Cell Number Variability during Early Embryogenesis

(A) Maximum projection of confocal images of a Tg[sox17:GFP] embryo at 75% epiboly stage showing the endoderm (red), DFCs (green), and nuclei (blue). Scale bar, 80 μm.

(B) Graphical representation of an 8-somite stage embryo, the KV, and the organ laterality at later stages.

(C–E) Cell number in endoderm (n = 49), DFCs (n = 49), and KV (n = 37), respectively, for individual embryos. The coefficient of variation (C_V) is indicated at the bottom right; (C) and (D) also show the total cell number on the x axis (C_V = 4.8%) and the coefficient of determination (R²). Total cell numbers are in good agreement with a previous publication (Kobitski et al., 2015).

See also Figure S1.

Fluctuations of DFC Numbers Lead to Defects of Organ Laterality

(A) Defective heart laterality percentages observed at prim-22 stage, including reversed and no heart loop observed in embryos incubated at 28.5°C (blue) and 33°C (orange). The column scatterplots show the percentage of embryos with defective heart laterality per individual clutch analyzed at 28.5°C (marked by the horizontal light blue bar) and 33°C (orange bar). The smaller circles indicate a clutch size of 50–150, and the larger circles indicate a range of 151–250 embryos.

(B) Confocal z-projection of the dorsal side of a Tg[sox17:GFP] (green) live embryo injected with H2B-mRFP (histone H2B—monomeric red fluorescent protein) (pink). The plot shows the DFC number distribution at 60% epiboly and the resulting heart and liver laterality in the individual embryos (n = 31) assessed by in situ hybridization at long-pec stage: myl7 and amhc for the heart and foxA3 for the liver (L). Scale bar, 100 μm.

(C) Graphical representation of the different temperature shift treatments: the first third of the gray bar represents the period between 1-cell and bud stage, the second until the 14-somite stage, and the third until the period of collection (prim-22 stage). Circle size and incubation temperature as in (A). Treatments: (I) incubation at 28.5°C until bud stage, change to 33°C; (II) 33°C until 14-somite and then transferred to 28.5°C; (III) 28.5°C until bud stage, shift to 33°C until 14-somite (4.5 h) and then back to 28.5°C; (IV) 33°C until bud stage, change to 28.5°C; and (V) 28.5°C until 14-somite and then transferred to 33°C.

(D) Relative frequency of different spaw expression patterns in embryos incubated at 28.5°C and 33°C. In situ hybridization photographs of normal (green), reversed (dark blue), and bilateral (gray) spaw expression in 18-somite stage embryos are shown to the right.

(E) Relative frequency of embryos with normal, reversed, or bilateral liver, separated into embryos with normal or defective heart laterality at long-pec stage (incubated at 28.5°C or 33°C).

See also Figure S2, Table S1, and Video S1.

Heart Laterality Defects Are a Stochastic Phenomenon That Is Linked to Maternal Effects

(A) Percentage of embryos with defective heart laterality in the progeny of individuals that showed either normal or reversed heart laterality at prim-22 stage. Incubation temperature 28.5°C (light blue) or 33°C (orange). The smaller points indicate a clutch size ranging of 50–150, and the larger points indicate a range of 151–250.

(B) Percentage of embryos with defective heart laterality per individual clutch analyzed at 28.5°C and 33°C in TL (same data as Figure 2A) and AB embryos.

(C) Number of DFCs/KV cells at shield and 8-somite stage: for shield stage, n = 78 and 75 for AB and TL, respectively, p < 0.001; for 8-somite stage, n = 94 and 97 AB and TL, respectively, p < 0.001.

(D) KV cell numbers at 8-somite stage in crosses between individual AB and TL males and females, AB/TL versus TL/AB, p < 0.001 (n = 76 for both); AB versus AB/TL, p = 0.461; TL versus TL/AB, p = 0.01; AB versus TL/AB, p < 0.001; TL versus AB/TL, p < 0.001. The boxplots display the median, the hinges represent the first and third quartiles, and the whiskers represent 1.5 of the inter-quartile range from the hinge.

(E) Summary of reported embryonic expression patterns for the down- and upregulated genes.

(F) Expression levels (in counts per million) for genes that are differentially expressed at all stages.

(G) Heatmap showing the pairwise Pearson correlation between the downregulated genes at 2.25 hpf for TL embryos. Color scale at the bottom. For (F) and (G), gene names are color coded to show the embryonic structure in which they are expressed. Three randomly selected genes with similar expression levels were included as an outgroup (gray).

See also Figures S3 and S4 and Tables S2 and S3.