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When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.
FIG. 1. Dorsal and ventral mesendodermal explants differ in their spreading behaviour. Small clusters of cells were explanted at stage 10.5
and placed on fibronectin after 1 h. (A) Sketch of an early gastrula embryo. Black areas (explanted regions): dorsal mesendoderm (dm), ventral
mesendoderm (vm). Grey area: prospective ectoderm and posterior mesoderm. (B) Time lapse sequence of a dorsal and a ventral explant of
the mesendoderm. After 2 h the borders between cells become visible in dorsal explants (arrow), but not in ventral explants. Bar: 100 mm.
FIG. 1. Dorsal and ventral mesendodermal explants differ in their spreading behaviour. (C) Increase in area covered by the explants shown in (B). The increase is plotted as the ratio of the size at different times [A (t)] to the initial
size [A (0h)]. (D) Spreading behaviour of dorsal and ventral groups of cells. For each region the sizes of 45 explants (five pooled experiments)
were measured at t 5 0h and t 5 3h and the area ratios were calculated.
FIG. 2. Xwnt8 increases spreading of ventral mesendodermal
explants. Four-cell stage embryos were injected on the ventral side
with both Xwnt8 and EGFP mRNA or with EGFP mRNA alone.
Mesendodermal explants from stage 10.5 embryos were placed on
fibronectin after 1 h of reaggregation. (A) Control-injected ventral
explants only show slight spreading in the periphery. Xwnt8
mRNA-injected ventral explants spread extensively within 3 h like
untreated dorsal explants. Bar: 100 mm. (B) Area ratio of 45 EGFP
mRNA injected and 85 Xwnt8/EGFP mRNA injected ventral
explants (three experiments).
FIG. 3. Autonomy of spreading behaviour. Embryos were either
dorsally or ventrally injected with Alexa Fluor coupled hydrazide.
Dorsal and ventral mesendoderm was obtained at stage 10.5. Pairs
of injected and noninjected dorsal and ventral explants were
allowed to fuse on agar for 1 h, transferred to fibronectin and their
behaviour evaluated after 3 h. (A) Recombinate of an unlabelled
dorsal and a labelled ventral explant. (B) Recombinate of a labelled
dorsal and an unlabelled ventral explant. The parts of dorsal origin
flatten on the substrate, the ventral parts stay compact. Bar: 50 mm.
FIG. 4. Morphology of cells from different regions. (A) Sketch of an early gastrula embryo (stage 10.5), illustrating the regions of
explantation. The approximate position of mesoderm and ectoderm is indicated (shaded area). ae 5 animal ectoderm (inner layer); de 5
dorsal ectoderm (inner layer); dl 5 dorsal blastopore lip; dm 5 dorsal (head) mesendoderm; vm 5 ventral mesendoderm; vl 5 ventral
blastopore lip. Explants were dissociated and the cells seeded on fibronectin. Typical morphology: (B) animal and (C) dorsal ectodermal cells
rounded, with hyaline protrusions (arrow) or filopodia (insert in B, arrow). (D) dorsal and (E) ventral mesendodermal cells of bipolar,
spindle-like shape, exhibiting lamellipodia (arrow) and retraction fibre (arrowhead). Bar: 20 mm. (F) Percentage of spreading cells on
fibronectin. Cells which showed lamellipodia-formation and a resulting change in shape within an hour after plating were counted as
“spreading”. In five independent experiments, 400–800 cells/region were evaluated. Error bars represent standard deviation.
FIG. 5. Migration of mesendodermal cells on fibronectin. The path covered by individual cells in 1 h was determined and the distance
measured to calculate the average velocity in mm/min. At least 60 cells/region were traced in three experiments. (A-C) stage 10.5: (A) animal cells do not migrate. (B) dorsal mesendodermal cells and (C) ventral mesendodermal cells are highly migratory. (D and E) stage 12: (D) dorsal mesendodermal cells and (E) ventral mesendodermal cells have not changed their migratory behaviour.
FIG. 6. (A) Adhesion of inner animal cap cells from embryos injected with activin mRNA and from noninjected embryos. Activin reduces
the adhesion to CEC and increases the adhesion to fibronectin. (B) The adhesion properties of dorsal and ventral mesendodermal cells are
similar and mirror the properties of activin-induced animal cap cells. Noninduced animal cap cells were included as a control sample in
this experiment (C) Morphology of cells at 30 min after seeding onto BSA-, CEC-, or FN-coated substrate. Bar: 50 mm.
FIG. 7. Contact formation of reaggregating cells. Dorsal and ventral mesendodermal cells from stage 10.5 embryos were seeded on
fibronectin and followed for 1.5 h by time-lapse recordings. The contact behaviour of individual cells, marked by numbers in A and B was
evaluated. (A) Pictures of a time-lapse sequence of dorsal mesendodermal cells. 10 min: Cells start to spread and move. 50 min: Cells have
formed contacts. 90 min: Contacts have been released and new contacts with other cells have been established. Bar: 50 mm. (B) Ventral
mesendodermal cells. 10 min: Single, spreading cells. 50 min: Cell-cell contacts. 90 min: The original contacts have been retained. New
cells have joined the aggregate. (C) The percentage of cells which had 1,2, . . . , 6 transient contacts before forming a stable contact is plotted.
The last set of bars shows the percentage of cells, which did not form a stable contact at all during the 90 min of observation. The data were
derived from observing 100 dorsal and ventral cells each in a total of five experiments
FIG. 8. Spreading dynamics on fibronectin (A and B): (A) Spread
dorsal explant with lamellipodia extending from peripheral cells
(arrows). (B) Explant 1 min later, after a collapse, with fragments of
torn lamellipodia on the substrate (arrowheads). Phase-contrast,
bar: 50 mm. Cell intercalation in bottom layer (C and D): (C) Dorsal
explant after 30 min. Cells in contact with the substrate marked in
red. (D) Explant after 2.5 h showing the distribution of the marked
cells. Epi-illumination, bar: 50 mm
FIG. 9. Actin cytoskeleton of dorsal and ventral mesendodermal explants. Explants on fibronectin, stained with Alexa-green coupled Phalloidin. Confocal images were obtained in stacks with 1 mm intervals and merged. (A and B): Bottom view, substrate-level of explants. (A) Dorsal explant with patches of actin in the periphery. (B) Ventral explant with continuous peripheral actin ring. Bar: 25 mm. (C-E): Top view. (C) Dorsal explant with lamellipodia, 1–3 mm from substrate-level. (C9) Same explant, 5–8) mm from substrate-level, with actin fibres. (D) Dorsal explant with actin network (1–25 mm stack). (E) Ventral explant with actin ring (1–35 mm stack). (F and G) Bottom view, atsubstrate-level. (F) Dorsal explant with lamellipodia (arrows). (G) Ventral explant without large protrusions. Bar: 25 mm.
FIG. 10. Differences in morphology of dorsal and ventral mesendoderm. Embryos with part of the blastocoel roof removed. View on the
involuted mesendoderm. (A) overview dorsal, stage 10.5; (B-F): involuted mesendoderm at higher magnification. (B) dorsal, stage 10.5. (C)
dorsal, stage 11.5. (D) ventral mesendoderm of Xwnt8 mRNA-injected embryo, stage 11.5. (E) ventral stage 10.5. (F) ventral, stage 11.5.
Arrows mark lamellipodia-like protrusions. Bars: 200 resp. 20 mm
