Espeseth A et al. (1998), The role of F-cadherin in localizing cells duri...

XB-ART-15374

Development 1998 Jan 01;1252:301-12. doi: 10.1242/dev.125.2.301.

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The role of F-cadherin in localizing cells during neural tube formation in Xenopus embryos.

Espeseth A , Marnellos G , Kintner C .

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The cell adhesion molecule F-cadherin is expressed in Xenopus embryos at boundaries that subdivide the neural tube into different regions, including one, the sulcus limitans, which partitions the caudal neural tube into a dorsal and ventral half (alar and basal plate, respectively). Here we examine the role of F-cadherin in positioning cells along the caudal neuraxis during neurulation. First, we show that ectopic expression of F-cadherin restricts passive cell mixing within the ectodermal epithelium. Second, we show that F-cadherin is first expressed at the sulcus limitans prior to the extensive cell movements that accompany neural tube formation, suggesting that it might serve to position cells at the sulcus limitans by counteracting their tendency to disperse during neurulation. We test this idea using an assay that measures changes in cell movements during neurulation in response to differential cell adhesion. Using this assay, we show that cells expressing F-cadherin localize preferentially to the sulcus limitans, but still disperse when located away from the sulcus limitans. In addition, inhibiting cadherin function prevents cells from localizing precisely at the sulcus limitans. These results indicate that positioning of cells at the sulcus limitans is mediated in part by the differential expression of F-cadherin.

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Species referenced: Xenopus
Genes referenced: cdh20 grap2 myc tbx2 tubb2b

