ROCK enrichment in the skeletogenic cells increases with time and depends on VEGF signaling.

(A-R) ROCK immunostaining at different times points in control and under VEGFR inhibition (150 nM Axitinib). In each condition and time point, the left image shows ROCK immunostaining and the skeletogenic cell marker, 6a9 (A, D, G, J, M and P). The middle image shows ROCK immunostaining alone in the whole embryo (B, E, H, K, N and Q). The right image shows the enlargement of the white rectangle region of the middle image (C, F, I, L, O, R). Scale bar in whole embryo is 50µm and in enlargements is 10µm. E – ectoderm, SC – skeletogenic cells. (S) quantification of the ratio between ROCK signal/area in the skeletogenic cells compared to the ectodermal cells (see methods for details). Each box plot shows the average marked in x, the median, the first and the third quartiles (edges of boxes) and all experimental measurement (dots). Experiments were performed in three independent biological replicates and in each condition at least 33 embryos were measured. Statistical significance was measured using paired 2-tailed t-test where, * indicates P<0.05, ** indicates P<0.005, and *** indicates P<0.0005.

ROCK activity is essential for spicule formation, normal elongation and branching in whole embryos and in skeletogenic cultures.

(A-E) genetic perturbation of ROCK translation using two different MASOs observed at 2dpf. (A) control embryo injected with Random MASO. (B) embryo injected with ROCK MO-2 shows ectopic spicule branching. (C, D) embryos injected with ROCK MO-1 or MO-2 show spicule initiations. (E) summary of MASO injection phenotypes based on 4-6 independent biological replicates. (F-K) pharmacological perturbations of ROCK activity using 10µM and 80µM of the inhibitor Y27632 observed at 2dpf. (F) representative control embryo with normal skeletal rods, B, body; AL, anterolateral; PO, post-oral and MV, midventral. (G) complete skeletal loss in embryo treated continuously with 10µM ROCK inhibitor. (J) reduced skeletal growth and enhanced ectopic branching in embryo where 10µM ROCK inhibitor was added at 25hpf. (J) small spicules with enhanced ectopic branching in embryo where 80µM ROCK inhibitor was added at 25hpf. (K) summary of perturbation phenotypes based on 3-8 biological replicates for each treatment. See additional concentrations, phenotypes and summary in Fig. S4 and Table S1. (L-O) representative spicules from skeletogenic cell cultures in control and under 30µM Y27632 at 72hpf. (L) linear spicule in control culture, (M) Y27632 addition at 16hpf, before spicule initiation, completely blocks spiculogenesis. (N, O) Y27632 addition after spicule initiation, at 48hpf, reduces spicule elongation (N), and enhances branching (O). (P) quantification of spicule length in control and ROCK inhibition (>48hpf) at 72hpf. *P < 0.05, **P < 0.001, Kruskal-Wallis non-parametric test. Results are based on three biological repeats for each treatment, except from 120µm that was done in two biological repeats. Scale bars are 50µm. In L, N and O, the numbers at the bottom indicate the number of spicules that show this phenotypes (left) over all observed spicules (right).

SR-µCT studies of ROCK inhibited spicuels show reduction in skeletal volume, surface area and total length, but not thickness and 2-pulse calcein shows loss of tip-dominance.

(A) Exemplary 3D-renderings of control spicules (top, blue) and spicules where 40µM of ROCK inhibitor was added at 25hpf (bottom, orange), dissected at 48hpf and 72hpf. Arrowheads point to tip splitting and arrow at 48hpf point to spicule dripping at the back. (B) Spicule volume vs. area for control and ROCK-inhibited spicules at 48 and 72 hpf. Each data point represents a single spicule. (C-D) Frequency distributions for volume and surface area of control and ROCK-inhibited spicules at 48 hpf and 72 hpf (left and right violin plots, respectively). (E) Spicules’ total branch length and average thickness for control and ROCK-inhibited spicules at 48 and 72 hpf. (F-G) Frequency distributions for spicule lengths and thickness of control and ROCK-inhibited spicules dissected at 48hpf and 72hpf. (H, I) Calcein two-pulses experiment. Embryos were exposed to blue calcein at 29-30hpf, to green calcein at 44-45hpf and stained with FM4-64 membrane marker (red) prior to image acquisition at 48hpf. (H) Control embryo, (G) Embryo where 30µM of Y27632 was added at 25hpf. The experiments were done in three biological replicates and the numbers at the bottom indicate the number of embryos that show this phenotype out of all embryos scored.

Actin polymerization and myosin activation affect skeletal growth and branching.

(A-F) representative embryos showing the effect of actomyosin perturbations at 2df. (A) control embryo (B) embryo treated with 2μM Blebbistatin >25hpf , (C) embryo treated with 2μM Blebbistatin >20hpf, (D) embryo treated with 2nM Latrunculin-A and 1.5μM Blebbistatin >25hpf, (E) embryo treated with 2nM Latrunculin-A >20hpf, (F) embryo treated with 2nM Latrunculin-A >25hpf, arrow pointing to the additional spicule rod. (G) Statistics of Latrunculin-A and Blebbistatin phenotypes, color code of phenotype is indicated in the representative images. Error bars indicate standard deviation. All treatments were conducted in at least three biological replicates and exact number of replicates and scored embryos are provided in table S4. (H-J) representative spicules recorded at 72hpf from (H) control skeletogenic cell culture, (I) skeletogenic cell culture were 2nM Latrunculin-A was added at 48hpf and (J) skeletogenic cell culture were 2μM Blebbistatin was added at 48hpf. (K) Quantification of spicule length in the different treatments at 72hpf **P < 0.001, Kruskal-Wallis non-parametric test. Results are based on three biological repeats for each treatment. Scale bars are 50μm.

