Fig. S1

Analyses of Convergence Extension, Apoptosis, and Cell Proliferation in itgα5^mo;αV^mo Embryos, Related to Figure 1

(A) Wild-type, fn1^mo;fn1b^mo, itgα5^mo;αV^mo and Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos were fixed at the tailbud stage and in situ hybridization for her1 was performed. We then measured the medial-lateral width of the tailbud at the level of the posterior tip of the notochord. The fn1^mo;fn1b^mo embryos (n=30) exhibited a 17.5% increase in width of the tailbud (unpaired t-test; p-value <0.05) which is consistent with previous observations [1]. In contrast, neither itgα5^mo;αV^mo (n=37) nor Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos (n=24) displayed a change in tailbud width relative to wild type (n=55) (p-value >0.05). Error bars show standard error. The fn1 and fn1b morpholinos have been previously described [2].
(B) Cell death was assayed in wild-type, itgα5^mo;αV^mo and Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos. We fixed embryos at the 13-somite stage and assayed apoptosis via TUNEL. Briefly, embryos were fixed overnight in 4% PFA in PBS at 4°C and stored in 100% methanol at 20°C. Embryos were rehydrated and permeabilized in 100% acetone at 20°C for 20 min followed by several washes in 0.5% Triton X-100 and 0.1% sodium citrate in PBS and then treated for TUNEL assay using the in situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland) according to the manufacture’s instructions. There was no clear difference in apoptosis in itgα5^mo;αV^mo embryos (n=12) nor in Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos (n=10) compared to wild type (n=12).
(C) Cell proliferation was assayed in wild-type, itgα5^mo;αV^mo and Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos. We fixed embryos at the 13-somites stage and assayed for cell proliferation via immunohistochemistry for Phosphorylated Histone H3 (PHH3) as previously described [3]. Compared to wild-type (n=5), itgα5^mo;αV^mo embryos (n=5) exhibited a 36% decrease in cell proliferation (unpaired t-test, p-value <0.05). Proliferation in Tg(tbx6l:itgα5-RFP);itgα5^mo;αV^mo embryos (n=5) was no different than in itgα5^mo;αV^mo embryos (p>0.05). Scale bars equal 200 microns.