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Figure 1. Pak1 inhibition affects movement of the mesendoderm relative to the blastocoel roof (BCR). A-D: DiI (1,1 prime , di-octadecyl-3,3,3 prime ,3 prime ,-tetramethylindo-carbocyanine perchlorate) labeling. Label of an initially (stage 10.5) horizontal streak is shown 2 hr later in sagittally sectioned embryos. A: Control. B,C: KD-Pak1. D: Rescue of KD-Pak1 with wt-Pak1. White arrowheads, initial labeling sites; black arrowheads, positions of mesendoderm after 2 hr. E-J: Pak1 inhibition and region-specific gene expression. Expression of the posterior mesodermal marker Xbra (E,G) and the dorsoanterior mesoderm marker gsc (F,H-J) was compared between control gastrulae (E,F,I) and embryos injected with KD-Pak1 (G,H,J) by in situ hybridization. I,J: Sagittally fractured embryos, dorsal is to the right. Blue arrowhead points to blastopore and red arrowhead points to archenteron. K: Archenteron length, measured (Axiovision) from fractured stage 11 embryos after injection of KD-Pak1 or KD-Pak1 with wt-Pak1 (rescue) mRNAs at doses indicated (pg per blastomere). Each column represents data from 22 embryos from 2-3 batches of eggs. Archenteron length was normalized to water-injected controls. The Welch test confirmed that KD-Pak1 (130 pg) was significantly shorter than control archenterons, at significance level alpha = 0.02; and KD-Pak1 (260 pg, 400 pg and 90 pg) at alpha = 0.005. Likewise, the rescue is significantly higher than KD-Pak1 (400 pg) at alpha = 0.05. L,M: Convergent extension. Explants of dorsal blastopore lip from control (L) and KD-Pak1 (400 pg) -injected embryos (M) stage 10 gastrulae were cultured overnight. |
