Polarity protein Par3/Bazooka follows myosin-dependent junction repositioning

Abstract

The polarity protein Par3/Bazooka (Baz) has been established as a central component of the apical basal polarity system that determines the position of cell-cell junctions in epithelial cells. Consistent with that view, we show that shortly before gastrulation in Drosophila, Baz protein in the mesoderm is down-regulated from junctional sites in response to Snail (Sna) expression. This down-regulation leads to a specific decrease in adherens junctions without affecting other E-Cadherin pools. However, we further show that, interactions between Baz and junctions are not unidirectional. During apical constriction and the internalization of the mesoderm, down-regulation of Baz is transiently blocked as adherens junctions shift apically and are strengthened in response to tension generated by contractile actomyosin. When such junction remodeling is prevented by down-regulating myosin, Baz is lost prematurely in mesodermal epithelium. During such apical shifts, Baz is initially left behind as the junction shifts position, but then re-accumulates at the new location of the junctions. On the dorsal side of the embryo, a similar pattern of myosin activity appears to limit the basal shift in junctions normally driven by Baz that controls epithelium folding. Our results suggest a model where the sensitivity of Baz to Sna expression leads to the Sna-dependent junction disassembly required for a complete epithelium-mesenchymal transition. Meanwhile this loss of Baz-dependent junction maintenance is countered by the myosin-based mechanism which promotes an apical shift and strengthening of junctions accompanied by a transient re-positioning and maintenance of Baz proteins.

Fig. 1. Baz shifts apically in mesoderm during apical constriction

(A) A schematic of a Drosophila embryo during cellularization showing orientation of cross-sections, apical-basal axis of the epithelium, the positions of the future ectoderm and mesoderm and the localization of adherens junctions. (B) Adherens junctions appear as clusters at the subapical position during cellularization. (C–F) Schematics of cross-sections of embryos at different stages: early to mid cellularization (C), late cellularization to early gastrulation (D), bending of mesodermal epithelium (E) and internalization of mesoderm (F). (G–L) Distributions of Baz and myosin during the corresponding stages. (G) Baz localizes at the subapical positions during early to mid cellularization. (H–J) As myosin starts to accumulate, Baz moves from subapical position to apical position. (K) Baz localizes to apical position at low levels when myosin drives the tissue shape change. (L) Baz fades as the mesoderm is internalized.

Fig. 2. Baz follows adherens junctions during their apical shift

(A) Tracking of an individual junction (GFP) and Baz (mCherry) cluster in wild type mesoderm during apical constriction. Tiles of 3-D reconstructed images show that the junction cluster moves apically while Baz is first left behind, diminishes and then recovers at the junctional region at lower levels (yellow arrows). (A′) The enlarged image of the boxed area. Tiles are 7.5 s apart in time. (B–C) Junctions in wild type ectoderm stay at subapical position from cellularization to gastrulation. (D) When myosin is activated in ectoderm cells by expressing Fog, there is a transient separation of junctions and Baz. (E) When junctions reach the most apical edge of the cell, Baz again overlaps with junctions. B′ and D′ are high-magnification images from B and D. Images are maximum projections from 6 μm thick image stacks. Scale bar: 10 μm.

Fig. 3. Sna downregulates Baz in mesoderm cells prior to gastrulation

(A–D) Wild type embryos at different stages of development from mid-cellularization (A), late cellularization (B), to gastrulation (C–D). Zoom-in images show cells from the dorsal (A′–D′), lateral (A″–D″) and ventral (A‴–D‴) regions. Images have been rotated so that the cells are of the same orientation with the apical side up. All zoom-in images are of the same setting to allow comparing the ratio between Baz and Arm in the three regions of the same embryo. Compared to dorsal and lateral sides, the merged images of ventral cells become increasingly greener indicating the Baz:Arm ratio becomes lower as the embryos develop. Scale bars: 20 μm (A–D) and 10 μm (A′–D′, A″–D″, A‴–D‴). (E) Ratios between Baz and Arm fluorescent intensity in mesoderm and ectoderm of fixed embryos at similar stages as embryos in A–D. For the ratios at each stage, at least three embryos are quantified. (F) Kymograph of Baz-mCherry (F) and E-Cad-GFP (F′) in ventral cells from late cellularization to gastrulation. (G) Total intensity of Baz-mCherry and E-Cad-GFP in ventral cells from later cellularization to gastrulation. Average values from 3 embryos are shown. (H) In wild type embryos, Baz is downregulated in mesoderm cells identified by their Sna staining (H′). (I) In sna mutant embryos Baz pattern is indistinguishable between ectoderm and mesoderm, in this cases, identified by Twist (Twi) staining (I′). Scale bar: 50 μm.

