ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Switch-like Behavior of Motile EC Selection in Angiogenesis In Vivo

(A and B) Time-lapse images of EC nuclei in ISVs of control (A) and dll4 KD (B) Tg(kdrl:nlsEGFP)^zf109 embryos from 19 h post-fertilization (hpf). Brackets indicate dividing cells. Nuclei are pseudocolored.

(C–E) Quantification of the number of ECs that are selected to branch (C), undergo proliferation (D), or the total number of ECs per ISV (E) in control, dll4 KD, flt1 KD, and 0.3 μM SU5416-treated embryos (n = 47 ISVs from 16 control, 78 ISVs from 24 dll4 KD, 28 ISVs from 8 flt1 KD, and 81 ISVs from 23 0.3 μM SU5416-treated embryos).

(F) Illustration of the biphasic nature of the selection of motile ECs in angiogenesis. Vegf signal levels define the number of ECs selected to branch, and Dll4-mediated LI prevents further selection of motile ECs. Increased Vegf (flt1 KD) or decreased Vegf (0.3 μM SU5416) signaling results in the selection of more or less motile ECs, respectively. In the absence of dll4, motile ECs continue to be selected.

Data are mean ± SEM. ^∗p < 0.05, two-way ANOVA test. Scale bars, 25 μm.

See also Figure S1.

Identification of Putative Positive-Feedback Modulators of Vegf Signaling

(A) Fold change in the indicated transcript levels by microarray following inhibition of Vegfr signaling (2.5 μM SU5416), Notch activity (100 μM DAPT), or both, from 22 to 30 hpf.

(B and C) Fold change in tm4sf18, kdrl, flt4, and dll4 transcript levels by qPCR in embryos incubated with 2.5 μM SU5416 for the indicated times (B) and tm4sf18 and kdrl transcript levels by qPCR upon dll4 KD (C; n = 3 separate experiments).

(D) Illustration of the putative transcriptional regulation of tm4sf18 by Vegf-Notch and proposed function as a positive-feedback modulator of Vegfr signaling.

(E) Lateral views of sprouting ISVs in Tg(kdrl:GFP)^s843 embryos (left) or WT embryos following whole-mount in situ hybridization analysis of tm4sf18 expression (right). Blue brackets indicate nascent ISVs; red brackets indicate anastomosed ISVs; arrows indicate tm4sf18-expressing ISVs; and arrowheads indicate tm4sf18 expression at regions of future angiogenic remodeling.

(F) Whole-mount in situ hybridization analysis of tm4sf18 expression in npas4l^s5 mutant embryos showing loss of expression, as well as upon dll4 KD showing ectopic expansion of tm4sf18 expression to the DA, consistent with de-repression of Vegfr signaling.

Data are mean ± SEM. Scale bar, 100 μm.

See also Figure S2.

TM4SF1/Tm4sf18 Expression Feeds Back to Amplify VEGF/Vegf Signaling

(A) Relative expression levels of TM4SF1 by qPCR in HUVECs transfected with control or TM4SF1-targeted siRNA (n = 4 separate experiments).

(B and C) Western blot analysis of pERK/ERK levels in HUVECs after VEGF-A stimulation following transfection with control or TM4SF1-targetting siRNA (B) and quantification of pERK/ERK ratios (C) (n = 3 separate experiments).

(D) Lesions introduced into the tm4sf18 loci by TALEN and CRISPR gene editing. A 19-bp deletion of tm4sf18 exon-1 and a 16-bp deletion and 2-bp insertion of exon-2 were generated using TALENs and CRISPR/Cas9, respectively. Genomic target sites for the TALENs, gRNA target site, and PAM sequence are indicated by blue, red, and green highlights, respectively.

(E) Strategy for assessing Vegfr signaling dynamics in vivo.

(F–I) Lateral views of pErk immunostaining in ECs of WT (F) or tm4sf18^−/− (H) Tg(kdrl:nlsEGFP)^zf109 embryos at 0 and 2 h after inhibitor washout and quantification of pErk fluorescence intensity in WT, tm4sf18^+/− (G) or tm4sf18^−/− (I) embryos. Arrowheads in (F) indicate pErk in neuronal cells (n = at least 39 ECs from 8 WT, 129 ECs from 20 tm4sf18^+/−, and 74 ECs from 13 tm4sf18^−/− embryos at each time point).

Data are mean ± SEM. ^∗p < 0.05, two-tailed t test. Scale bars, 25 μm.

Tm4sf18 Modulates the Magnitude and Timing of the Angiogenic Response

(A and B) Quantification of the number of ECs selected to branch (A) or the percentage of ECs that undergo proliferation (B) in WT, tm4sf18^+/−, and tm4sf18^−/− embryos (n = 62 ISVs from 16 WT, 58 ISVs from 15 tm4sf18^+/−, and 31 ISVs from 8 tm4sf18^−/− embryos).

