Variation in apparent stiffness and displacement (at 450 seconds following suction) during gastrulation. (A) Displacement (D450) versus Time and (B) Stiffness (E450) versus Time for the between-clutch (filled symbols) and within-clutch (open symbols) experiments. Note in a single measurement, there was a strong contraction resulting in negative displacement, hence an infinite apparent stiffness. (C–D) Variation in D450 versus Ln-transformed time in the Within-Clutch experiment (C) and in the Between-Clutch experiment (D). (E–F) Variation in Ln-transformed stiffness versus Time in the Within-Clutch experiment (E) and in the Between-Clutch experiment (F). Best-fit lines for each embryo (C and E) or clutch (D and F) are shown (thin lines), as well as the best-fit line for the whole data set for each experiment (thick lines). Measurements were made in the region on the dorsal quadrant between the blastopore and a position midway from the vegetal to the animal pole (Fig. 1B–C).

Environmental and drug effects on apparent stiffness and success of gastrulation. (A, C, and E) Effects of treatments on displacement at 450 s following suction. Data were separated by clutch. Error bars indicate ±1 standard deviation. Measurements were made in the region on the dorsal quadrant between the blastopore and a position midway from the vegetal to the animal pole (Fig. 1B–C). (B, D, and F) Frequency of gastrulation defects (failure to close blastopore or failure to internalize endoderm) assessed between late neural fold and post-neural tube closure stages (st. 17 to 21, respectively). Sample sizes are in parentheses. S: standard salt and pH media (0.33 × MBS + BSA, pH 7.4); H: high salt media (1.3 × MBS+BSA, pH 7.4); L: low pH media (0.33 × MBS + BSA, pH 5.4); M: culture media (0.33 MBS); D: DMSO control (0.006 % DMSO in 0.33 × MBS + BSA); and La: 0.3 μM Latrunculin B, in 0.33 × MBS + BSA).

(A) Micro-aspiration apparatus: an embryo (em) was pressed against a channel (ch) cast in PDMS connecting the high-pressure (hp) and low-pressure (lp) reservoirs. Tissue displacement into the channel was viewed through a microscope objective below. Pressure was adjusted by adding or removing media from the low-pressure reservoir. (B–C) Diagrams of gastrula stage embryos in vegetal view (B) and in cross section (C): arrows indicate directions of tissue movement; bg: blastopore groove; ect: animal cap ectoderm; m: deep-layer mesoderm; en: endoderm. (D–E) Representative embryos before and after application of −5.4 Pa suction showing smooth (D) or lumpy (E) appearance of tissue (arrows). The bulk of the embryo was to the right of the channel opening (o), but obscured by reflection off the PDMS surface. (F–G) Apparent stiffness (F) and displacement (G) versus time following suction at two different pressures: suction was ended at 800 seconds. Embryos in F and G were aspirated on the ventral side between the end of blastula stages (late st. 9) and early gastrula stage (st. 10.5). Baseline pressure was −2.4 Pa. Error bars indicate ±1 standard deviation (n = 5 embryos for −3.6 Pa, and n = 6 for −7.2 Pa). A, B and C modified from von Dassow and Davidson (2007).

Contraction and relaxation behavior. (A) Displacement versus time following suction (−5.4 Pa) for 6 embryos showing the variety of contraction and relaxation behaviors observed. Arrows indicate beginning and end of the period shown in panel C for the embryo shown in the gold curve. Note that for this embryo some leakage occurred following the contraction (between asterisks). (B) Displacement during the first 10 minutes (gray region in panel A) following suction (−5.4 Pa) in 19 embryos. Embryos in panels A and B were at the end of blastula stages (late st. 9) or beginning of gastrulation (st. 10) at the start of measurement. (C) The edge of partially aspirated tissue (colored lines) in an embryo undergoing a localized contraction while under suction. Arrowhead indicates site of initiation of contraction; the color bar (bottom) indicates the times associated with each colored curve; and the vertical bars are artifacts from the channel wall. Images from a time-lapse video were processed to outline the boundary of the tissue, and false color values were assigned to the different, superimposed frames. (D) Time series showing strain in cells (*) adjacent to cells undergoing division (d). Images were centered on the dividing cells by eye, and processed to enhance contrast and reduce noise. (E) An embryo in which most of the dorsal marginal zone underwent a rapid contraction. Top series: a sequence of frames from a time-lapse video (contrast enhanced, and image smoothed to reduce noise). Bottom series: Difference images between frames taken 1 minute apart show regions undergoing displacement from minute to minute. Blue: increase in brightness; red: decrease in brightness. Between minutes 0 and 1, a large region on the dorsal side contracted towards a point just above the blastopore (bp); little movement occurred between minutes 1 and 2; and a small amount of relaxation occurred near the blastopore between minutes 2 and 3. This embryo was at late blastopore closure stages (st. 12).