Figure 1.
Sry-α regulates cortical F-actin levels.
(A) Schematic of cellularization. In wild-type embryos (WT), furrows ingress between nuclei (purple) to form mononucleate cells. In mutants with reduced F-actin, some furrows regress, resulting in multinucleate cells (orange nuclei). (B) Surface views of furrow canals stained for Myosin-2 in embryos of indicated genotypes. Multinucleate cells highlighted (orange nuclei) in corresponding segmented images. (C) Cross sections show F-actin levels, as detected by phalloidin staining. Arrow indicates furrow canal position. (D) Quantification of F-actin levels in furrow canals. Each point represents one embryo with ≥75 furrow canals measured (mean ± s.d.). (E) Quantification of Amphiphysin (Amph) tubules. Each point represents one embryo with ≥100 furrows analyzed.
Figure 2.
Sry-α and Spt are paralogs with redundant F-actin binding activity.
(A) Domain structure of Vinculin/α-Catenin Superfamily members. Vinculin Homology domains 1, 2, and 3 (VH1, VH2, VH3) are orange, purple, and blue, respectively. The green and gray domains indicate sequences shared between α-Catenin, Sry-α, and Spt. White areas indicate unique sequences. Dashed lines are guides to show how the different proteins align with each other. (B) Cladogram of Vinculin/α-Catenin Superfamily. Bootstrap statistics at branch points (1000 iterations). Outgroup is M. brevicollis Vinculin. Shading highlights division of the clades. (C, D) Cross sections show (C) Sry-α and (D) Spt-HA co-localization with F-actin in furrow canals. Arrows indicate furrow canal position. (E) Western blots for an F-actin co-sedimentation assay show Sry-α full-length protein (GST-Sry-α) and Spt truncate (GST-SptΔ; amino acids 250–461) pellet with F-actin. α-Actinin and GST are positive and negative controls, respectively. S, supernatant; P, pellet. (F) Quantification of percent proteins bound to F-actin from three independent co-sedimentation assays (mean ± s.e.m.). Student's t-test was performed to calculate P values as shown.
Figure 3.
sry-α and spt induce the same loss of function phenotype.
(A) Surface views of furrows and nuclei, detected in living embryos (membrane Gap43-mCherry, green; nuclear Histone-GFP, purple), following the indicated sry-αRNAi or sptRNAi treatment. Multinucleate cells highlighted (orange nuclei) in corresponding segmented images. (B) Quantification of multinucleation phenotypes. Each point represents one embryo with ≥150 nuclei analyzed (n = 12 embryos per condition). (C) Frequency of multinucleation for embryos following the indicated RNAi treatment (n≥6,000 nuclei from 30 embryos per treatment; mean ± s.e.m.). Student's t-test was performed to calculate P values as shown in (B, C).
Figure 4.
Sry-α and Spt share redundant function during cellularization.
(A) Surface views of furrow canals stained for Myosin-2 in embryos of indicated genotypes. Multinucleate cells highlighted (orange nuclei) in corresponding segmented images. (B) Quantification of multinucleation phenotypes. Each point represents one embryo with ≥150 nuclei analyzed (n = 12 embryos per condition). (C) Quantification of F-actin in furrow canals of furrows of length 3–5.5 µm (n = 9 embryos per genotype, with 15 furrows analyzed per embryo; mean ± s.e.m.). Student's t-test was performed to calculate P values as shown in (B, C).
Figure 5.
Spt is maternally provided, while Sry-α is zygotically expressed in a pulse for cellularization.
(A) RT-PCR and (B) Western blots show Sry-α and Spt expression profiles. Developmental stages are (1) mitotic cycles 1–9; (2) mitotic cycles 10–11; (3) mitotic cycles 11–13; (4) cellularization (shaded); (5) ventral furrow formation; (6) segment formation. Profiles were obtained from three independent RT-PCR or Western Blotting experiments (mean ± s.e.m.).
Figure 6.
Sry-α is not required for cellularization at an optimal temperature of 18°C, but is required as conditions approach an extreme of 32°C.
(A) Surface views of furrow canals stained for Myosin-2 in embryos of indicated genotypes. Multinucleate cells highlighted (orange nuclei) in corresponding segmented images. (B) Quantification of multinucleation phenotype for indicated genotype and temperature. Each point represents one embryo with ≥150 nuclei analyzed (n = 12 embryos per condition). Two-way ANOVA analysis was performed to calculate P values as shown.
Figure 7.
Two copies of sry-α are required for reliable cellularization at the extreme temperature of 32°C or when the embryo is challenged by a genetic perturbation.
(A, C) Frequency of multinucleation for heterozygous embryos reared (A) at 32°C, or (C) with a reduced dose of maternal Profilin (½profilin; mean ± s.e.m.). (B, D) Quantification of multinucleation phenotype for heterozygous embryos reared (B) at 32°C, or (D) with a reduced dose of maternal Profilin (½profilin). Each point represents one embryo with ≥150 nuclei analyzed (n = 23 embryos per condition). Two-way ANOVA analysis was performed to calculate P values as shown in (A–D).