New movie showing marginal zone contraction in a plane, w/o vegetal plate (maybe):

Link to movie 1

Link to movie 2

I did get at least one nice result out of last weeks movies;

Basically, if I "unwrap a marginal zone" (cut through one side in the

An-Vg direction, then cut off vegetal plate and "animal cap" material,

such that you are left with the circumferential extent of the marginal

zone, but cut in one place and laid flat, like a map of the world),

and put it under a relatively loose glass coverslip, it first tries to

roll up (curving in the An-Vg direction), but then eventuall and very

abruptly contracts in the circumferential direction. The primary flaw

with this experiment is that I can't say exactly where I'm cutting animally

and vegetally, so I don't really know if I've gotten rid of all the

vegetal plate. With more embryos, and especially if the nile blue is

more localized, I can do some controls to see if I can see the crease

forming along one edge when I delibrately try to include vegetal plate.

And if we had a marker, we could do in situs too. Oh, I suppose that

markers that specifically *didn't* label the vegetal plate would also

be useful.

 

 

 

 

OLDER STUFF BELOW

 

 

Here's the description of all the movies, with links for most (they're really too big to all be sitting on the server...). My conclusions, model and questions can be found here.

 

You'll need Quicktime to view these movies, Quicktime can be downloaded here

Be patient; some of these movies will take a while to load. You may want to do this using two browser windows, one for looking at movies and one for reading text.  

 

Saccoglossus 040923 explants

Each set described left to right, top to bottom

AS = animal cap sandwich

VS = vegetal plate sandwich

SR = Sagittal ring = a torus including both animal and vegetal

SH = Sagittal Hemisphere = half an embryo cut sagittally

VP = vegetal plate; the part that invaginates

MZ = marginal zone (arbitrarily define as the region next to the VP)

AC = animal 1/2 or so

BP = Blastopore

 

Sacco sand group 2: Upper row: AS, VS, VS; lower row: AS, VS, AC. The AS's don't do much; somewhat remarkably, they neither swim away nor rotate; ciliary beat in the opposite direction from the two halves? The VS's don't do much either, though the one in the middle of the bottom row shows an odd behavior; this VS is not well matched with its other half, underneath (esp. at lower right); notably, in the region toward the center from this a hole spontaneously opens up and many cilia or perhaps filopodia appear on the inner side of the hole. This hole pretty clearly forms at the interface between the vegetal plate and the marginal zone, where one imagines the cleft that forms on the basal surface would be. The other two VS's grow some cilia, and wind up with lots of loose cells on the surface (which may be unhappy or dying cells). The animal cap closes it's open, basal side a bit, spins around for a while, then seems to stop and flatten out (maybe it's dying?). Not a real exciting experiment.

Sacco set 1: This is actually Ray's handiwork. Upper row: SR cut animally, SR cut vegetally, and an SH; Lower row: two uncut SR's, and another SH. Everything starts vegetal down. First frame is 4.5x, then all 5x. At frame 8, I've pried the two cut SR's apart; they show that the buckling of the VP is pretty autonomous. Not entirely clear why the vegetally cut SR opens as it does; perhaps the AC region generates tension apically? Note that the animally cut SR makes a serious attempt to actually close it BP on all sides. Not sure why the first SR shows fairly good BP closure, while the second does not. The two SHs manage to close their blastopores fairly effectively; although the details aren't particularly clear, it looks like they do so fairly evenly, and with about the some time course as an intact embryo, suggesting that the contraction of the marginal zone will opperate to close the BP almost as effectively as in an intact embryo, i.e. the continuity of a ring of contraction within the marginal zone is not necessary. On the other hand, the two SRs do a lousy job of closing their BP, so maybe half an embryo worth of MZ is enough, where as two small (1/8th circumference?), separated bits of MZ are not. The SHs also appear to undergo some animal-vegetal elongation.

Sacco set 4: My attempt to play with SR's; obvious what's what; all start VP down. Not real interesting. Don't see the opening of the vegetal side in the vegetally cut SRs, as in set 1; the ends of the vegetal plate seem to want to come back together when pried apart. Maybe I need to make these earlier during BP formation, as in set 1. Especially in the third vegetall cut SR, the bending vegetal plate actually seem to bend the adjacent MZ outward. Animally cut SRs consistently show BP closure behavior, showing that an intact AC is not required.

Sacco set 5: Top row: 1/2 VP, with MZ attached (top); 1/2 AC; whole VP with MZ attached; Bottom Row: Whole embryo; animal cap; vegetal plate.

The first explant is notable because the MZ appears to contract, or converge toward the top, in the circumferential direction; this behavior begins about frame 50 is strongest as the blastopore is contracting in the whole embryo. The VP begins moderately dimpled, but then flattens out and never invaginates. This and other experiments suggest that the entire MZ is programmed to converge circumferentially as the VP invaginates.

The second explant balls up and swims off.

The third explant invaginates nicely, and tries to close the BP, but is much delayed w.r.t. the whole embryo. With the first explant, this suggests that a circumferentially intact marginal zone is required for invagination.

The whole embryo shows the normal progression of blastopore closure, as a controls for the behavior of explants in position 1, 3 and 6.

The fifth explant contracts a bit, perhaps becoming moderately convex; it eventually swims off.

