of Notch4* (B). Within 24hrs of Notch4* repression (C), the AV shunt was greatly reduced in diameter, with a reduction in blood velocity. Note that the same number of cells were present, but grouped closer. Scale bars = 100μm.
Fig. S11. Tie2-tTA; TRE-H2B-eGFP marks brain endothelial cells.
Imaging of GFP+ endothelial cells from sectioned brain specimens of Tie2-tTA; TRE-GFP; TRE-Notch4* mice perfused with Cy5 bound tomato-lectin. Endothelial nuclei were identified by co-localization of DAPI staining and Cy5 signal in cells along the wall of patent vessel lumens. Nuclei fully enveloped by Cy5 signal were considered endothelial cells (closed arrow heads) and other vascular cells were not (open arrow heads). Triple-color merged image showing DAPI, GFP+ nuclei, and Cy5-labeled endothelium. Two-color merged imaged showing GFP+ nuclei and Cy5-labeled endothelium. Scale bars = 20μm. Quantification of GFP+ endothelial cells from 446 analyzed nuclei in 3 mice.
Fig. S12. Loss of endothelial cells is not required for AV shunt regression
(A) The table shows the number of AV shunts, over time (columns), binned by the percentage of starting diameter (rows). The bottom panel shows average diameter of all 38 AV shunts and corresponding s.e.m. by percentage of starting diameter. (B) Loss of TRE-H2B-eGFP marked cells in AV shunt regression. The table shows the number of AV shunts, over time (columns), binned by the percentage of retained cells (rows). While individual AV shunts displayed variability, initiation of regression by 12hrs usually exhibited no cell loss. Cell number trended downward over time, though many AV shunts retained most or all of their GFP+ endothelial cells. (C) There was no apparent correlation between the percentage of cell loss and the degree of shunt regression by 28hrs. (D) There was no apparent correlation between the percentage of cell loss by 28 hours and the starting AV shunt diameter.
Fig. S13. Endothelial cells are narrowed in regressing AV shunts
(A–D) Fluorescent immunostaining of VE-cadherin in Notch4* mutant mice, imaged in whole mount. Areas shown in A&B were imaged by confocal microscopy at high magnification. Note that the dimensions of endothelial cells were reduced both axially and paraxially along the AV shunt. EC area was reduced (n=4 shunts in one 0hrs Notch4*-Off mouse: 546μm2 ± s.d. 193μm) vs. (n=4 shunts in each of two 48hrs Notch4*-Off mice: 239μm2 ± s.d. 68μm2 and 211.4μm2 ±s.d. 60μm2).