Fig. 6

tmem33 knockdown reduces endothelial Notch and ERK signalling during angiogenesis. a–c Notch reporter expression is unaffected by injection of 0.4 ng tmem33 morpholinos at 26 hpf. Notch signalling activity in control embryos is present in neural tube (blue arrowhead) and DA (red arrowhead) (c, unpaired t-test; t = 0.5553; DF = 20; 2 repeats; n = 5 or 6 embryos per group). Scale bars 50 µm. d–f Injection of 0.4 ng tmem33 morpholinos reduces Notch reporter expression at 48 hpf. Notch expression in control Tg(csl:venus) embryos is present in the neural tube (blue arrowhead), DA (red arrowhead) and SeAs (white arrowheads) (f, unpaired t-test; p = 0.0451; t = 2.138; DF = 20; 2 repeats; n = 6 and 4 embryos per group). Scale bars 50 µm. g Expression of notch1b, dll4, hey2 and her12 are not significantly altered by tmem33 CRISPRi at 26 hpf (one-way ANOVA using post hoc Tukey’s comparison test. n = 3 repeats). h Expression of notch1b, dll4, hey2 and her12 are significantly reduced at 48 hpf by tmem33 CRISPRi (one-way ANOVA using post hoc Tukey’s comparison test. *p = < 0.05; **p = < 0.01. n = 3 repeats). i–p In comparison with control morphants (i–l), embryos injected with 0.4 ng tmem33 morpholino (m–p) display reduced expression of notch1b within the DA (i, m, red arrowheads) and SeAs (i, m, white arrowheads), dll4 within SeAs (j, n, white arrowheads), hey2 within the DA (k, o, red arrowheads) and her12 within the DA (l, p, red arrowheads) and SeAs (l, p, white arrowheads). Scale bars 100 µm. q–t tmem33 knockdown by CRISPRi reduces endothelial ERK phosphorylation (q, r, SeAs highlighted by yellow outline and white arrowheads). Intensity plot of ERK staining is shown (s, t, SeAs highlighted by yellow outline and white arrowheads). Scale bars 50 µm. u tmem33 crispants display significantly reduced levels of pERK phosphorylation. Pixel intensity normalised to neural tube ERK fluorescence (unpaired t-test, **p = 0.0097, t = 2.993 DF = 14. n = 2 repeats, 4 embryos per group). Source data are provided as a Source Data file