Descriptions of Selected E. coqui movies for Rick

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I first discuss my general conclusions, with links to the best examples of the movie(s) they are drawn from. This is followed by a list of all included movies, and their notable features. Some movies may not be referenced in the general conclusions, but have some feature I wanted you to see.

 

General conclusions:

(note: I will refer to the traditional dorsal/lateral/ventral sides of the blastopore, with no implication about the actual fate of these regions.)

(Disclaimer: The opinions expressed below are subject to re-evaluation and confirmation and should by no means be taken as dogma.)

Blastopore Formation, Involution & Closure:

Blastopore formation is rather bizarre, with an apparent "pre-blastopore" (pre-BP) forming first, followed by a more normal looking "blastopore-proper" (BPP) that closes over the pre-BP and shows involution. The best place to see this is in Ecoqui041103_SideGast3.mov. These two phases are very clear laterally and ventrally (dorsally, I don't have a good movie from the true onset of gastrulation, but it doesn't look much different from Xenopus).

First, beginning dorsolaterally, at the juncture between the smaller, presumably supra-blastoporal marginal zone cells on the animal side and the larger, presumably vegetal endodermal cells on the vegetal side, cells appear to flatten their apices in the An-Vg direction, constrict toward each other and disappear, somewhat like in subduction. This can again be best seen in Ecoqui041103_SideGast3.mov. A view of this occurring in a fluorescently labeled cell can be seen in Ecoqui041006_epi07ShadSW.mov. As cells on the vegetal side constrict their apices and/or disappear, the pre-BP moves more vegetally and as cells on the animal size constrict, more distant animal cells move both more vegetally and closer to the pre-BP. I assume the motive force for all this vegetal-ward movement is the apical contraction of the cells, but this is an open question. The flattening and disappearing is clearest on the vegetal side, although many of the more animal cells also clearly disappear early, and at least constrict their apices later. It's not clear what's happening to the cells; there is no clear cleft or invagination. Perhaps it's simply a very narrow, compressed cleft of tightly constricted bottle cells, perhaps there is some form of ingression. It would be very useful to look at this in SEM and/or confocal of RDA labeled embryos.

Subsequent to the formation of this "pre-blastopore" (pre-BP), an invagination finally does form, tending to inflect right at the pre-blastopore dorsolaterally but inflecting more animally of the pre-blastopore as the fold moves ventrally. The blastopore proper (BPP) rapidly covers the pre-BP, which probably winds up at the tip of the invagination, at least dorsolaterally and laterally, just at the bottle cells that initiate the BP in Xenopus do. Movies of giant sandwiches Ecoqui050217_GiantEpi05.mov, Ecoqui050217_MiniSandEpi07.mov and Ecoqui050217_GiantEpi0304.mov suggest that bottle cell formation continues throughout gastrulation after the formation of the BPP, such that cells apically constrict just after involuting while cells at the most vegetal edge of the pre-BP progressively re-expand theirs.

Some questions: Are the two apparent BPs really different from what happens in Xenopus, or is the initial formation of the bottle cells just drastically expanded over a much broader An-Vg area? Is there really an independent second lip, or is it just that the mass of constricted lateral cells can't fold inward anymore? What drives the BPP? There is lots of epiboly going on (see below); is that what drives the BPP? Or is it a consequence of some post-involution convergence behavior (e.g. by the previously constricted bottle cells)?

There is a strong dorsal to ventral progression of both kinds of blastopore formation, and cells appear to be pulled toward dorsal as the lip forms. See Convergence and Extension, below.

There is rapid involution around the BPP as it forms, and involution continues, perhaps at a slower rate, as the blastopore closes. This is most obvious in Ecoqui041103_SideGast3.mov and also in Ecoqui040902_epi12.mov. Involution is strongest dorsally, tending to little, and later perhaps no involution ventrally. Involution is very strong w.r.t. to blastopore closure, indicating both a strong epiboly, and that cells must either be piling up just inside the lip, or moving animally equally rapidly.