ROCK inhibition effect on F-actin organization and MyoII activity at 27hpf.

Representative images showing normal embryo (A-J) and embryos treated with ROCK inhibitor from fertilization (K-T, 80µM), at 27hpf. Phalloidin (green) was used to stain F-actin, MyollP (red) was used to stain phosphorylated myosinII and 6a9 (blue) was used to mark the skeletogenic cells. Right panels show enlargements of the rectangle sections marked in the left most panels. Green arrows point to regions that show enriched F-actin signal, blue arrows point to the pseudopodia cable. The blue line in B, C and E marks a region of the pseudopodia cable that is stained by 6a9 but has low F-actin signal. The experiments were done in 3 biological replicates, the numbers at the bottom left of (A, K) indicate the number of embryos that show this phenotype out of all embryos scored. Scale bar in A, F, K and P is 50μm, and in B, I, N and S is 10μm.

F-actin filaments are enriched at the tips of the spicules and the tip to back ratio is unaffected by ROCK inhibition

(A-J) representative images at 33hpf, showing normal embryo (A-E) and embryos treated with 30µM ROCK inhibitor from the gastrula stage (>25hpf, F-J). Embryos are stained with Phalloidin (green), MyollP antibody (red) and 6a9 (blue). (B-E, G-J) enlargement sections of the spicule area marked with rectangle in A and F. White arrowheads point to the enriched F-actin signal at the tips. Blue arrows point to the region of the pseudopodia cable that is not filled with the spicule cavity. (K, L) quantification of the tip to back F-actin signal (number of green pixels per area) at 33hpf in control embryos and ROCK inhibition >25hpf. Each box plot shows the average (x), median (middle line), the first and the third quartiles and all the points measured. Asterisks indicate statistical significance in paired t-test, where * is p<0.05 and *** is p< 0.0005, NS-not significant. (M-V) similar experiments to (A-J), at 48hpf, 40µM of ROCK inhibitor added at 25hpf. Red arrows point to non-skeletogenic cells enriched with MyoIIP. The experiments were repeated in 3 biological replicates and the numbers at the bottom left of (A, F, M, R) indicate the number of embryos that show this phenotype out of all embryos scored. Scale bar in A, F, M, R is 50µm and in B, G, N, S, is 10µm. (W, X) representative spicules out of three biological replicates from control skeletogenic cell culture (W, n=60), and skeletogenic cell treated with 30µM ROCK inhibitor added at 48hpf and recorded at 72hpf (X, n=52). Left panel is phase image, middle panel is phalloidin staining and right panel shows the overlay. Green arrows point to the enhanced F-actin signal at the tips. Scale bar is 20μm.

ROCK activity is essential for normal gene expression in the skeletogenic cells.

(A, B) the effect of 80µM ROCK inhibition on gene expression. (A) continuous ROCK inhibition at 27hpf and 50 hpf (n=4). (B) addition of ROCK inhibitor at 25hpf and the wash of ROCK inhibitor at 25hpf, measured at 50hpf (n=3). Asterisks indicate p<0.001, one tailed z-test. Error bars show standard deviation. (C-F) Representative images of control embryo (top panels), embryos where ROCK inhibitor was added at 25hpf (middle panels), and embryos that were exposed to continuous ROCK inhibition (bottom panels), at the pluteus stage (∼48hpf). (C) skeletogenic cell marker, 6a9. MV, midventral; AL, anterolateral, and PO, Post-oral rods, D, dorsal skeletogenic chain. (D-F) spatial expression of skeletogenic genes. Gene names are indicated at the top of each panel. Numbers at the bottom of each image indicate the number of embryos that show this phenotype (left) out of all embryos scored (right), conducted in at least three independent biological replicates.

The role of ROCK and the actomyosin network in sea urchin skeletogenesis, summary.

(A) the spicule forms at about 24hpf in P. lividus and at 27hpf, the triradiate spicule is coated with F-actin. (B) ROCK activity is required for spicule initiation and for F-actin organization around the spicule. (C) model of the functional links between the skeletogenic GRN, ROCK, F-actin and their skeletogenic outcomes, during spicule initiation (∼24-27hpf in P. lividus). Cian arrows indicate links discovered in this work. (D) during skeletal elongation F-actin is detected around the spicule cavity and is enriched at the tips of the rods. The expression of SM50 and some other skeletogenic genes, is localized to the tips of the rods. (E) under the addition of ROCK inhibitor, skeletal growth rate is reduced and ectopic spicule branching is observed. The expression of skeletogenic genes is localized to the vicinity of the growing rods. (F) the functional links between the skeletogenic GRN, ROCK, F-actin and MyoII and their skeletogenic outcomes during skeletal elongation. Cian arrows indicate links discovered in this work.