(C) Quantification of the distribution of ISV cellularity in WT, tm4sf18^+/−, tm4sf18^−/−, and HU/Ap-treated embryos (n = 65 ISVs from 16 WT, 62 ISVs from 15 tm4sf18^+/−, 31 ISVs from 8 tm4sf18^−/−, and 88 ISVs from 22 HU/Ap-treated embryos).

(D) Quantification of the total number of ECs per ISV in WT, tm4sf18^+/−, and tm4sf18^−/− embryos. n is the same as in (A).

(E) Predicted shift in the level of VEGF signaling required to achieve a selection threshold in the absence of positive feedback.

(F and G) Quantification of the number of ECs selected to branch in 40 nM ZM323881-treated WT, tm4sf18^+/− and tm4sf18^−/− embryos (F) and corresponding time-lapse images of sprouting ISVs in 40 nM ZM323881-treated WT and tm4sf18^−/− embryos from 20 hpf (G). Embryos were incubated with 40 nM ZM323881 from 18 hpf onward. Nuclei of sprouting ECs emerging from the DA are pseudocolored (n = 26 ISVs from 10 WT, 50 ISVs from 20 tm4sf18^+/−, and 21 ISVs from 10 tm4sf18^−/− embryos).

Data are means ± SEM. ^∗p < 0.05, two-way ANOVA or two-tailed t test. Scale bar, 25 μm.

See also Figure S3.

Hypocellular Vessels in tm4sf18^−/− Mutants Fail to Extend Appropriately

(A) Time-lapse images of sprouting ISVs in WT and tm4sf18^−/−Tg(kdrl:nlsEGFP)^zf109 embryos from 19 hpf. Brackets indicate dividing cells. Nuclei are pseudocolored. ISVs appear shorter in the absence of tm4sf18.

(B–D) Quantification of the dorsal movement of tip (cell 1) or stalk (cell 2) ECs in WT and tm4sf18^+/− (B), tm4sf18^−/− (C), or HU/Ap-treated (D) embryos (n = 71 ISVs from 16 WT, 69 ISVs from 15 tm4sf18^+/−, 39 ISVs from 8 tm4sf18^−/−, and 89 ISVs from 22 HU/Ap-treated embryos).

(E) Quantification of the dorsal movement of tip ECs in non-proliferating ISVs consisting of 1, 2, and 3 or more ECs and comparison with the motility of tip ECs in tm4sf18^−/− embryos (n = 39 ISVs from 8 tm4sf18^−/− embryos, as well as 17 ISVs with 3 cells, 53 ISVs with 2 cells, and 16 ISVs with 1 cell from 22 embryos).

(F) Quantification of the number of ECs that reach the DLAV position in WT, tm4sf18^+/−, tm4sf18^−/−, and HU/Ap-treated embryos (n = 63 ISVs from 16 WT, 56 ISVs from 15 tm4sf18^+/−, 31 ISVs from 8 tm4sf18^−/−, and 86 ISVs from 22 HU/Ap-treated embryos).

(G) Illustration of the causes of vessel hypoplasia and phenotypic effect on vessel extension.

Data are means ± SEM. ^∗p < 0.05, two-way ANOVA or two-tailed t test. Scale bars, 25 μm.

See also Figure S4.

Robustness of Tip Identity Is Lost in tm4sf18^−/− Mutants

(A) Lateral views of sprouting ECs in ISVs of WT and tm4sf18^−/−Tg(kdrl:nlsEGFP)^zf109 embryos immunostained for pErk. Prior to fixation, embryos were incubated with DMSO or 40 nM ZM323881 from 22 hpf for 3 h.

(B) Quantification of pErk fluorescence intensity in WT, tm4sf18^+/−, and tm4sf18^−/− embryos following incubation with DMSO or increasing concentrations of ZM323881 (n = at least 32 ECs from 8 WT, 87 ECs from 22 tm4sf18^+/−, and 35 cells from 8 tm4sf18^−/− embryos at each concentration).

(C) Putative role of positive-feedback-generated bistability and hysteretic dynamics in the control of VEGFR signal level robustness in angiogenesis. Bistability ensures that higher levels of VEGF are required to induce tip patterning than to reverse this active state, conferring robustness on tip identity against fluctuations in VEGF levels.

(D) Impact of Tm4sf18-mediated positive feedback on the magnitude, speed, and robustness of motile EC selection during ISV branching. Tm4sf18 drives quick and robust decision making but also ensures delicate modulation of the magnitude of EC selection by Vegf levels. In the absence of Tm4sf18, the magnitude of EC selection is diminished, and both the speed and robustness of EC selection are highly variable and more dependent on Vegf level.

Data are means ± SEM. ^∗p < 0.05, two-way ANOVA or two-tailed t test. Scale bar, 25 μm.