Fig. 5

Chondroitin sulfate is required for the hydration of Versican-hyaluronate ECM. (A) 3D-rendered OVs showing hyaluronic acid (HA), chondroitin sulfate (CS), and F-actin using HA-binding protein (HABP), anti-chondroitin sulfate antibody, and phalloidin, respectively, in embryos injected with control buffer or chase (10 U/ml), stained at 3 hpi. Scale bar: 50 μm. (B) Quantification of fluorescence intensity of HABP in embryos injected with control buffer or chondroitinase (Chase) at 3 h post-injection (hpi). Maximum intensity projection images (20 μm-thickness) of HABP in the lateral bud region were used for the quantification. Data are mean±s.e.m. n denotes the number of embryos measured from two independent experiments. P-value as labeled (paired, two-tailed Student's t-test). (C) Composites of 2D sections from membrane-NeonGreen-expressing embryos before injection or at 3 hpi of control buffer or Chase. Pre-injection and post-injection images were acquired from the same embryo. The bud-ECM region is labeled with 3 kDa Texas-Red Dextran. Anterior, posterior, and lateral buds from different z-depths are framed in blue, yellow, and white boxes, respectively. Scale bar: 50 μm. (D) Quantification of bud length (left), bud-ECM volume (right) of the anterior bud in embryos before injection or at 3 hpi of control buffer or Chase. Bud-ECM volume was measured using the bud-ECM region labeled by Texas-Red Dextran. n denotes the number of embryos measured from two independent experiments. P-values as labeled (paired, two-tailed Student's t-test). (E) Representative 2D sections of anterior bud in membrane-NeonGreen-expressing embryos injected with control buffer or chase at 3 hpi. (F) Quantification of cell height (F) and cell aspect ratio (height/width) (F′) of the anterior bud in the membrane-NeonGreen-expressing embryos before injection or at 3 hpi of control buffer or chase. n denotes the number of embryos measured from two independent experiments. P-values as labeled (unpaired, two-tailed Student's t-test). (G) Composite of 2D sections of OVs showing percolation of dextran from periotic region into bud-ECM region at 2 hpi. Control buffer or Chase was co-injected with Texas-Red dextran (magenta; 3, 10, or 70 kDa) and aBt (green) in embryos expressing membrane-NeonGreen (blue). aBt colocalizes with all three dextran sizes in the periotic region (white). Contrast is the same across embryos. Experiments with 10 kDa dextran were performed on different days than experiments with 3 kDa and 70 kDa dextrans. Scale bar: 50 μm. (H) Quantification of fluorescence intensities of different sizes of dextran in the bud-ECM region normalized to their intensities in the periotic space. Data are mean±s.e.m. n denotes the number of embryos measured from two independent experiments. P-values as labeled (one-way ANOVA with Tukey's test). (I) A schematic model illustrating the three classes of bud-ECM behaviors regulated by Versican and its CS side chains: In vcana and vcanb knockdown (KD) conditions, reduced HA and CS accumulation lead to decreased bud-ECM volume; selective inhibition of CS [Chondroitinase (Chase) treatment] results in unaffected HA levels but increased ECM density and reduced ECM hydration and volume; and an intermediate regime, where Versican facilitates both HA and CS accumulation, promotes intermediate ECM density, significant hydration, and bud extension. Created in BioRender. Munjal, A. (2024) https://BioRender.com/d61d142. Representative images from two independent experiments are shown.