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In both invertebrate and vertebrate embryonic central nervous systems, deep cells differentiate while superficial (ventricular) epithelial cells remain in a proliferative, stem cell state. The conserved polarity protein PAR-1, which is basolaterally localised in epithelia, promotes and is required for differentiating deep layer cell types, including ciliated cells and neurons. It has recently been shown that atypical protein kinase C (aPKC), which is apically enriched, inhibits neurogenesis and acts as a nuclear determinant, raising the question of how PAR-1 antagonises aPKC activity to promote neurogenesis. Here we show that PAR-1 stimulates the generation of deep cell progeny from the superficialepithelium of the neural plate and that these deep cells have a corresponding (i.e. deep cell) neuronal phenotype. We further show that gain- and loss-of-function of PAR-1 increase and decrease, respectively, the proportion of epithelial mitotic spindles with a vertical orientation, thereby respectively increasing and decreasing the number of cleavages that generate deep daughter cells. PAR-1 is therefore a crucial regulator of the balance between symmetric (two superficial daughters) and asymmetric (one superficial and one deep daughter) cell divisions. Vertebrate PAR-1 thus antagonises the anti-neurogenic influence of apical aPKC by physically partitioning cells away from it in vivo.
Fig. 1. PAR-1 promotes the generation of deep cells from the superficialepithelium. (A) Schematic of layer-grafting experiment showing homotopic, homochronic transplant to an untreated host of manually dissected superficial ectodermal epithelium from a Xenopus embryo injected with GFP-CAAX plus or minus PAR-1TA mRNA. (B,C) Whole-mount live fluorescence images of GFP-expressing superficial layer grafts in the dorsal ectoderm at stage 14 (B) and stage 24 (C), revealing graft integrity and integration into the elongating and neurulating host embryos. (D) GFP-labelled control grafts at stage 14, showing that nearly all superficial-layer-derived cells remain within the superficial epithelial layer. Infrequently, individual cells outside the layer could be seen (asterisk). (E) GFP-CAAX-labelled, PAR-1TA-expressing grafts at stage 14 (open neural plate stage) showing numerous labelled deep layer cells (arrows). (F) Quantification of the highly significant increase in the number of labelled deep cells derived from superficial layer grafts with PAR-1 activation. Cells were scored as deep if they were not part of the superficial layer.
Fig. 2. PAR-1 promotes neurite differentiation in superficial layer progeny. (A) Control-grafted stage 25 Xenopus embryos showing that labelled cells lack neurites. (B) Lineage-labelled cells from PAR-1TA-expressing superficial layer grafts at stage 25 showing numerous cells bearing long neurite-like processes (arrows). (C) Quantification of superficial layer graft progeny showing significant induction of differentiation in PAR-1TA-expressing, but not control-grafted, embryos. Neurite-like processes were scored conservatively as labelled cell extensions that extended at least 5 μm from the cell body. *, P=0.025; Student's t-test.
Fig. 3. PAR-1 is necessary and sufficient to promote vertical spindle orientation in the early neuroectoderm. (A-D) Maximum z-projections (main panels) and selected orthogonal yz (left) and xz (above) sections (with the plane of yz and xz sections indicated by the vertical and horizontal white lines, respectively) of confocal image stacks. Superficialneuroectoderm at early gastrula stage was stained for alpha-tubulin (spindles, red) and GFP (membranes and nuclei, green). Tissues injected with GFP-CAAX only (A), PAR-1A MO (C) or control MO (D) show numerous metaphase spindles in the xy plane, whereas with Myc-PAR-1TA-injection (B), many spindles are seen end-on (circled).