 

Blastopore closure appears to be fairly isotropic (circumferentially equal). This can be seen in Ecoqui041103_SideGast3.mov, Ecoqui040730_epi1n2.mov, Ecoqui040902_epi3n4.mov and Ecoqui040902_epi9n10.mov. While the blastopore clearly forms dorsally first and then progresses laterally and ventrally, the blastopore appears to converge on and close over the center of the vegetal endoderm. However, this is not easy to measure, given plane of focus issues. It is also not clear how important or reliable a measure this is, as the vegetal endoderm can move w.r.t. the BP, e.g. the dorsal-ward shear of the Vegetal endoderm under the DBPL (see movies above), which occurs (much less dramatically) between stage 10.5 and 11 in X. laevis. There is however a remarkable amount of closure over the vegetal endoderm from the ventral side in E. coqui. In Nile blue stained embryos (e.g. Ecoqui041103_SideGast3.mov), the ring of involuted material is fairly equal for about 240 degrees centered on the ventral side but then there is a much broader dorsal bulge. This correlates with the apparent disparity in involution dorsally & laterally vs. ventrally, and with the apparent dorsal convergence of cells prior to involution. The dorsal bulge seems to lie entirely under the presumptive brain region, suggesting that it is composed of head mesoderm, rather than axial tissues. Thus it is not clear whether it really represents the result of active dorsal convergence or simply migration of pre-chordal plate. The dorsal bulge appears to arise from the dorsal-most 120 degrees, without obvious convergence; it is simply left behind more animally after involution as the BP closes vegetally; thus it could simply reflect the earlier onset of involution dorsally, rather than any actual dorsal convergence.

 

Vegetal Endoderm Behavior:

Whereas I see apical contraction in the cells just vegetal of the blastopore as they form the pre-BP, there is nothing obvious in the more vegetal endoderm, providing no evidence for a vegetal rotation type mechanism operating vegetally. This can be seen in Ecoqui041006_epi07ShadSW.mov and Ecoqui041006_epi03.mov, although in the later movie the central vegetal cells do appear to begin shrinking toward the end of gastrulation. See also Ecoqui040902_epi3n4.mov and Ecoqui040902_epi9n10.mov.

It is still worth asking whether there any reduction in apical area of the more vegetal endoderm before or during gastrulation. It may be possible to map some of this from existing movies, although resolution and focal plane issues are problematic.

 

Emboly & Epiboly:

Emboly, or mesendoderm migration, appears very similar to every other anuran I've ever looked at. This can be seen very nicely in Ecoqui041103_SideGast3.mov. Epiboly is also seen very clearly in this movie. The animal cap roof becomes very thin, as in G. riobambae. The cells undergoing emboly appear to meet at the animal pole perhaps slightly in advance of blastopore closure, but the angle is not good. The enormous amount of involution seen in some movies (above), also indicates that a great deal of epiboly is occurring. A movie of the animal hemisphere would probably be useful for quantitating the timing and amount of epiboly; perhaps a double movie, so I can correlate it with blastopore formation & closure.

 

Convergence and extension:

There does appear to be some convergence toward the dorsal midline; this is especially obvious in the vegetal endoderm early, as the dorsal blastopore forms (see Ecoqui040730_epi1n2.mov, Ecoqui040902_epi12.mov, Ecoqui040902_epi1n2.mov, Ecoqui040902_epi3n4.mov, Ecoqui040902_epi9n10.mov), and in conjunction with involution around the dorsal to lateral BPP (see Ecoqui041103_SideGast3.mov). In some movies, convergence appears stronger in the cells just above, compared to those just below the blastopore lip; if there is in fact less below, this would indicate some kind of shear between supra-blastoporal and sub-blastoporal cells. It's not clear how significant convergence during dorsal through lateral blastopore formation really is, or how restricted to involution proper it is, i.e. it may be a passive response to bottle cell formation and the D-V progression of involution, as opposed to being some kind of ongoing, autonomous, active MIB based convergence.

 

Giants also show a lot of convergence, especially along what I assume is the BP indent, which becomes the limit of involution. See Ecoqui040730_Giant1.mov, Ecoqui041006_Giants2n3abc.mov and Ecoqui041103_Giant4ab.mov. It seems that if much endoderm is left attached to the giant, convergence is less obvious, as the endoderm spreads out vegetally while the animal portion is converging (see in particular Ecoqui050217_GiantEpi0304.mov). There is some extension dorsally, but it is local (meaning that points within the tissue move apart, without necessarily showing overall explant elongation) and weak. At least some extension must be due to dorsal bottle cell respreading, but there may be some additional extension from other causes, including radial or mediolateral intercalation behaviors. The giants I have are useful for quantitating convergence during gastrulation, but are not great; I could definitely benefit from some more, especially those made early (at 10- to 10), with little endoderm, and left under glass to heal for long enough that they extend outward rather than curling in.

