DEV031815 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1.

• Movie 1 -

  Movie 1. DIC movie of wild-type embryo. Syncytial divisions are synchronous and cellularization progresses uniformly.

• Movie 2 -

  Movie 2. DIC movie of smg mutant embryo. Rapid syncytial division leads to mitotic failure and ‘nuclear drop-out’. The embryos do not cellularize.

• Movie 3 -

  Movie 3. DIC movie of embryo expressing Smaug in a gradient. Mitosis starts at the posterior pole and progresses in a wave towards the anterior. Cellularization initiates synchronously between 100% and 75% EL, and progresses in a gradient toward the posterior pole between 75% and 0% EL.

• Movie 4 -

  Movie 4. DIC movie of embryo expressing Smaug in a gradient. Mitosis start at the posterior pole and progress in a wave towards the anterior. Nuclear drop-out at the posterior leads to uneven nuclear distribution. Cellularization initiates and division stops at the anterior pole, whereas the center of the embryo progresses through an additional mitosis and cellularizes later, in an anterior-to-posterior gradient.

• Movie 5 -

  Movie 5. DIC movie of wild-type embryo, focusing on the posterior pole. Syncytial divisions are synchronous and cellularization progresses uniformly. Note that development is somewhat faster than in Movie S1, owing to higher ambient temperature. Movie generated under identical conditions to those in Movie 6.

• Movie 6 -

  Movie 6. DIC movie of embryo expressing Smaug in a gradient. Cellularization progresses in a gradient toward the posterior pole and is completed ∼24 minutes later than in wild type (compare with Movie 5). Development is somewhat faster than in Movies 3 and 4, owing to higher ambient temperature. Movie generated under identical conditions to those in Movie 5.

• Supplemental Table S2 -

  Table S2. Zygotic genes identified by microarray. Genes are listed in the same order as they appear in Fig. 2A.

• Supplemental Table S3 -

  Table S3. Expression of maternal genes downregulated by the mir-309 cluster in wild type and smg mutants. The data are in the same order as they appear in Fig. 2D. Our arrays contain 406 out of the 410 maternal mRNAs reported to be stabilized in miR-309 mutants. Mean Log 2 Ratios are given.

• Supplemental Figure S1 -

  Fig. S1. Mutations in mnk do not suppress the cellularization defects associated with smg. Wild-type (A) and mnk mutant (D) embryos cellularize during interphase 14, with uniformly distributed nuclei (red) surrounded by membranes (green). Mutations in both grp (B) and smg (C) block cellularization, resulting in disorganized nuclei without membrane invagination. (E) Embryos mutant for both grp and mnk cellularize, despite defects in nuclear organization. (F) However, embryos double mutant for smg and mnk fail to cellularize and arrest as disorganized syncytia. Nuclei were labeled with TOTO-3 and membranes were labeled with anti-phospho-Tyrosine antibodies. Optical cross sections are shown.

• Supplemental Figure S2 -

  Fig. S2. maternal transcript destruction in mnk grp and mnk; smg double mutant embryos. (A-F) Maternal cyclin B mRNA destruction in wild-type (A,D), mnk grp (B,E) and mnk; smg (C,F) double mutant embryos. Embryos are orientated with their anterior pole facing leftwards. In both wild type and mnk grp double mutants, maternal cyclin B mRNA is expressed in syncytial blastoderm embryos (S, A,B) and degraded during interphase 14 (14, D,E). By contrast, the transcript is stable in mnk; smg double mutants (S and 14, C, F). Maternal transcript destruction is therefore independent of signaling through the Chk2 kinase encoded by mnk.

• Supplemental Figure S3 -

  Fig. S3. miR-309 cluster is transcribed at high levels in embryos. (A,B) Results are shown of miRs purified from staged active unfertilized eggs (A) or embryos (B) hybridized to Exiqon microarrays that probe for all known miRs. The miR-309 cluster is transcribed at low levels or not at all in activated eggs but at very high levels in 2- to 4-hour-old embryos. The units on the y-axis are arbitrary. Standard deviation is shown from two to four replicates of each time point.

• Supplemental Figure S4 -

  Fig. S4. Timing of cellularization as a function of Smaug expression. The time from interphase 10 (NEF) to ‘mid-cellularization’ (when membrane invagination reaches the base of the cortical nuclei) was measured by time-lapse DIC microscopy. For each embryo, times were determined at three points along the anteroposterior axis (black bars for anterior, grey bars for middle and white bars for the posterior). Average time and standard deviation (brackets) are indicated. The number of individual embryos scored is given. Overexpression of Smaug in an anterior to posterior gradient (USB/NGV) or uniformly over the entire embryo (smg^A105) does not alter the timing of cellularization. However, when the Smaug gradient drops below wild-type levels in smg¹ USB/smg¹ NGV embryos, cellularization is delayed over center and posterior regions.

• Supplemental Figure S5 -

  Fig. S5. Low levels of Smaug delay cellularization and gastrulation. Time-lapse DIC images of a wild-type embryo (top row) and an embryo expressing Smaug in an anterior to posterior gradient (bottom row). The sequences focus on the posterior pole. Time in minutes from nuclear migration is shown at the top left of each panel. The white arrows indicate the cellularization front and the red arrows indicate the position of the pole cells as germband extension is initiated. Note that cellularization and germband extension are delayed by ∼20 minutes at the posterior of embryos expressing the Smaug gradient.