JCS043471 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Hinge point and ZO-1 defects are produced by translation-blocking zic morpholinos and are not apoptosis-dependent. (A,B) ZO-1 staining is patchy and fails to separate correctly in the dorsal midbrain of zic2a morphants (B, n=7 of 7). (C,D) Z0-1 staining is unaffected, or affected very mildly, more ventrally in the morphants (D). (E,F) Hinge points fail to form in translation-blocking zic morphants (n=18 sections from four morphant embryos). (G,H,I) Apical F-actin seam (labeled with phalloidin) is formed in p53 MO-injected embryos (G, 14 of 14 total embryos develop normally), but fails to form in zic morphants generated using splice-blocking MOs (H, n=10 of 11 embryos) or embryos co-injected with morpholinos against p53 and zic2a and zic5 (I, n=6 of 6 embryos). Images show horizontal confocal sections through the midbrain (A-D, G-I) or cross-sections through the midbrain (E,F). In A-F, uninjected controls are shown on the left, Zic morphants on the right. Scale bar: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Active myosin II is reduced in zic morphant midbrains. All images are midbrain cross-sections of embryos immunostained for rMLC-P. Control morphants are on the left, Zic morphants on the right. (A,B) 19-somite stage. (C,D) 23-somite stage.

• Supplemental Figure S3 -

  Fig. S3. F-actin localization is disrupted in living zic morphants. Still images were extracted from movies of embryos labeled with mCherry-Utrophin (actin, red) and Histone2B-GFP (nuclei, green). (A-C) Control morpholino-injected embryo. (A) t=0 minutes, approximately 14-somite stage. (B) t=120 minutes, approximately 18-somite stage. (C) A high magnification view of the boxed area in B showing strong, uniform apical F-actin localization. (D-F) Zic morpholino-injected embryo. (D) t=0 minutes, approximately 14-somite stage. (E) t=120 minutes, approximately 18-somite stage. (F) A high magnification view of the boxed area in E showing weak, disorganized F-actin. All images are horizontal confocal sections through the dorsal midbrain with anterior to the left. Scale bars: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. Apical actin and ZO-1 are affected in 24 hour Zic morphants. All images are single Z-planes through the dorsal midbrain of embryos stained for ZO-1 (red) and F-actin (green). A, D and E are the dorsal most sections, approximately 15 µm into the embryo. C, F and I are the most ventral sections, approximately 35 µm into the embryo. Scale bar: 50 µm. Arrowheads mark the apical actin seam. (A-C) Control morphant embryo. (D-F) Zic morphant embryo scored as having a small lumen before fixation. (G-I) Zic morphant embryo scored as having no lumen before fixation.

• Supplemental Figure S5 -

  Fig. S5. Real-time PCR analysis shows equivalent levels of ccnB1 transcript in standard control morpholino and zic2a and zic5 morpholino-injected embryos. Preparation of cDNA: total RNA was extracted with Trizol (Invitrogen) from 15 morphant and 15 control embryos at 18-19S and reverse-transcribed in duplicate with an iScript Select cDNA Synthesis kit (BioRad) using oligo-dT primers. Duplicate reactions were pooled for use as RT-PCR template. Real-Time PCR: Primer design: Primers were designed as described by Shepard et al., 2005 against sequences that spanned an intron of the corresponding gene and produced a ∼200 bp amplicon. Primer specificity was ensured by agarose gel electrophoresis and melting curve analysis after qPCR. Reaction set-up: each 20 µl reaction was comprised of the following: 1× Power SYBR Green Master Mix (Applied Biosystems), 200 nM of each primer, and 2 µl cDNA template. Four reactions were made for each template analyzed during a single technical replicate. No RT and no template control reactions were performed for each sample. Reaction conditions: Reactions were run on an Applied Biosystems ABI 7500. Data were analyzed using the dCt method.

• Movie 1 -

  Movie 1. Formation of apical F-actin seam in control morphant. Horizontal confocal section through the dorsal midbrain, anterior is to the left. Movie begins at 14-somite stage and ends at 18-somite stage. Images were taken every minute for 2 hours.

• Movie 2 -

  Movie 2. Formation of apical F-actin seam in zic2a and zic5 morphant. Horizontal confocal section through the dorsal midbrain, anterior is to the left. Movie begins at 14-somite stage and ends at 18-somite stage. Images were taken every minute for 2 hours.