Somitogenesis Period Increases after Reduction in Delta-Notch Coupling

(A) Delta-Notch coupling between two oscillating PSM cells. Delta is the ligand for the Notch receptor, which can be inhibited via the small molecule DAPT. Mib is a ubiquitin ligase required for Delta trafficking and activation.

(B) Time-lapse movies of wild-type (wt) and mib embryos. Bars indicate formed somite boundaries; arrowheads indicate forming boundaries. Dorsal view is anterior to top. The scale bar represents 50 μm.

(C) Time versus somite number plot for (B). Linear fits of data (R² [wt and mib] = 0.999) yield somitogenesis periods of 20.5 min (wt) and 25.0 min (mib).

(D) Distribution of somitogenesis periods in the experiment from which (B) and (C) were taken (n [wt] = 12, n [mib] = 11). Blue bars indicate mean somitogenesis period. Temperature = 28.2 ± 0.1°C.

(E) Box-and-whisker plots of somitogenesis period: n ≥ 37 total embryos, more than six independent trials per experimental condition, except ace/fgf8, mbl/axin1, and nof/raldh2, for which n ≥ 16 total embryos, two independent trials per experimental condition. het denotes heterozygote. ^**p < 0.001, Student′s t test. Figures S1 and S2 show that general developmental rate is unaffected in the conditions with slower period. The central box covers the interquartile range with the mean indicated by the small square and the median by the line within the box. The whiskers extend to the 5^th and 95^th percentiles, and small bars depict the most extreme values.

Segment Length Increases after Reduction in Delta-Notch Coupling

(A) Somites of six-somite-stage live embryos. Bars indicate formed somite boundaries; arrowheads indicate forming boundaries. Dorsal view is anterior to top. The scale bar represents 25 μm.

(B) Somite lengths (mean ± 95% CI), n ≥ 40 total embryos per experimental condition, except n = 14 for somite five in aei/deltaD, six independent trials per experimental condition. For control population, the largest CI detected is displayed.

(C) In situ hybridization of mespb (arrowheads) and isl1 (interneurons, Rohon-Beard neurons, n). The scale bar represents 50 μm.

(D) Box-and-whisker plots of segment length, n ≥ 40 total embryos, more than three independent trials per experimental condition. ^*p < 0.01, ^**p < 0.001, Student′s t test. Figure S3 shows that the position of the arrest front in the PSM is unchanged in the conditions with increased segment length. The central box covers the interquartile range with the mean indicated by the small square and the median by the line within the box. The whiskers extend to the 5^th and 95^th percentiles, and small bars depict the most extreme values.

Determination of Segmentation Clock Collective Period from Oscillating Gene Expression Patterns

(A) In situ hybridization: deltaC and isl1 (interneurons and Rohon-Beard neurons, n) in representative wild-type and mib embryos. The scale bar represents 50 μm.

(B) Measurements of normalized gene expression wavelength λ and position x; wild-type is black and mib is pink. Data points: n (wt) = 65, 28 embryos, n (mib) = 70, 28 embryos, two independent trials. Curves indicate fit of Equation 3 to data (Experimental Procedures).

(C) Values of frequency profile decay length ρ, and normalized segment length s parameters from the fit to the data. Error bars show 95% CI from bootstrap analysis (Supplemental Experimental Procedures 2.2.5). ^**p < 0.001, Student′s t test.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Coupling Time Delays Regulate the Stability of the Segmentation Clock

(A) Schematic of how the autocorrelation function (Equation 6, Experimental Procedures) is computed. A magnification of the gene expression pattern from (B) is shown, together with the reference axes x and y in pixels, and the distance δ between two sample points of gene expression intensity I.

(B) Representative cyclic dlc expression in PSM of wild-type embryo and DCT simulation with wild-type parameters defined in this work, black dot in Figure 5C.

(C) Average autocorrelation function of spatial patterns in red box from (B): n (embryos) = 15, black line; n (simulations) = 20, gray line. Error bars show standard error of the mean (SEM).

(D) Representative experimental Mib overexpression (400 pg mRNA) and corresponding DCT simulation with reduced coupling delay, blue dot in Figure 5C.