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	Fig. 1. Ectoderm mixing assay. (A) One blastomere of a 16-32 cell stage embryo was injected with nLacZ RNA. At stage 12, the embryo was fixed and processed for X-gal staining. Shown are X-gal-stained cells derived from the injected blastomere within the ventral ectoderm. Note that these cells have intermingled with descendants of neighboring blastomeres. (B) One blastomere of a 16-32 cell stage embryos was injected with RNA encoding F-cadherin. At stage 12, the embryo was fixed and stained for the myc epitope. Shown are myc antibody-stained cells derived from the injected blastomere within the ventral ectoderm. Note that these cells have not intermingled to the same extent as in A. Asterisks indicate cells in the deep layer of ectoderm where mixing is not suppressed by Fcadherin expression. Arrowheads indicate the enrichment of expressed protein at sites of cell-cell contact. (C) Same analysis as in B except that the embryos were injected with RNA encoding ÆFcadherin. Note that these cells mix as well as control cells. (D) Fcadherin and ÆF-cadherin RNAs were mixed at different ratios and injected into a blastomere of a 16-32 cell stage embryo. At stage 12, embryos were fixed and stained for the myc-epitope. The number of isolated cells per unit area at the edge of a patch of labeled cells was analyzed for 6-12 embryos, and their mean and standard error calculated. Note that cell mixing is significantly reduced with both high (5 ng) and low (2.5 ng) concentrations of F-cadherin RNA relative to b-gal controls (P<0.001 and P<0.01, respectively, using one-tailed t-tests). Co-injection of ÆF-cadherin RNA with Fcadherin RNA inhibits the effects of F-cadherin on cell mixing: note that cell mixing is significantly increased when ÆF-cadherin (7 ng) is included with F-cadherin, relative to when 5 ng or 2.5 ng of Fcadherin RNA is injected alone (P<0.005 in both cases, using a onetailed t-test). Embryos injected with 7 ng DF-cadherin and b-gal controls were not statistically different for P<0.10 (using a t-test).
	Fig. 2. Expression of F-cadherin RNA during neural tube formation in Xenopus embryos. Embryos at stage 16 (neural plate stage) and stage 22 (early neurulae) were stained for the expression of Fcadherin RNA using whole mount in situ hybridization. (A,B) Stage 16 embryo stained for F-cadherin expression showing the stripe of F-cadherin-expressing cells within the caudal neural anlage, either in whole mount (B, arrow) or in a transverse section through the neural plate (A, arrow). Note that F-cadherin-expressing cells are already localized to the prospective sulcus limitans, based on their position within the neural plate (np) along the mediolateral axis. (C) After neural tube formation, F-cadherin is expressed in cells localized to the sulcus limitans, which divide the neural tube (nt) along the D-V axis into the basal and alar plate. Note in panel C that the cells with F-cadherin staining lie adjacent to the ventricle, but that this staining is probably confined to the cell body. Thus, it is likely that the cells expressing F-cadherin RNA are neuroepithelial, or radial glial cells, that span the neural tube from the lumen to the pial surface.
	Fig. 3. Effects of F-cadherin and ÆF-cadherin on convergentextension. One blastomere at the two-cell stage was injected twice with (A) nLacZ RNA, (B) a mixture of nLacZ and F-cadherin RNA or (C) a mixture of nLacZ and ÆF-cadherin RNA. At late neural plate stages the embryos were fixed and stained with X-gal to reveal the injected side (arrow in panel B), and for for N-tubulin expression using whole mount in situ hybridization to reveal the position of the primary neurons (Chitnis et al., 1995). Note that injection of Fcadherin, but not nLacZ or ÆF-cadherin RNA significantly alters CE as revealed by the effects on the position of primary neurons. The embryo in panel B is slightly younger in age than those shown in Panels A and C.
	Fig. 4. Bin analysis of cell mixing during convergent-extension. (A) Donor tissue, marked by injection of nLacZ RNA was transplanted into the prospective neural plate of host embryos at the beginning of gastrulation. (B-D) Host embryos were stained for b- galactosidase expression to reveal the location of the transplanted cells soon after transplantation (st. 10.5, B), at midgastrulation (st. 11, C), or at neural tube stages (st. 26, D). An arrow points to the dorsal blastopore lip in panels B and C, while transplanted cells are indicated by arrowhead. Note that the transplanted cells incorporate and interact with host cells during convergent-extension. (E,F) Tissue section of a host embryo after staining for b-galactosidase to mark the nuclei of transplanted cells, and whole-mount in situ hybridization for F-cadherin expression. In D, the arrowhead points to a transplanted b-galactosidase-expressing cell. In F, the bins used to determine the D-V distribution of transplanted cells are diagrammed. The SL bin was positioned at the site of F-cadherin expression. Cells that crossed the roof plate (RP) or floor plate (FP) were assigned to bins designated with a negative sign.
	Fig. 5. Average distribution of donor nLacZ cells in the posterior neural tube after convergent-extension. (A) Distribution of transplants into the alar plate with a peak location in the bin adjacent to the roof plate (Bin 1). (B) Distribution of transplants into the alar plate with a peak location at the sulcus limitans (Bin SL). (C) Distribution of basal transplants with a peak location at the bin midway between the sulcus limitans and the floor plate (Bin 6). (D) Distribution of basal transplants with a peak location at the sulcus limitans (Bin SL).
	Fig. 6. Results of a computer simulation designed to model the distribution of cells during neural plate elongation. (A,B) Simulation performed with the patch of cells having only default adhesion. (C,D) Simulation performed with the patch of cells adhesive for itself as well as the border of the array. (E,F) Simulation performed with the patch of cells adhesive to the border but not to itself. (A,C,E) Distribution of a patch of cells placed away from the border of the starting array. (B,D,F) Distribution of a patch of cells placed near the border of the starting array.
	Fig. 7. Average distribution of donor F-cadherin-expressing cells in the posterior neural tube after convergent-extension. (A) Distribution for transplants into the alar plate with a peak location in the bin adjacent to the roof plate (Bin 1). (B) Distribution for transplants into the alar plate with a peak location at the sulcus limitans (Bin SL). (C) Distribution of basal transplants with a peak location at the bin midway between the sulcus limitans and the floor plate (Bin 6). (D) Distribution of basal transplants with a peak location at the sulcus limitans (Bin SL).
	Fig. 8. Average distribution of donor ÆF-cadherin-MT-expressing cells in the posterior neural tube after convergent-extension. (A) Distribution for transplants into the alar plate with a peak location in the bin adjacent to the roof plate (Bin 1). (B) Distribution for transplants into the alar plate with a peak location at the sulcus limitans (Bin SL). (C) Distribution of basal transplants with a peak location at the bin midway between the sulcus limitans and the floor plate (Bin 6). (D) Distribution of basal transplants with a peak location at the sulcus limitans (Bin SL).
	Fig. 9. Average distribution of donor CBR-MT-expressing cells in the posterior neural tube after convergent-extension. (A) Distribution of transplants into the alar plate with a peak location in the bin adjacent to the roof plate (Bin 1). (B) Distribution of transplants into the alar plate with a peak location at the sulcus limitans (Bin SL). (C) Distribution of basal transplants with a peak location at the bin midway between the sulcus limitans and the floor plate (Bin 6). (D) Distribution of basal transplants with a peak location at the sulcus limitans (Bin SL).
	Fig. 10. Computer simulation of cell dispersion during neurulation. (A) The diagram illustrates a rearrangement step of the model, where the dark squares represent the grid of positions of the tissue at the very beginning of the simulation, when each grid position is occupied by a single cell, and the light gray squares represent the tissue after the first rearrangement step when the tissue has shrunk by one column and elongated. The numbers in the light gray squares indicate how many cells have ended up in those grid positions; there are grid positions with no cells and others with more than one cell, the total number of cells on the grid being of course the total number before the rearrangement (8 in this example). Note that cells in the dark squares can move to a grid position either up or down and to the left or right (except for cells at the borders which can move up or down, but to one side only ). After this rearrangement step of the algorithm, comes a ‘diffusion-adhesion’ step which moves cells around on the (light gray) grid (as described in the Methods), then another rearrangement step (from the light gray grid), and so on till the grid has converged and extended to the desired final size. (B-C) Initial and final frames from a simulation run. Patch cells are represented in green. Grid points may contain more than one cell. (D) Final distribution of patch cells on the grid, for a patch that was initially placed towards the middle of the DV extent of the beginning grid (pooled data from 20 simulations).