DEV016337 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. SRF-VP16 expression does not cause cell shape, AJs or actin changes. Embryos with anterior leftwards, genotypes, GAL4 drivers and antigens indicated. (A-F) Dorsal closure. (A-D) Expression of SRF-VP16 does not alter epidermal cell shape, or levels or localization of AJ proteins. (E,F) Expression of SRF-VP16 does not cause changes in actin. (F) In some cases, embryos expressing SRF-VP16 have slightly deeper segmental grooves (arrowheads). (G) Immunoblot showing Myosin and DE-cad in wild type, Dia^CA × e22c-GAL4 and SRF-VP16 × e22c-GAL4 (this GAL4 driver drives in all ectodermal cells). Tubulin is used as a loading control. (H) Northern blot showing MHC total RNA levels in wild type, Dia^CA × e22c-GAL4 and SRF-VP16 × e22c-GAL4. RP49 is useed as a loading control. Scale bars: 20 µm

• Supplemental Figure 2 -

  Fig. S2. The dia⁵ allele is not null. (A) Diagram of dia⁵ genomic region, showing position of P-element in first intron of one dia transcript and 5′ UTR of the other. (B) Dia levels in dia⁵M/Z (embryo on right) versus zygotically rescued dia⁵M (embryo on left). (C) Immunoblot, hand-selected wild-type and dia⁵M/Z mutant embryos probed for Dia and α-tubulin as a loading control. Embryos identified using a twist-GFP-Balancer. (D) Embryonic lethality of progeny of dia⁵ germline mutant females crossed to dia²/+ males is temperature sensitive. Scale bars: 20 µm

• Supplemental Figure 3 -

  Fig. S3. Cortical actin and Myosin are not dramatically altered by Dia reduction. (A-F) Embryos with anterior leftwards, genotypes and antigens indicated. Control embryos are from same slide. (A,C,E) Wild-type embryos. (B,D,F) dia⁵M/Z mutants. Scale bars: 20 µm

• Supplemental Figure S4 -

  Fig. S4. Dia and Rho exhibit non-allelic non-complementation. Cellularizing embryos with genotypes and antigens indicated. (A-C) Surface view. (D-J) Cross-sections. (A,D,G) Wild type. (B,E,H) Progeny of dia +/+ Rho1 mothers. (C,F,I) Maternal dia⁵ mutants. (J) Progeny of dia²+/+ RhoGEF2⁰⁴²⁹¹ mothers. (B,C) Multinucleate cells (arrows). (E,F) Nuclear fallout (arrows). (G-J) Pole cells or lack thereof (white bar). Scale bars: 20 µm

• Supplemental Figure S5 -

  Fig. S5. β-Integrin levels and localization are not dramatically altered in dia/Rho1 mutants. Embryos with anterior leftwards, genotypes and antigens indicated. (A-D) Germband retraction. Scale bars: 20 µm.

• Supplemental Figure S6 -

  Fig. S6. UAS-EGFP::Dia map. UAS-EGFP::Dia vector. UAS-EGFP::Dia was made using Gateway technology (http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html). Dia cDNA was amplified using a forward primer led with CACC for directional TOPO cloning followed directly by Dia ORF. The resulting entry vector was used to recombine Dia into pPGW, which is the UASg destination vector for N-terminal GFP tagging.

• Movie 1 -

  Movie 1. Wild-type ventral furrow formation showing Moesin::GFP. Images were collected every 15 seconds but this movie displays only one frame/45 seconds. Video is displayed at 10 frames/second. Frames are in Fig. 6A.

• Movie 2 -

  Movie 2. dia⁵M ventral furrow formation showing Moesin::GFP. Images were collected every 15 seconds but this movie displays only one frame/45 seconds. Video is displayed at 10 frames/second. Frames in Fig. 6B.

• Movie 3 -

  Movie 3. Wild-type germband retraction showing Moesin::GFP. Images were collected every 15 seconds but this movie displays only one frame/45 seconds. Video is displayed at 10 frames/second. Frames are in Fig. 8I.

• Movie 4 -

  Movie 4. dia²/Rho1 germband retraction showing Moesin::GFP. Images were collected every 15 seconds but this movie displays only one frame/45 seconds. Video is displayed at 10 frames/second. Frames are in Fig. 8H.

• Movie 5 -

  Movie 5. dia²/Rho1 germband retraction (Embryo#2, tail view) showing Moesin::GFP. Images were collected every 15 seconds but this movie displays only one frame/45 seconds. Video is displayed at 10 frames/second.