An actin-based viscoplastic lock ensures progressive body-axis elongation

Abstract

Body-axis elongation constitutes a key step in animal development, laying out the final form of the entire animal. It relies on the interplay between intrinsic forces generated by molecular motors^1-3, extrinsic forces exerted by adjacent cells^4-7 and mechanical resistance forces due to tissue elasticity or friction^8-10. Understanding how mechanical forces influence morphogenesis at the cellular and molecular level remains a challenge¹. Recent work has outlined how small incremental steps power cell-autonomous epithelial shape changes^1-3, which suggests the existence of specific mechanisms that stabilize cell shapes and counteract cell elasticity. Beyond the twofold stage, embryonic elongation in Caenorhabditis elegans is dependent on both muscle activity⁷ and the epidermis; the tension generated by muscle activity triggers a mechanotransduction pathway in the epidermis that promotes axis elongation⁷. Here we identify a network that stabilizes cell shapes in C. elegans embryos at a stage that involves non-autonomous mechanical interactions between epithelia and contractile cells. We searched for factors genetically or molecularly interacting with the p21-activating kinase homologue PAK-1 and acting in this pathway, thereby identifying the α-spectrin SPC-1. Combined absence of PAK-1 and SPC-1 induced complete axis retraction, owing to defective epidermal actin stress fibre. Modelling predicts that a mechanical viscoplastic deformation process can account for embryo shape stabilization. Molecular analysis suggests that the cellular basis for viscoplasticity originates from progressive shortening of epidermal microfilaments that are induced by muscle contractions relayed by actin-severing proteins and from formin homology 2 domain-containing protein 1 (FHOD-1) formin bundling. Our work thus identifies an essential molecular lock acting in a developmental ratchet-like process.

Figure 1:. Muscle contractions deform the epidermis to their mechanical coupling

(a) C. elegans embryonic elongation from comma to 2-fold stages depends on a ROCK-promoted actomyosin force in seam cells (cyan) and actin-promoted stiffness in dorso-ventral cells (orange); elongation beyond the 2-fold stage requires repeated muscle contractions (red flash), which induce a PAK-1-dependent mechano-transduction pathway. Open cross-sections (bottom) show muscle positions. (b-b’’’) Epidermis actin filament (green) and muscle nucleus (red) tracking in a wild-type 2-fold embryo. (b’) Kymographs from the yellow rectangle area (b) showing the concurrent displacement of epidermal actin and muscle nuclei. (b’’) Resulting displacement curves; (b’’’) quantification of the area between them; its low value underlines the tight mechanical coupling between both tissues. Scale bar, 10 μm. (c-c’’) A muscle contraction/relaxation cycle illustrating its local impact on epidermal actin filaments in a wild-type 2-fold embryo (timing in left corner). Yellow (relaxation), red (compression) green (stretching) distances between landmarks denoted 1–4: (c) [1–2], 7.8 μm; [2–3], 19.8 μm; [3–4], 24.6 μm. (c’) [1–2], 9.4 μm; [2–3], 13.6 μm; [3–4], 26.2 μm. (c’’) [1–2], 8.0 μm; [2–3], 19.2 μm; [3–4], 25.0 μm. In (b-c) the Pepidermis promoter is Pdpy-7. (d) Hysteresis graph of an idealized elastic material returning to its initial shape after deformation (top), or showing permanent deformation (bottom).

Figure 2:. Loss of PAK-1 and SPC-1 triggers a muscle-dependent retraction of embryos

