August 1, 2009;
PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis.
Generation of neurons in the vertebrate central nervous system
requires a complex transcriptional regulatory network and signaling processes in polarized neuroepithelial progenitor cells. Here we demonstrate that neurogenesis in the Xenopus neural plate
in vivo and mammalian neural progenitors in vitro involves intrinsic antagonistic activities of the polarity proteins PAR-1
. Furthermore, we show that Mind bomb
), a ubiquitin ligase that promotes Notch
ligand trafficking and activity, is a crucial molecular substrate for PAR-1
. The phosphorylation of Mib
results in Mib
degradation, repression of Notch
signaling, and stimulation of neuronal differentiation. These observations suggest a conserved mechanism for neuronal fate determination that might operate during asymmetric divisions of polarized neural progenitor cells.
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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.
Figure 5. PAR-1 Does Not Affect Signaling of a Mib-Independent Form of Dll1(A and B) Embryos were injected with indicated RNAs and processed for in situ hybridization with N-tubulin probe as described in Figure 1. RNAs were injected at 0.3 ng. Dorsal views are shown, with anterior to the left; the injected side is up (light blue). Arrows point to changes in N-tubulin expression. (A) PAR-1 suppressed the inhibitory effect of untagged Dll1 on neurogenesis, but had no effect on the activity of Dll1δC-Ub. (B) Quantification of PAR-1 effects on Dll1 and Notch signaling is shown. Numbers of embryos per group are indicated above bars. Data are pooled from several independent experiments. RNA doses are indicated per embryo.(C) PAR-1 does not affect Notch reporter activation by Dll1δC-Ub. Signal-sending cells were transfected with PAR-1, Dll1, or Dll1δC-Ub constructs and cocultured with signal-receiving cells that were transfected with Notch and pGL3-11CSL-Luc, as described in Figure 2E. *p <0.01 and #p <0.01 indicate significant differences from control- or the Dll1-transfected group, respectively. Results are presented as the means ± SD of three independent experiments, each carried out with triplicate samples per group.
Notch signalling regulates stem cell numbers in vitro and in vivo.