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. 2022 Dec 1:10:992371.
doi: 10.3389/fcell.2022.992371. eCollection 2022.

Mechano-biochemical marine stimulation of inversion, gastrulation, and endomesoderm specification in multicellular Eukaryota

Ngoc Minh Nguyen  1 , Tatiana Merle  1 , Florence Broders-Bondon  1 , Anne-Christine Brunet  1 , Aude Battistella  2 , Emelie Britt Linnea Land  1 , Florian Sarron  3 , Aditya Jha  4 , Jean-Luc Gennisson  5 , Eric Röttinger  6   7 , María Elena Fernández-Sánchez  1 , Emmanuel Farge  1
Affiliations

Mechano-biochemical marine stimulation of inversion, gastrulation, and endomesoderm specification in multicellular Eukaryota

Ngoc Minh Nguyen et al. Front Cell Dev Biol. .

Abstract

The evolutionary emergence of the primitive gut in Metazoa is one of the decisive events that conditioned the major evolutionary transition, leading to the origin of animal development. It is thought to have been induced by the specification of the endomesoderm (EM) into the multicellular tissue and its invagination (i.e., gastrulation). However, the biochemical signals underlying the evolutionary emergence of EM specification and gastrulation remain unknown. Herein, we find that hydrodynamic mechanical strains, reminiscent of soft marine flow, trigger active tissue invagination/gastrulation or curvature reversal via a Myo-II-dependent mechanotransductive process in both the metazoan Nematostella vectensis (cnidaria) and the multicellular choanoflagellate Choanoeca flexa. In the latter, our data suggest that the curvature reversal is associated with a sensory-behavioral feeding response. Additionally, like in bilaterian animals, gastrulation in the cnidarian Nematostella vectensis is shown to participate in the biochemical specification of the EM through mechanical activation of the β-catenin pathway via the phosphorylation of Y654-βcatenin. Choanoflagellates are considered the closest living relative to metazoans, and the common ancestor of choanoflagellates and metazoans dates back at least 700 million years. Therefore, the present findings using these evolutionarily distant species suggest that the primitive emergence of the gut in Metazoa may have been initiated in response to marine mechanical stress already in multicellular pre-Metazoa. Then, the evolutionary transition may have been achieved by specifying the EM via a mechanosensitive Y654-βcatenin dependent mechanism, which appeared during early Metazoa evolution and is specifically conserved in all animals.