(E) Average autocorrelation function of spatial patterns in red box from (D): n (embryos) = 8, dark blue line; n (simulation) = 20, light blue line. The arbitrary units in the correlation axis are multiplied by 10^-3. Figures S5 and S6 show the autocorrelations for decreasing delays in numerical simulations and increasing levels of Mib overexpression in the Figure S7 shows that the Delta-Notch loss-of-coupling mutants have autocorrelation functions distinct from Mib overexpression. Error bars show SEM.

Expression of mesoderm and ectoderm differentiation markers in mib embryos
In situ hybridization with ectoderm (A) and mesoderm (B, C) markers in the PSM of wildtype/heterozygous and mib embryos at the 10 somite stage. Elevated expression of isl1 in (B) in interneurons and Rohon-Beard neurons (n, out of focal plane) and reduced expression of mespa (arrow in C) is observed in mib embryos, either of which was used to distinguish wildtype/heterozygous from mib embryos. Dorsal view with anterior to the top. Expression patterns were assessed in at least five embryos per experimental condition. Scale bars = 25 μm.

Axial elongation is not changed in Delta-Notch mutant and DAPT-treated embryos
(A) Stills from time-lapse movies [8, 9] of wildtype and mib embryos. White lines: line along which axial length was measured. Arrow: bulge in mutant brain due to neuronal hyperplasia. Bracket: region containing irregular somite boundaries in the mutant. Lateral view with anterior to the left. Scale bar = 100 μm. (B-E) Plots of embryo length (mean ± 95% confidence interval, CI) vs. elapsed recording time for different experimental conditions, n ≥ 4 embryos for each data point. The time points at which wildtype embryos reached the 4 or 10 somite stage are indicated. Values for control and mutant or DAPT-treated embryos were not significantly different (p > 0.01 in all cases as assessed by Student’s t-test) at any time point or for any of the experimental conditions.

Position of arrest front is unchanged within the PSM of Delta-Notch mutants and DAPT-treated embryos
(A) In situ hybridization for mespb (blue) and isl1 (blue; expressed in interneurons and Rohon-Beard neurons, n) and myoD (red) in the PSM of wildtype/heterozygous and mib embryos. Bracket: expression pattern of mespb, striped in wildtype and in a salt-and-pepper pattern in mib. Double-headed arrow: d, distance between the posterior boundary of mespb expression domain and the posterior tip of the embryo. Dorsal view with anterior to the top. Scale bar = 25 μm. (B-E) Plots of d (mean ± 95% CI) vs. developmental stage in different experimental conditions, 10 ≤ n ≤ 59 (mean = 22.5) embryos from at least two independent trials for each data point (Table S1). Each experimental condition had three trials for at least one of the data points. Somite stages are given for the wildtpye siblings or clutch mates. Values of arrest front position for control and mutant or DAPT-treated embryos were not significantly different (p > 0.01 in all cases as assessed by Student’s t-test) at any time point or for any of the experimental conditions.

Cyclic gene expression patterns resulting from elevated Mib levels are distinct from loss of coupling mutants
Spatial organization of oscillating deltaC expression in the PSM of representative embryos shown for (A) wildtype at 3-4 ss, (B) 400 pg mib mRNA at 3-4 ss, (E) wildtype at 10 ss, and the loss of coupling mutants (F) after eight (aei/deltaD) at 8 ss, (G) deadly seven (des/notch1a) at 8 ss, and (H) mind bomb (mib) at 10 ss. Corresponding average autocorrelation functions of oscillatory gene expression patterns in the anterior PSM of (C) wildtype at 3-4 ss (n=15), (D) Mib over-expression (n=8), (I) wildtype at 10 ss (n=13), (J) aei/deltaD (n=38), (K) des/notch1a (n=36), (L) mib (n=26). The arbitrary units in the correlation axis are multiplied by 10^-3. The left/right asymmetry of the pattern in Fig. S7B reflects the asymmetric distribution of the injected mRNA. Autocorrelation functions were always calculated from the most strongly affected side.