 

Based on one or two giants, it appears that Dorsal extension occurs primarily during last third of gastrulation, then pauses for about 24 hours, then extends more later (during late neurulation).

 

Timing issues:

When does Gastrulation start? How long is it? What are recognizable points along gastrulation? What does early stage 10 look like? Does the dorsal lip progress more like Xenopus (forming and spreading laterally fairly linearly) or like Ambystoma (forming, contracting, pulling lots of cells in via bottle cell formation, then much later eventually expanding)?

How long does neurulation take? Other interesting timing features?

 

I've tried to estimate some of these from movies (see notes in individual movie descriptions, below). I have no movies clearly showing the initial formation of the dorsal lip, so I can only guess when it actually forms; based on some of the blastopore movies, I see the first signs of dorsal convergence about 4-6 hours earlier than I actually see any sign of a dorsal lip (bad angle, near equator, bad lighting), so I'm guessing it takes about 5 hours to get to something like 10+, where many of my movies start, or the dorsal lip become apparent. With a good movie, I think you'd see a Xenopus-like stage 10 lip about 2 hours after the earliest onset of DBC formation, and another 3 hours to the stage 10+ like stage. The expansion of the lip away from dorsal is hard to define clearly, and my estimate is very an estimate; this could probably be refined from current movies. I define this and other lip formations as the onset of involution around the BPP, which is of course substantially later than the formation of the pre-BP, but the pre-BP is generally very hard to resolve. Certainly laterally, there is a very long gap between the onset of cells converging vegetally and animally toward the pre-BP and the onset of BPP involution (12+ hours). Dorsolateral is about 45 degrees, lateral about 90. "Dorsal vegetal endoderm slides up" is the onset of the animal-ward movement of the dorsal vegetal endoderm through the dorsal BPP at a faster rate than the BPP lip is closing. The onset of convergence is defined from giants, as is the extension of dorsal tissues. I find it interesting that vegetal endoderm sliding and convergence start within an hour or two of each other, just as the blastopore is being completed. This seems to be consistent for the stage 11 equivalent in many species, from Xenopus to Ambystoma.

 

Event

Time (hours)

10- (onset of dorsalward convergence)

"0"

10 (Evident dorsal lip?)

2 (1-3)

10+ (stage many of my movies start)

5 (4-6)

expansion of BPP from initial dorsal lip

6.5

Dorsolateral BPP involution starts

11.25 (11-13)

Dorsal vegetal endoderm slides up

15 (15-16)

lateral BPP Involution begins

16 (15-17)

Convergence begins

17 (16-18)

Ventral BPP involution begins

18

Dorsal Bottle cell re-expansion / extension

19

Blastopore closure

26 (25-28)

Neurulation finished

57 (56-60)

 

 

The Movies

 

Blastopore Formation Movies

In most cases these embryos were stained with nile blue; some (the 041006 series) were injected in a few vegetal cells with Rhodamine Dextran. All were from the Metamorph at 4X.

 

Intended to show vegetal dynamics prior to and during blastopore closure. Some of this can be seen with extensive processing (LUT changes and shadowing). Many of these movies are reconstructed from two focal planes (e.g. epi1n2).

 

If I refer to a position around the blastopore as "3 O'clock", I am referring to its position around a clock face.

 

The bottom line seems to be that nothing to dramatic happens though following some vegetal cells through a few divisions might show some shrinkage. Doesn't show BP closure very well.

 

Ecoqui040730_epi1n2.mov: Shows "inner" and "outer" lips, but doesn't reveal much about cells; some indication of vegetal cell behaviors. Hard to say what stage at start is (10+, 10.25?), but the dorsolateral lip appears very early in the movie (about 14:30); Lateral lip shows up about 17:30 (frame 46), BP closes about 6:00 to 7:00 (about 13 hours post lateral lip (BPP))

Ecoqui040730_epi3n4.mov: Decent composite view of just sub-blastoporal vegetal cell behaviors during blastopore formation

 

Ecoqui040902_epi12.mov: nice view of early BP lip formation/involution at bottom (with contrast cranked up). Nice with some shadowing (SW/NE); shows rapid rate of involution, at least early before focus is lost; it is hard to track individual features however. Dorsal lip starts forming about 19:30.