Keywords: beta-catenin-dependent mechanosensitivity; choanoflagellate Choanoeca flexa; cnidaria Nematostella vectensis; evolutionary emergence of first Metazoa organisms; hydrodynamic mechanical strains; mechanotransduction; myosin-dependent mechanosensitivity; primitive motor-sensorial behavioral mechanosensing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Hydrodynamic stimulation of Nematostella embryo gastrulation by the marine flow. (A) The most representative gastrulation state population of embryos at (i) 18 h and (ii) 21 h. (B) Setup: mimicking sea ocean stream by shaking a Petri dish with sand on its ground. Hydrodynamic deformation of jelled embryos with sand. (C) Quantification of the elliptic deformation of embryos. nControl = 43 and nWavelet = 57, p = 3.10–15. Mann–Whitney statistical test. (D) i,ii, iii: embryos representative of the majority of individuals submitted to the flow (see text). White arrows: invaginations. Orange arrows: apex constriction. Scale bar is 100 μm. (E) Quantitative characterization of the percentage of gastrulating embryos (in orange), submitted to the flow compared to control (in grey), based on significant normalized invagination depth d/D measurement higher to 18% (see text), nControl = 30 and nWavelet = 33, p = 7.10–4. Fisher statistical test. N = 2 biological replicates (embryo number inside columns).
FIGURE 2
FIGURE 2
Hydrodynamic stimulation of Nematostella invaginations is Myo-II and stb dependent. (A) 18 h hydrodynamically stimulated N. vectensis embryos in the presence of the ML7 MLCK of Myo-II inhibitor. Control: ethanol, vehicle of ML7 (see methods). ML7-treated embryos show a slightly higher thickness than controls. (B) Quantitative analysis. nHydroStim = 36 and nHydroStim+ML7 = 30 p = 1.9.10–3, nHydroStim+ML7 = 30 and nControl = 36 p > 0.05. Statistical test: Fisher. (C) 18 h equivalent hydrodynamically stimulated N. vectensis embryos in the presence of the blebbistatin Myo-II inhibitor. Control: DMSO, vehicle of blebbistatin. (D) Quantitative analysis. nHydroStim = 22 and nHydroStim+Blebb = 21, p = 6.7.10–4, nHydroStim+Blebb = 21 and nControl = 23, p > 0.05. Statistical test: Fisher. (E) Hydrodynamically stimulated Nematostella 21 h embryos injected with the stb-MO. Control: water injected. Control-MO: injected with the MO control sequence (see section materials and methods). (F) Quantitative analysis. nHydroStim = 22 and nHydroStimStb-MO = 26, p = 2.7.10–5. N = 2 biological replicates. Fisher statistic test. White arrows: invaginations. The scale bar is 100 μm.
FIGURE 3
FIGURE 3
Hydrodynamically induced and endogenous gastrulation of Nematostella embryos stimulates the endomesodermal fz10 gene expression downstream of mechanical stimuli in a Myo-II-dependent process. (A) fz10 expression in 18 h non-invaginating embryos (B) and in the invaginating endomesoderm of 21 h embryos. (C) fz10 expression in the invaginating tissue of 18 h hydrodynamically stimulated embryos (D) and in hydrodynamically stimulated non-gastrulating embryos treated with ML7. (E,F) Quantitative analysis. nControl = 45 and nHydroStim = 42, p = 9.10–5, and nControl = 52 and nHydroStim = 52, p = 5.7.10–3, nHydroStim = 52 and nHydroStim+ML7 = 72, p = 2.7.10–4. Control of (F) ethanol, vehicle of ML7. Fisher statistic test. (G) fz10 expression in non-gastrulating embryos ML7-treated 21 h embryos (H,I) and quantitative analysis. nControl = 14 and nML7 = 15, p = 5.10–5, nControl = 19 and nML7 = 20, p = 3.9.10–5. Control: ethanol, vehicle of ML7. Statistical test: Fisher. N = 2 biological replicates. The scale bar is 100 μm.
FIGURE 4
FIGURE 4
Mechanical induction of the endomesodermal fz10 gene expression is initiated by the mechanical activation of Y654-β-cat phosphorylation. (A) Y654-β-cat phosphorylation in blastulae (18 h), gastrulae (21 h), and 21 h gastrulae embryos treated with either InhibY654-βcat, the inhibitor of Y654 phosphorylation, or ML7, the inhibitor of gastrulation. Western blot: Anti-pY654 β-catenin protein detection by western blotting of N. vectensis embryos at 18 h and 21 h of development. GAPDH was used as the loading reference. (B) Quantification of the gradient ratio (in percentage) of Y654-β-cat phosphorylation in invaginating compared to non-invaginating domains and from domains of maximal intensity compared to the other domains in non-gastrulating embryos of (A). nDMS021h = 6 and nInhibY654bcat21h = 17, p = 2.3.10–4, nEth21h = 7 and nML721h = 13, p = 2.10–4. Mann–Whitney statistical test. (C) Expression of fz10 in hydrodynamically stimulated 18 h embryos and in 21 h gastrulae embryos: (up) treated with InhibY654-βcat–control is DMSO, (down) and injected with the N. vectensis Y654F-βcat RNA dominant negative–control is H20 injected. (D) Quantification of (C) (up) nControl = 24 and nHydroStim = 25, p = 4.10–3, and nHydroStim = 25 and nHydroStimInhibY654bcat = 25, p = 1.7.10–4 (down), nControl = 30 and nHydroStim = 37, p = 1.7.10–3, and nHydroStim = 37 and nHydroStimY654Fbcat = 31 1.7.10–3—Y654F-βcat 18h is not shown in (C). (E) Nuclear β-cat translocation state in the non-invaginating blastulae 18 h tissue, in the 21 h invaginating tissue of the gastrulating embryos, and in the hydrodynamically stimulated 18 and 21 h invaginating tissues of gastrulating embryos treated with the Y654-β-cat phosphorylation inhibitor. Control and hydrodynamically stimulated: DMSO, vehicle of Inhib-Y654-β-cat. Full embryos: sum of