Fig. 4. Sna expression is sufficient to disassemble junctions via downregulation of Baz

(A–D) Cross-sections of fixed embryos of different genotypes showing localization patterns of Baz and Arm. (A″-D″) High-magnification images showing different Cadherin/Catenin pools. Green arrowhead: adherens junctions in ventral mesoderm cells. Green arrow: adherens junctions in ectoderm cells. Yellow arrowhead: non-junctional membrane associated Cadherin/Catenin. Yellow arrow: basal junctions. Basal junctions are only present in the ectoderm at this stage as cellularization is finished in mesoderm cells. (A‴–D‴) High-magnification images showing Baz distributions. (E–F) Compared to wild type (E), depletion of Baz (F) leads to the loss of adherens junctions in ectoderm cells without affecting myosin activation (E–F) and other Cadherin/Catenin pools (E″–F″). It thus phenocopies ectopic Sna expression. Arrow annotations are the same with A–D. Scale bars: 20 μm (A–D, A′–D′, A⁗–D⁗, E–F, E′–F′) and 10 μm (A″–D″, A‴–D‴, E″–F″).

Fig. 5

Myosin is necessary to maintain Baz localization in mesodermal cells and ectopic activation of myosin relocates Baz to the apical surface. (A) Arm and Baz staining in the wild type embryo. (B) Loss of Sqh (the myosin regulatory light chain) leads to loss of Baz and junctions in the ventral mesoderm cells while ectoderm cells are largely unaffected. (C) Failure in activating myosin by removing cta and T48 leads to similar loss of Baz and junctions specifically in the mesoderm. (D) Activation of myosin by Fog overexpression leads to relocation of Baz and junctions to the apical edge of the cell. (E) Baz is maintained in the presence of Sna expression when Fog is expressed simultaneously. Scale bar: 20 μm.

Fig. 6. Fog expression between dorsal folds optimizes the morphology of the epithelial folds

(A) Fog is expressed at low levels between the future dorsal folds, whose positions are indicated by the second and fifth Runt stripes (yellow arrows). (B–C) Scanning EM images show two transverse furrows (yellow arrows) form in the wild type embryo (B) but are abolished in the embryos with Fog expressed throughout the dorsal ectoderm (C). (D–F) Dorsal folds are deeper in the fog mutant embryos carrying hkb-fog compared to the embryos expressing hkb-fog alone. Male embryos carrying a fog mutant X chromosome and a Y chromosome are identified by the absence of nuclear Sex-lethal (Sxl) staining. Scale bars: 50 μm (A–C, D–E, D″–E″) and 20 μm (D′–E′).

Fig. 7. Relationships between Baz, adherens junctions and myosin during cellularization and gastrulation

(A) During early to mid cellularization, Baz localization at the subapical position determines the position of adherens junctions. (B) At late cellularization, the expression of Sna leads to the downregulation of Baz which in turn results in lower level of adherens junctions. (C) Upon myosin activation, myosin-dependent junctions maintenance and strengthening leads to an increase in junction levels and delayed Baz loss in the presence of Sna expression. Due to myosin network engaged with adherens junctions, cells are apically constricted and apical-basally elongated (He et al., 2014). (D) As myosin fades after the internalization of mesodermal tissues, both adherens junctions and Baz are eventually lost.