The final explant tries to invaginates, but notably, does not do nearly as well as the VP with more MZ; it seems as though its trying to close its BP (see frames 80-150). There are many highly active ("loose"?) cells; perhaps VP cells have a more mesenchymal character than MZ and AC cells, or perhaps the VP cells are more unhappy about being exposed than MZ and AC cells, or maybe its just a generally unhappy explant. But see also the cells at the lower edge of the first explant, which also show a lot of activity.

 

Conclusions:

An intact AC is not required for VP invagination or (at least some) BP closure.

The marginal zone tends to converge circumferentially, and appears to be required for VP invagination

 

Questions:

1) what is the A-V extent of the contractile MZ?

 

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Saccoglossus movies 040924

all embryos nile blue labeled; all embryos pinned in place with coverslip bits. "sagittally" = cut along the AV (Animal-Vegetal) axis, rather than transversely, across the AV axis.

epi 01 VP basal** a vegetal plate, looking at the basal surface; note in particular the peri-plate tissue converging toward the lower left (between about 6 and 8 O'clock); also, between frames 10 and 20, a depression forms in this region between this tissue and the plate proper, but largely dissappears by 100. Lots of protrusvie activity/blebs at free edges.

epi 02 VP apical a vegetal plate from the animal side (or possibly it's an animal half?); a curious wave propagates inward from the lower right, from about frame 35 to 90; also more subtly from the upper margin of the explant, at the same time. Lots of cilia at the margins, especially upper left. Boring otherwise.

epi 03 VP basal*** The piece de resistance: A vegetal plate from the basal side; a very clear division/cleft forms around the vegetal plate, except on the right (presumably, I cut more vegetally there); clearly visible by frame 50 and persists throughout; the tissue outside the cleft converges to the left, and gets "thicker" in the Animal vegetal direction, presumably reflecting isotropic (circumferentially equal) convergence and extension in the non-involuting tissue. Doesn't really seem to drag a lot of the vegetal plate cells with it as it converges, though it does leave behind a free edge that seems to be more protrusive than the converging marginal zone.

epi 04 BP vegetal view of BP closure; embryo was held under glass; BP closure seems inhibited (perhaps pressed too hard? Suggests that Animal-vegetal extension is required as part of the process); perhaps some isotropic convergence (could try measuring it, but….), but no obvious rearrangement.

epi 05 BP as 04, but even less exciting

epi 06 BP as 04, but some weirdness going on just inside the blastopore

epi 07 BP ditto 05

epi 08 ring a "sagittal" ring (AP is at 11:00, VP at 5); looks too squished under the CS; does sort of flatten up and fold in on itself

epi 09 ring another ring, not as good as 08

epi 10 hemi A half embryo, cut "sagittally"; not pretty, probably under too much pressure from CS; vegetal plate sort of everts; does try to close up the opening, dunno what that means. Some animal-vegetal extension.

epi 11 hemi* as for 10; I don't really understand what the vegetal plate is doing, but it does it here too. There is also some fairly strong animal-vegetal extension evident as well.

 

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040925 Saccoglossus movies

 

These explants were made as the embryos became clearly dimpled. They are either "animal caps", which don't include any vegetal plate, and little of the region near it; "vegetal plates", usually partial plates with some attached "marginal zone" material; or "planula 1/2's", cut sagittally and also pinned under coverslips (but still wander off).

 

epi 01 planula 1/2: Anterior end thickens tremendously; looks like some overall C&E going on; embryos swims off…

epi 02 planula 1/2: as above, and looks like some interesting ingression/invagination action going on in the animal region

epi 03 planula 1/2's: Older, with some anterior cavity getting closed off; maybe cut off-sagittal?

epi 04 planula 1/2's: as above; epi 03 swims into view later…

epi 05 planula 1/2's: again with anterior cavity closing

epi 06: partial VP w/attached marginal zone, animal view (?); strong convergence of MZ on left; odd crease develops on right side of (apparent) VP… dunno what that's about.

epi 07 partial VP w/attached marginal zone; some convergence on left/upper-left side

epi 08: partial VP w/attached marginal zone, Basal view; crease visible early, but disappears by frame 50; a bit, but not a lot of obvious convergence of animal material; perhaps pressed too hard under cs.

epi 09: notes say "partial VP w/attached marginal zone, Basal view", but I think it's really animal cap, basal view; material in lower left looks like it's converging a bit; as below.

epi 10: Animal cap, Basal view; upper left region might be converging a bit at end; maybe that's marginal zone?

epi 11: partial VP w/attached marginal zone, Basal view; convergence visible in lower right; bit of crease visible from about frame 90-200 (not visible early because of poor focus?)

epi 12: partial VP w/attached marginal zone, Basal view; convergence visible in upper right; crease visible from frame 0 to 200/250; looks like marginal zone stuff had already contracted by the time movie started (crease not concave facing vegetal material).

epi 13: partial VP w/attached marginal zone, apical view(? apparently concave VP, no obvious crease); marginal zone material on left/lower-left converges strongly, especially early (up to about 100);

epi 14: AC only, basal surface; no obvious convergence. a wave of some cell behavior moving in from upper left at beginning of movie; no obvious consequence. Nice control

epi 15: AC only, basal surface(?); some minor convergence of entire left side; perhaps this represents MZ material?

epi 16*: Smaller chunk of VP, with cleft, in upper right, attached to larger bit of MZ & AC; not dramatic, but a very clear waist forms at the MZ region! Not sure which side is visible; cleft heals rapidly & early.