the optical cross-section on the half sphere. Zoom: a given optical crosscut corresponding to the white square location shown on full embryos. In invaginating embryos, observation is made in invagination lateral domains when signal intensity is lost in its middle. (F) Quantification of (E) nControl18h = 21, nHydroStim18h = 19, p = 6.10–6, and nControl21h = 24, p = 8.6.10–8. nHydroStim18h = 19 and nHydroStimInhibY654bcat18h = 20, p = 1.6.10–3, nControl21h = 24 and nInhibY654bcat21h = 25, p = 7.10–6. (G) Nuclear βcat translocation states in both non-gastrulating ML7-treated 18 h hydrodynamically stimulated and 21 h embryos. Control and hydrodynamically stimulated: ethanol, vehicle of ML7. (H) Quantification of (G). nControl18h = 13, nHydroStim18h = 15, p = 1.710–3, and nControl21h = 14 , p = 10–4, nHydroStim18h = 15 and nHydroStimML718h = 11, p = 6.9.10–3, nControl21h = 14 and nInhibML721h = 15, p = 2.10–4. Statistical tests. Fisher for embryo number counting and Mann–Whitney for quantitative data by embryos. N = 2 biological replicates. The scale bar is 100 μm.
FIGURE 5
FIGURE 5
Myo-II-dependent hydrodynamic stimulation of multicellular sheet curvature inversion is conserved in C. flexa choanoflagellates. (A) Multicellular C. flexa choanoflagellate before hydrodynamic stimulation (no flagellate outside) (B) and after hydrodynamic stimulation (white arrows: flagellate outside). (C) Inversion status of hydrodynamically stimulated C. flexa treated with the ethanol vehicle of ML7 alone (white arrows: flagellate outside) (D) and with the Myo-II inhibitor ML7 (no flagellate outside). Zooms: with enhanced contrast to check for flagellate presence. (E) Quantitative analysis. nControl = 41 and nStimulated = 57, p = 10–9, nStim+veh = 99 and nStim+ML7 = 79, p = 4.8.10–9. Scheme from Brunet et al. (2019). (F) Myo-II and actin in C. flexa before hydrodynamic stimulation (G) and after hydrodynamic stimulation. (H) Quantitative analyses of Myo-II expression per cell 20–30 min after hydrodynamic stimulation initiation. Immunofluorescence representative of n = 8 controls and n = 14 inverted structures of the hydrodynamically stimulated C. flexa. nControl = 8 and nStim = 14, p = 1.25.10–5. (I) 200 nm fluorescent beads internalization in cells in non-inverted (J) and inverted C. flexa after hydrodynamic stimulation. (K) Quantitative analyses of particle internalization per cell 60 min after hydrodynamic stimulation initiation. Immunofluorescence representative of n = 12 non-inverted and n = 10 fully inverted structures of the hydrodynamically stimulated C. flexa. nControl = 12 and nStim = 10, p = 9.9.10–5. N = 2 biological replicates. Statistical tests are Fisher for histograms and Mann–Whitney for quantitative analysis.
FIGURE 6
FIGURE 6
Mechanotransductive origins of first Metazoa animal emergence evolutionary: a possible scenario. (A) Results: C. flexa unclosed multicellular choanoflagellate inversion [scheme from Brunet et al. (2019)] are mechanotransductively stimulated by hydrodynamic flow mimicking soft waves on the sea shore in a Myo-II-dependent mechanotransductive process (Figure 5), like N. vectensis cnidaria gastrulation (Figure 1) and the mechanotransductive activation of Drosophila bilateria gastrulation (Mitrossilis et al., 2017). Evolutionary possible scenario: Myo-II mechanotransductive induction of constriction leading to multicellular inversion/gastrulation as possibly conserved along Metazoa evolution, from the hydrodynamically induced inversion of pre-Metazoa unclosed multicellular choanoflagellate. (B) Results: environmental hydrodynamic sea water hydrodynamic forces lead to mechanotransductive induction of gastrulation, with gastrulation strains mechanotransductively stimulating endomesoderm fz10 gene expression in the gastrulating tissue of the cnidaria N. vectensis in a Y654-β-cat phosphorylation activation mechanotransductive process conserved with bilateria (Brunet et al., 2013) (Figures 3, 4). Evolutionary possible scenario: pre-Metazoa inversion that led to hydrodynamic induction of gastrulation after closing multicellular choanoflagellates, with gastrulation strains mechanotransductively leading to endomesoderm gene expression in gastrulating tissue after evolutionary emergence of β-cat mechanosensitivity, as possible conditions of the ur-Metazoa emergence. (C,D) Results: internal morphogenetic movements of gastrulation lead to endomesodermal fz10 gene expression in the N. vectensis cnidaria embryo in a Y654-β-cat phosphorylation-dependent mechanotransductive process (Figures 3, 4). This is similar to the expression of Twist and brachyury endoderm and mesoderm genes in the early Drosophila and zebrafish bilateria embryos (Brunet et al., 2013). Evolutionary possible scenario: genetically regulated internal morphogenetic movements (gastrulation) possibly replaced external environmental (i.e., seawater) mechanical strains to stimulate gastrulation and endomesoderm specification in the Eumetazoan common ancestor of cnidaria and bilateria, thereby having initiated autonomous Metazoa embryogenesis. (E) The latter property has been possibly conserved in mesoderm and endoderm specification after endoderm/mesoderm divergence at the origin of the first bilateria emergence, as found in early Drosophila and zebrafish embryos that directly diverged from the common ancestors of bilateria (Brunet et al., 2013).
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