Ecoqui040902_epi1n2.mov: Good early/mid blastopore formation; lots of involution, or at least strong approach toward lateral lip; can also see a fair amount of dorsal convergence of cells in the marginal zone as the dorsal lip of the BP forms. First signs of dorsal convergence at about 19:00 or 20:00. First obvious signs of Dorsal lip formation (at about 10 O'clock) show up about 2:00 (frame 141). Lateral lip forms around 11-12:30. can see movements in yolk plug early, but not especially well or interesting.

Ecoqui040902_epi3n4.mov: dorsal convergence movements start about 20:00 or 21:00; first sign of dorsal lip (at 1 to 2 O'clock) about 1:00; gastrulation still not finished by 18:00 next day; lateral BPP forms around 11 or 12:00. can see a bit of the "inner-lip, outer-lip" action at top; decent view of vegetal endoderm; some dorsal convergence of dorsolateral veg. endo is seen; a dimple in "inner" BP lip region is seen, ventrally (or is that a funny reflection off the vitelline?)

Ecoqui040902_epi9n10.mov; vegetal endo initially good. The convergence of cells toward the forming DBPL is pretty striking, even before the lip become obvious; some cells appear move a good 60 degrees toward the midline. useful for looking at vegetal cell dynamics / apical surface change

 

Ecoqui041006_epi03.mov: last part of movie may show deformation of vegetal cells. Cells that end movie at about 11 O'clock and 1 O'clock around the BP are probably just fluorescent schmutz being pushed along by the lip, rather than actual cells.

Ecoqui041006_epi07.mov: In this version w/o shaddowing, the initial disappearance/contraction of the bright cell at the bottom (about 7 O'clock) illustrates the two blastopore lips rather nicely.

Ecoqui041006_epi07ShadSW.mov: w/ shadowing, can see inner BP lip wave of contracting (?) vegetal cells; can also follow several vegetal cells. Pretty nice. Most bright patches don't shrink prior to or as getting covered, though some do shrink (or otherwise change the shape of) their more animal edge. This shadowed version appears to be slightly sharper as an NIH Image file compared to QuickTime movie.

 

Gastrulation from the Side movies

 

Ecoqui041006_SideGast2.mov: water evaporates and embryos get moved all over, but is very complete view of gastrulation through neurulation & well beyond Starts at 041005, 11:10, dorsal blastopore lip formation (gastrulation probably started about 2-3 hours prior?). Gastrulation done ~ 10:00 -12:00 041006, for about 25-28 hours.

 

Ecoqui041103_SideGast3.mov: starts about stage 10+ (22:43). Nice view of dorsal through lateral BPP (spreading beyond initial dorsal invol at 0:18, true dorsolateral beginning about 5:00; lateral at 10:42) as well as of epiboly and BCR thinning and subsequent emboly. Blastopore closure about 20:15. Ends (early-?) mid neurulation, but neurulation not visible. The end of gastrulation looks a lot like a node receding back along a primitive streak. Early dorsal involution seems to lead to what there is of an AP axis, which becomes evident as the ventral BPP is formed (out of sight but about 12:00?). Trunk axis appears to extend starting around when the dorsal edge of the YP begins to flip inward, at about 8:37. Indicates that 10+ to BP closure is about 21 hours, so if 10+ is about 3 hours, then all of gastrulation about 24 hours. Assuming this, dorsal endoderm starts moving under BP faster than it is advancing at about 13 hours. Dorsolateral lip starts involuting about 9.25 hours, Lateral lip starts involuting about 15 hours. Estimate that ventral lip forms/starts involuting at about 16.25 hours.

 

Giant Sandwich Explant movies

 

Ecoqui040730_Giant1.mov: This is a giant made out of two cysteine/enzyme treated embryos; it was first pressed under a cover slip to heal, then, early in the movie (15:04), the cover slip is removed. Animal up, ventral edges laterally. Embryos are in DFA. A good deal of sub-blastoporal endoderm was included, though much of the internal vegetal endoderm (& mesendoderm?) was scraped out. The initial dorsally centered crease is the pre-BP, whereas the crease eventually running from side to side across the middle of the explant probably corresponds to the BPP or perhaps just the limit of involution.

The remarkable thing here is the dramatic amount of convergence, coupled with very little, and only dorsally located, extension, not of the whole explant, just dorsal tissue within the explant (could be just bottle cell respreading).

This experiment need to be repeated a few times, perhaps with less sub-blastoporal endoderm.

