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The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility. Using a kinase-dead mutant of xPak1, we show that during Xenopus gastrulation, the kinase activity of Pak1 is required upstream of Cdc42 for the establishment of cell polarity in the migrating mesendoderm. Overactivation of Pak1 function by the expression of constitutively active xPak1 compromises the maintenance of cell polarity, by indirectly inhibiting RhoA function. Inhibition of cell polarization does not affect the migration of single mesendoderm cells. However, Pak1 inhibition interferes with the guidance of mesendoderm migration by directional cues residing in the extracellular matrix of the blastocoel roof, and with mesendoderm translocation in the embryo.
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.
