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doi: 10.1242/10.1242/dev.00322

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Cell contact-dependent positioning of the D cleavage plane restricts eye development in the Ilyanassa embryo

Section of Integrative Biology, University of Texas, Austin, Texas, 78712
Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA

View larger version (33K): [in a new window]   Fig. 1. Clonal fate map of first-quartet micromeres (A) and origin of quadrant lineages (B) in the Ilyanassa embryo. (A) Eight-cell embryo and hatching-stage larva are seen from the animal pole with dorsal at the top; the different clonal territories are drawn schematically using the data of Render (Render, 1991). (B) First and second cleavages are shown in dorsal aspect with the animal pole (marked by polar bodies) at the top. PL, polar lobe.

View larger version (24K): [in a new window]   Fig. 2. (A-C) Schematic of operations used to isolate partial embryos in these experiments. Ablated cells are shaded. CD half-embryos are shown being isolated at the two-cell (A), four-cell (B) or eight-cell stage (C). In most experiments, the 1c cell was subsequently removed at the eight-cell stage, generating a CD-1c partial embryo (B,C, in box). To isolate analogous AD-1a and BD-1b partial embryos, the appropriate quadrant pairs were removed at the four-cell or eight-cell stage followed by ablation of the 1a or 1b micromere.

View larger version (40K): [in a new window]   Fig. 3. A representative CD-1c partial larva in which the labeled 1d cell formed an eye (arrows). Scale bar: 50 µm.

View larger version (8K): [in a new window]   Fig. 4. Results of 1d labeling experiments in CD half-embryos. Each oval represents an individual with the stated number of labeled and unlabeled eyes.

View larger version (22K): [in a new window]   Fig. 5. Frequencies of eye development in BD-1b partial embryos generated by removing the A and C quadrants during different time windows (x-axes). Each time point represents samples from at least three experiments (seven clutches of eggs used in all). Phases of mitosis (shown at the top) were determined by chromatin staining of samples fixed in parallel during two experiments.

View larger version (19K): [in a new window]   Fig. 6. Histograms showing first-quartet micromere volumes in normal embryos or partial embryos isolated at the four-cell stage. Micromeres were measured after isolation from whole or partial embryos. Quarter embryos, half embryos and three-quarter embryos were isolated by puncturing unwanted blastomeres between 35 and 45 minutes after second cleavage (30 to 40 minutes before micromere formation). One micromere was isolated from each whole or partial embryo; this was achieved by isolating the desired quadrant (either during the four-cell stage or immediately after third cleavage) and then ablating the sister cell of the desired micromere 30-40 minutes after third cleavage.

View larger version (27K): [in a new window]   Fig. 7. Effect of cell ablation before third cleavage on 1d division pattern. (A) Normal division chronology of first-quartet micromeres. Minutes after first cleavage are indicated on vertical axis; horizontal gray lines indicate the ages of embryos shown in B, F and G (bfg) and in C, D and E (cde). (B-G) Camera lucida drawings of nuclei in whole and partial embryos, shown at the same scale; first-quartet micromere derivatives are shaded. (B) Normal embryo, 500 minutes after first cleavage. The 1b12 nucleus is in prometaphase; the small 1d12 nucleus is in interphase. (C) Nuclei in a representative D quarter embryo isolated in the mid four-cell stage and fixed at 375 minutes after first cleavage; 1d1 is precociously in metaphase. (D) Another D quarter isolated in the mid four-cell stage, and fixed at the same stage as the specimen in C; both 1d1 and 1d2 are in metaphase. (E) Nuclei in a D quarter embryo isolated in the early eight-cell stage and fixed at 375 minutes after first cleavage. Interphase 1d1 and 1d2 nuclei exhibit their normal size difference. (F) Nuclei in a BD half-embryo isolated in the mid four-cell stage and fixed at 500 minutes after first cleavage, showing two abnormal pairs of nuclei apparently derived from 1d. The 1b lineage has a normal appearance, including one small nucleus (1b2) and two larger ones (1b11 and 1b12); the 1b12 nucleus is in metaphase. (G) Nuclei in a BD half-embryo isolated in the early eight-cell stage and fixed at the same stage as the embryo in F. The 1d cell has produced the normal complement of three nuclei. Uncharacteristically, the two daughters of 1b1 are dividing synchronously. In both F and G, the 3b nucleus has entered prophase earlier than normal, probably as a result of abnormal exposure to 3D signaling. 4d is in metaphase; 4D is not shown.

View larger version (46K): [in a new window]   Fig. 8. Ectopic eye development correlated with displacement of the D cell division plane. (A) Histograms showing 1d volume in embryos that were subsequently assayed for eye development. 1d cells were measured in situ following compression at third cleavage (compressed embryos) or ablation of neighboring cells during the mid four-cell stage (partial embryos). Black boxes represent individuals that developed one or more eyes; light-gray boxes represent eyeless individuals. Normal micromere volumes (data from Fig. 6; dark-gray boxes) are shown on the same scale for comparison. (B) A three-eyed larva that developed after compression during third cleavage. Scale bar: 100 µm.

View larger version (9K): [in a new window]   Fig. 9. Hypothesis to explain the difference in 1d division asymmetry when the polar lobe or the A/C cells are removed. Micromeres are shown with their apical pole towards the top; broken lines indicate the plane of their first division. (A) The normal 1a, 1b and 1c micromeres, which are presumably equivalent to the 1d micromere formed after polar lobe ablation. (B) In the normal 1d cell, a polar lobe-derived factor repositions the division plane with reference to the apical pole. (C) In partial embryos, the polar lobe-derived factor still positions the 1d division plane, but as the cell is bigger, division occurs proportionally higher on the axis of the cell. In this diagram, the resulting volume of the apical cell is intermediate between the normal volumes of 1d1 and 1a1/1b1/1c1.