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Fig. 6. Lrig3 attenuates Fgf signaling. (A) Lrig3 inhibited ERK phosphorylation. Embryos were injected with active Ras (HaRas, 100 pg), eFgf (1 pg), Lrig3 (1 ng), zebrafish sef (500 pg) or mkp3 (500 pg). Animal caps were treated with bFgf (20 ng/ml) for 3 hours as indicated. Cell lysates were analyzed for diphosphorylated (dp)-ERK and total (pan)-ERK. AC, untreated. (B) Overexpression of Lrig3 inhibits Xbra induction by bFgf. Animal caps were treated with bFgf (50 ng/ml) for 4 hours. Xbra expression was induced by bFgf treatment in uninjected (lane 4), but not in Lrig3 (1 ng) injected caps (lane 5). (C) Lrig3 did not attenuate Xbra expression induced by activin (250 pM). Injection of Smad7 (500 pg), but not of Lrig3 (1 ng), inhibited Xbra induction. (D) Lrig3 inhibits Fgf8 activity in animal caps. Embryos were injected with the indicated dose of Fgf8a alone or with 1 ng of Lrig3. Expression of Xbra, Xwnt8 and Xmsx1 was examined at equivalent stage 11.5. (E) Differential effect of Lrig3 on Slug and Twist induction by different doses of Fgf8a. Fgf8a at increasing doses, as indicated, was injected alone or with a constant amount (1 ng) of Lrig3, and induction of Slug and Twist was assayed at equivalent stage 22.

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