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Fig. 1. Cell size asymmetries in early sea star embryos. A Representative images of sea urchin (Lytechinus pictus) and sea star (Patiria miniata, Patiriella regularis) embryos at the 4-, 8- and 16-cell stages. Embryos were injected with mRNA coding for a membrane-bound fluorescent protein (mYFP, mGFP) and fluorescently tagged histone (H2B-RFP, H2B-mCherry) and subsequently imaged live on a confocal microscope. The datasets were segmented using the Fiji plugin Limeseg and individual blastomeres rendered as 3D meshes. Scale bars: 50 μm. B Volumes of individual blastomeres normalized to embryo volume, calculated as the sum of the volumes of the 4 blastomeres at the 4-cell stage. For sea star embryos, animal and vegetal poles were assigned according to the position of the polar bodies, and for sea urchin they were assigned according to the position of the micromeres. An, animal; Vg, vegetal; Me, mesomere; Ma, macromere; Mi, micromere. C Ratios of largest to smallest cells’ volume at 16-cell stage in sea star embryos. L. pictus: n=119 cells, 5 embryos. P. miniata: n=180 cells, 8 embryos; P. regularis: n= 91 cells, 4 embryos
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Fig. 2. First and third cleavages predict the anteroposterior axis in P. miniata sea star embryo. A Representative images of P. miniata embryos injected with a lineage tracer at the 2- or 8-cell stage. One blastomere was injected with a mixture of Dextran-Alexa488 and mRNA coding for Histone-BFP either at the 2- or at the 8-cell stage. At the 8-cell stage, either one animal or one vegetal blastomere was injected, scored according to the position of the polar bodies. Embryos were then raised at 16C and imaged on an epifluorescence microscope at 30, 48 and 72 hpf. Scale bars: 50 μm. B Alignment of the first cleavage with the animal-vegetal axis. Embryos injected at the 2-cell stage were stained with cell mask orange and imaged in toto on a confocal microscope. The images were rendered in 3D and the angle formed between the clone and the animal-vegetal axis was measured. n= 27 embryos. Rayleigh test, ***: p-value < 0.001. C Quantification of the lineage-tracing experiments. One blastomere of P. miniata embryos was injected either at the 2- or 8-cell stages, discriminating between animal and vegetal blastomeres at the 8-cell stage based on the position of the polar bodies. Embryos were raised at 16C and the position of the injected clone scored at 26 hpf. n= 161 embryos, 4 experiments
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Fig. 3. The position of smaller cells at 16-cell stage is biased toward the ventral side in P. miniata sea star embryos. A–D First and third cleavage in relation to the dorsoventral axis. Representative confocal image of a sea star larvae injected at the 2-cell stage (A) or 8-cell stage (C). One blastomere was injected with a mixture of Dextran-Alexa488 and mRNA coding for Histone-BFP at the 2-cell stage, raised at 16C, fixed at 72 hpf, stained with Draq5 to visualize all nuclei and imaged in toto on a confocal microscope. Quantification of the angles formed by the injected clone and the sagittal plane of the larva at 72 hpf is shown in B (2-cell; n= 30 embryos) and D (8-cell; n= 24 embryos). E–L Position of small cells in relation to the dorsoventral axis. (E) Representative confocal image of a sea star larva showing the clone derived by a small cell at the 16-cell stage. Oocytes were injected with mRNA coding for the photoconvertible protein Kaede (
) and incubated ON at 16C. Oocytes were subsequently activated, fertilized and incubated until the 16-cell stage, when one of the 16-cell was photoconverted (
) on a confocal microscope (405 nm laser). Embryos were raised at 16C for 72 hpf and then imaged live in toto on a confocal microscope. F Schematic representation of the clone shown in (E). Several views of the same embryo are drawn, to fully represent the position of the labelled clone in 3D. G–J Quantification of the positions of clones observed after the photoconversion of a random cell (G) or a small cell (I) at 16-cell stage and the angles of the same clones formed with the sagittal plane at 72 hpf (H, J). Random cells: n= 32 embryos; Small cells: n=29 embryos. Rayleigh test, *: p-value < 0.05; ns, not significant. Scale bars: 100 μm
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Fig. 4. The position of animal clones is not biased with respect to DV axis due to morphogenesis. Representative confocal image of a sea star larvae injected at the 8-cell stage (A). Oocytes were injected with H2B-CFP to mark nuclei, fertilized and raised until the 8-cell stage, when one animal blastomere was injected with DiI. Embryos were imaged in toto on a confocal microscope at three different developmental stages (26, 50 and 72 hpf). Orientation of the embryo is indicated in the upper left corner of each image and a schematic representation of the clone is provided in the upper right corner. B Quantification of the angles formed by the injected clone and the sagittal plane of the larva at 72 hpf. n= 55 embryos. Rayleigh test, ns, not significant. Scale bars: 50 μm
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Fig. 5. Cell size asymmetries are not necessary to establish the dorsoventral axis in P. miniata embryos. Representative DIC images of 72 hpf larvae generated by blastomeres of dissociated embryos. A CTRL embryos (not manipulated). B, E, and F Larvae generated by Individual blastomeres of embryos dissociated at the 2- (B), 4- (E) or 8- (F, F’) cell stages. C, D Larvae generated by vegetal (C) or animal (D) quartets of blastomeres isolated at the 8-cell stage. This corresponds to the vegetal and animal halves of an embryo. Vegetal or animal identity was established according to the position of the polar bodies. G Phenotypes of larvae formed by isolated blastomeres at between 48 and 120 hpf. Blastula: no invagination; Gastrula: evident gut; Bipinnaria: mouth formed. n= 94 isolated blastomeres; 35 embryos; 2 experiments. H, I Removal of one small cell at the 16-cell stage. Zygotes were denuded of their fertilization envelope and raised at 16C until the 16-cell stage. No cell (H) or one small cell (I) was removed by micropipette aspiration. Embryos were raised at 16C to 72 hpf, fixed, stained with Draq5 and Phalloidin and imaged on a confocal microscope. Phenotype quantification shown on bottom right. n=33 embryos, 2 experiments. J–M Cell size reduction. Representative confocal images of CTRL (J) or manipulated (L) larvae. One blastomere at the 8-cell stage was injected with DiI and immediately reduced in size by micropipette aspiration of cytoplasm. CTRL embryos were injected but not aspirated. Quantification of the angles formed by the injected clone and the sagittal plane of the larva at 72 hpf is shown in K (CTRL; n=18 embryos) and M (reduced; n= 11 embryos). Rayleigh test, ns, not significant. Scale bars: 50 μm
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Fig. 6. Differences in ectoderm morphogenesis between sea urchin and sea star. Schematic representation of the known and hypothesized fate maps of sea urchin and sea star ectoderm. In the sea urchin, the ciliary band marks the boundary between dorsal/aboral and ventral/oral ectoderm (scheme adapted from Davidson et al., 1993). In the sea star, the position of the precursors of the ciliary band (Yankura et al., 2013) and the location of pre-oral and post-oral ciliary bands are known (Nakajima et al. 2004; Hinman and Burke 2018). Our lineage tracing data suggests that the boundary between dorsal/aboral and ventral/oral anterior ectoderm lies between the two ciliary bands in the sea star
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