Movie starts at 14:30, with only dorsal lip obvious, spreading laterally fairly early on. Convergence starts between about 19:24 and 20:14, getting more noticeable by 22:00; Dorsal extension (DBCs unfolding?) starts between about 22:40 and 23:05 and continues through about 7:49 (so about 9 hours, which would roughly correspond with the expected time to the end of gastrulation from the start of dorsal extension). There isn't really enough resolution to say much about when the lateral and ventral parts of the lip form.

 

Ecoqui041006_Giants2n3abc.mov: Giant 2 (top, vegetal edge up) looks pretty good; dorsal extension starting about 11:00. Actual image quality mediocre; Gastrulation probably ends about 20:00, about 2/3 of the way through; convergence and extension both slow or stop during this time. Explant elongates 42% by 24 hours. Giant 3 (bottom, vegetal edge down) looks less good. Lots of convergence, very little extension (possibly because it's running into giant 2?? Or more likely just because it's a poorly made/healed explant…).

Ecoqui041006_Giants2n3de.mov: more of the same two giants (2 on right, 3 on left); both show more convergence and extension, especially giant 2, but mostly in the last 1/3 of the movie.

 

Ecoqui041103_Giant4ab.mov: about 18 hours; starts shortly before lateral-ventral involuting BP formation (looks like lateral lip starts about frame 30 (about 14 hours into gastrulation) and ventral most starts around frame 50-60, which would be about 16 hours into gastrulation; based on this, gastrulation ends about frame 150, which is the time the dorsal lip is about done unfolding). Extension of dorsal "post involution" material starts about frame 60; not sure how this corresponds to external gastrulation movements (e.g. dorsal endo "rolling in"); may reflect true dorsal axial convergence and extension, may just reflect dorsal bottle cell re-expansion, but I'm guessing this plays a limited role here; it does look like there is some sort of true C&E in the posterior region.

Ecoqui041103_Giant4c.mov: not much going on, including no extension. Explant begins falling apart shortly hereafter.

 

E.coqui giants 050217

A set of Giant sandwiches I made with Sorrel. These don't behave very well, perhaps because I left on too much endoderm (see Giants 4 & 5 in E. coqui 041103), or perhaps because they weren't allowed to heal long enough under glass. They do seem to suggest that bottle cell formation and re-expansion might be a continuous process, operating across a wide (An-Vg) swath of the marginal zone.

Movies start about an hour past 1/4 to 3/4 crescent BP; end at mid-late neurula (judging from neural folds on flip of first explant); Nile blue stained

Ecoqui050217_GiantEpi0304.mov: mostly interesting for observing blastopore dynamics (see also Epi05 below…); some convergence is obviously occurring, but it's hard to say anything about extension.

Ecoqui050217_GiantEpi05.mov: not great as a giant (too much endoderm?? Also, mesodermal tissue go off the top), but the view of cells constricting and then releasing as they go through the erstwhile blastopore ("rolling") is quite interesting and suggests that a Lateral Marginal Zone type explant might be very interesting (an LMZ explant is what I used in Ambystoma to look at progressive de-epithelialization behavior; movies can be found under figure S5 at http://faculty.virginia.edu/shook/uro1/supp_figs.htm).

Ecoqui050217_MiniSandEpi07.mov: a miniature version of epi 05, showing the same bottle cell "rolling" behavior (a chunk of giant that got cut off by accident that I filmed anyway). I think what we are seeing is cells rolling over the BPP (at top), contracting into the pre-BP (the dark base of the inflection), then re-expanding below it. More precisely, some cells are re-expanding below the BPP, but they may well be the vegetal cells that initially formed it, rather than any newly involuted cells.

 

Neurulation and Beyond movies

Ecoqui040730_Neur1NW.mov: Set of views of neural tube selected to be in more or less the same orientation. Shows neural tube closing fairly well.

 

Ecoqui041006_NeurToTBLat.mov: Embryo is being spun by ciliary movement at this point, so tried to select frames with a lateral view of the embryo

Ecoqui041006_NeurToTBPost.mov: tried to select orientation to watch posterior end of embryo (same embryo and time span as above). Big "depression" of lateral tissue between 8:00 and 10:00. Neural tube closure about 16:00 (56 hours after the start of gastrulation). Tail bud forms before this, about 12:00. Posterior limb buds are distinct lateral swellings by 15:45.

Ecoqui041006_PostTailbud1.mov: 20, then 40 minute snap shots of post-neurulation development

Ecoqui041006_PostTailbud2.mov: continuation of above, all 40 minute intervals. Nearing ventral body wall fusion by end.