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Figure 1. PAR-1 and aPKC Regulate Neurogenesis in Xenopus Embryos(A–H) Four-cell embryos were unilaterally injected with LacZ RNA (50–100 pg) and indicated RNAs or MOs, and subjected to whole-mount in situ hybridization with N-tubulin (A–D) or Sox2 (E–H) anti-sense probes at neurula stages. The injected area is identified by β-galactosidase activity (light blue). Dorsal views are shown, anterior is to the left, and the injected side is at the top. Arrows point to altered gene expression at the injected side. Three stripes of primary neurons (medial, m; intermediate, i; lateral, l) are indicated (A). (A and B) PAR-1 RNA (300 pg, doses are per embryo) increased (A), whereas PAR-1BY MO (5–10 ng) decreased (B), N-tubulin-positive cells on the injected side. (C and D) aPKC-CAAX RNA (30–100 pg) inhibited (C), and aPKC-N RNA (4 ng) expanded (D), N-tubulin-positive cells. (A–D) Dotted lines indicate midline. (E–H) Modulation of PAR-1 and aPKC does not influence the pan-neural marker Sox2 (doses are as in [A]–[D]).(I and J) Phenotypically active doses of PAR-1 ([I], 300 pg) and aPKC-CAAX ([J], 100 pg) RNAs do not alter the number of Sox3-positive progenitor cells. GFP-CAAX RNA was used as a lineage tracer. Stage 15 embryos were cryosectioned and immunostained with anti-Sox3C (red) and anti-GFP-antibodies (green). Dotted lines demarcate neural plate (np) and notochord (nc). Scale bar, 100 μm.(K) Summary of data combined for several independent experiments presented in (A)–(D). Frequency of embryos with a change in N-tubulin-positive cells is shown for each experimental group. Numbers of embryos per group are indicated above bars.(L) Sox3+ progenitor analysis in cryosections. Summary of data for embryos injected with aPKC-CAAX (100 pg), PAR-1 (300 pg), tBR (1 ng) RNAs, and PAR-1BY MO, COMO (5–10 ng). tBR RNA strongly upregulated the number of Sox3-positive progenitors. Mean ratios of Sox3+/DAPI+ cells ± SD are shown. At least 8 embryos were used in each experimental group. *p < 0.01 indicates significant difference from uninjected cells.(M) Lack of effect of PAR-1 and aPKC manipulation on Sox3 protein levels in neurula stage (stage 15) embryos (doses are as in [A]–[D]). BMP7 RNA (500 pg) dramatically reduced Sox3 protein levels. |