(a) RNAi screen in a pak-1 mutant identified spc-1 as an enhancer (Table S1). (b) DIC micrographs of newly hatched wild-type, pak-1(tm403) (scale bar: 10 μm), spc-1(RNAi) and spc-1(RNAi) pak-1(tm403) (scale bar: 25 μm). Quantification of L1 hatchling body length: wild-type (n=38); pak-1(tm403) (n=32); spc-1(RNAi)(n=26); spc-1(RNAi) pak-1(tm40) (n=36). (c) A yeast two-hybrid screen using the PAK-1 N-term domain as a bait identified the SPC-1 SH3 domain as a prey (orange background) (Table S2). (d) Elongation profiles and corresponding terminal phenotypes of wild-type (n=5), pak-1(tm403) (n=5), spc-1(RNAi) (n=8), spc(RNAi) pak-1(tm403) (n=8). (e) Elongation profiles in a muscle defective background. unc-112(RNAi) (n=5); spc-1(RNAi) (n=8); unc-112(RNAi); pak-1(tm403) spc-1(ra409) (n=5); spc-1(RNAi) pak-1(tm403) (n=8). Right bracket (d, e), extent of retraction for spc-1(RNAi) pak-1(tm403) embryos. Scale bars, 10 μm. Error bars, SEM.

Figure 3:. Actin filament defects in SPC-1 and PAK-1 defective embryos

(a-d) Epidermal actin filaments visualized with the Pdpy-7::LifeAct::GFP reporter construct in wild-type (a-a’’’), pak-1(tm403) (b-b’’’), spc-1(RNAi) (c-c’’’) and spc-1(RNAi) pak-1(tm403) (d-d’’’) embryos at mid-elongation (2-fold equivalent) stage. Yellow rectangle, region of interest (ROI). Scale bar, 10 μm. (a’-d’) ROI after binarisation (green) and major axis detection (red), based on (a’’’) three steps of image treatment for continuity and orientation analysis. (a’’-d’’) Actin continuity: distribution of actin segments based on their length. Wild-type (n=16); pak-1(tm403) (n=21); spc-1(RNAi) (n=21); spc-1(RNAi) pak-1(tm403) (n=17) (b’’’-d’’’) Actin filament orientation: the curves represent the number of actin filaments oriented perpendicular to the elongation axis (90° angle in wild-type) based on the Fast Fourier Transformation (FFT in a’’’). Wild type (n=18); pak-1(tm403) (n=20); spc-1(RNAi) (n=18); spc-1(RNAi) pak-1(tm403) (n=18). Scale bars, 10 μm (c, d, e, f), or 1 μm (c’, d’, e’, f’). P values, *<0,05; **<0,001; ***<0,0001; ns not significant.

Figure 4:. An actin-remodeling network providing mechanical plasticity ensures embryo elongation

(a-b’) Cellular model of embryo elongation. (a-a’) In control embryos, muscle contractions (red arrows) provoke actin filament shortening in the dorso-ventral epidermis, probably through sliding or shortening, followed by SPC-1/PAK-1-dependent actin stabilization. Whether spectrin is found along (scenario 1) or between (scenario 2) actin filaments is unknown (a). (b-b’) In spc-1 pak-1 deficient embryos, actin remodeling goes uncontrolled. (c-f) Viscoplastic mechanical model of embryo elongation. The embryo is represented as a Kelvin-Voigt solid (spring stiffness k, resting length λ, viscosity η) submitted to the forces F_epid and F_muscle. System equations for the model. (d) Wild-type case: an increasing resting length during stretching phases imparts mechanical plasticity. (e) spc-1 pak-1 mutants: F_epid progressively decreases. (f) Comparison of experimental and predicted elongation curves taking the constitutive equations shown in (c). (g) A retraction screen in a spc-1 mutant identifies fhod-1. (h) Snapshot at three time-points of spc-1 deficient embryos in control, pak-1 or fhod-1 backgrounds; (i) terminal body length at hatching: spc-1(ra409) after feeding on L4440 control (n=21), or fhod-1(RNAi) (n=25) bacteria. (j) Pdpy-7 driven epidermis expression of truncated FHOD-1 variants and terminal body length at hatching: spc-1(RNAi)(n=26); spc-1(RNAi)pak-1(tm403) no transgene (n=36), FHOD-1(full length) (n=16), FHOD-1(ΔDAD) (n=17), FHOD-1(ΔFH2-DAD) (n=38) and non-transgenic siblings (n=78), FHOD-1(ΔFH1-FH2-DAD) (n=18). Scale bar, 15 μm. Error bars, SEM. P values: *<0,05; **<0,001; ***<0,0001; ns, not significant.