(A,B) Influence of acvr1b-a and acvr1b-b on the Nodal signaling range at shield stage. The range of Nodal signaling in shield-stage wild-type, knockdown and rescued embryos was determined by counting the maximum number of nuclei tiers positive for pSmad2/3 immunostaining from the embryonic margin towards the animal pole (i.e. the number of pSmad2/3 positive nuclei tiers at the dorsal side). acvr1b-at03pm/t03pm mutants and 0.4 ng acvr1b-b transcriptional start site-targeting morpholino (MO-1) were used for receptor loss-of-function conditions. Receptor loss-of-function was rescued with 50 pg of acvr1b-a or 25 pg of acvr1b-b mRNA. Data was obtained from three independent replicate experiments. (A) Maximum intensity projections show dorsal views. Scale bar represents 200 µm. (B) n indicates the number of analyzed embryos. Averages are displayed in red, and error bars show standard deviation. (C) Rescue of Type I receptor function after combinatorial mutation/knockdown using acvr1b-a and acvr1b-b mRNA. To deplete the Type I receptors, the acvr1b-at03pm/t03pm mutant was used in combination with 0.4 ng acvr1b-b transcriptional start site-targeting morpholino (MO-1). mRNA amounts are given in pg. n indicates the number of analyzed embryos. Note that strong overexpression of Acvr1b-a receptor-encoding mRNA leads to high lethality or tissue aggregates that eventually disintegrate (termed ‘Nodal gain-of-function’). See the Figure 4—source data 1 file for source data.
