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doi: 10.1038/ncomms3821.

Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria

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Free PMC article

Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria

Thibaut Brunet et al. Nat Commun.2013.
Free PMC article

Abstract

The modulation of developmental biochemical pathways by mechanical cues is an emerging feature of animal development, but its evolutionary origins have not been explored. Here we show that a common mechanosensitive pathway involving β-catenin specifies early mesodermal identity at gastrulation in zebrafish and Drosophila. Mechanical strains developed by zebrafish epiboly and Drosophila mesoderm invagination trigger the phosphorylation of β-catenin-tyrosine-667. This leads to the release of β-catenin into the cytoplasm and nucleus, where it triggers and maintains, respectively, the expression of zebrafish brachyury orthologue notail and of Drosophila Twist, both crucial transcription factors for early mesoderm identity. The role of the β-catenin mechanosensitive pathway in mesoderm identity has been conserved over the large evolutionary distance separating zebrafish and Drosophila. This suggests mesoderm mechanical induction dating back to at least the last bilaterian common ancestor more than 570 million years ago, the period during which mesoderm is thought to have emerged.

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Figures

Figure 1
Figure 1.ntlexpression is β-cat dependent but Wnt independent in the zebrafish embryo mesoderm at the onset of epiboly.
(a) Zebrafish embryos at sphere stage (4 hpf). (b) Dome-stage zebrafish embryos at the onset of epiboly (4.3 hpf). (c) β-cat labelling, margin cells (left) and dorsal pole (right, white arrows: nuclear β-cat) at sphere. (d) β-cat labelling around the margin (left and right) at dome (ubiquitous nuclear β-cat). (e) β-cat labelling at animal pole (upper pole) at sphere stage. (f) β-cat labelling at animal pole (upper pole) at dome stage. Scale bar, 20 μm (white bars). (g)ntl expression initiation in dorsal pole at sphere. (h)ntl expression initiation in margin zone at 30% epiboly (79/79). (i)ntl labelling at 30% epiboly in heat-shocked HS-TcfΔN-GFP embryos (26/26). (j)ntl labelling indkk-injected embryos (8/8 controls, 8/8dkk), as well as in heat-shocked HS:Dkk-GFP embryos, at 30% epiboly (14/14 WT siblings, 7/7 HS:Dkk-GFP). (k) PIV analysis with no significant cell deformation at sphere. (l) PIV analysis with marginal cell dilation at dome, coinciding with epiboly initiation. (m) Quantification ofntl-expressing embryos in HS-TcfΔN (n=35) and Dkk (n=15) compared with WT (n=37) at 50% epiboly in zebrafish.P<2.2E−16 by theχ2-test. All experiments were replicated two times. Scale bar, 100 μm (black and white bars).
Figure 2
Figure 2. Mechanical induction of nuclear translocation of β-cat in the zebrafish embryo mesoderm.
(a) 4.8 hpf embryos at 30% epiboly. (b) 4.8 hpf embryos blocked at sphere after treatment with blebbistatin. (c) Blebbistatin-treated embryo compression and the resumption of epiboly movements and marginal cell dilation. (d) Blebbistatin washing and resumption of epiboly movements and marginal cell dilation. Deformations are assessed by PIV analysis. Note that velocity fields differ betweenc andd, but the dilations of the marginal cells in blue are the same. Scale bar, 100 μm (black and white bar). (e) β-cat labelling around the margin in dome to 5.7 hpf non-treated 50% epiboly embryos. (f) β-cat labelling around the margin in blebbistatin-treated embryos (nuclear in the dorsal pole only, see Supplementary Fig. S4a). (g) β-cat labelling around the margin after global deformation of blebbistatin-treated embryos. (h) β-cat labelling after blebbistatin washing upon resumption of endogenous movements. Scale bar, 20 μm (white bar). (i) Quantification of marginal β-cat-positive nuclei in controls (n=16), blebbistatin-treated (n=22), blebbistatin-treated and globally compressed (n=16), blebbistatin-treated and washed (n=10), and blebbistatin treated embryos with epiboly rescue by magnetic forces (n=17). Differences between control and blebbistatin-treated embryos, and between treated embryos and rescued embryos, are statistically significant according to Mann–Whitney’s exact test (P<0.001). Error bars are s.d. Note that ectopic positive nuclei in blebbistatin-treated and compressed individuals (Supplementary Fig. S5c) are not taken into account in this quantification.
Figure 3
Figure 3.Nuclear translocation of β-cat and ofntlexpression by magnetically induced mimicking of the onset of epiboly.
(a) Embryos injected with UML (in red, green counter colour in transmission, surface lateral view). (b) Magnetic force applied to magnetically loaded margin cells by the ring micromagnet. (c) Margin tissue in blebbistatin-treated UML-injected embryos at time zero of magnetic field application, (d) and after 100 min of application. In the two later cases, the loss of resolution due to the ring micromagnet setup impaired PIV analysis (see Methods). (e) Labelling of β-cat in blebbistatin-treated UML-injected embryos in the absence of ring micromagnet. (f) Labelling of β-cat in blebbistatin-treated UML-injected embryos after exposure to the ring micromagnet (see Supplementary Fig. S9b for UML-injected embryo controls and Fig. 2i for quantification). (g)ntl Labelling in blebbistatin-treated UML-injected embryos in the absence of ring micromagnet (representative ofn=12 embryos onn=15 injected embryos). (h)ntl Labelling after the ring micromagnet applied forces to UML-injected embryos (see Supplementary Fig. S9b for UML-injected embryo controls and Fig. 4e for quantification). (i) Quantification of nuclear translocation of β-cat in controls (n=5), UML-injected controls (n=5), blebbistatin-treated embryos (n=35), blebbistatin UML-injected embryos (n=4) and blebbistatin UML-injected embryos submitted to the ring micromagnet (n=17). All data are characterized byP<0.001 using Mann–Whitney’s exact test. Error bars are s.d. All experiments were replicated at least two times. Scale bars, 100 μm (black bars) and 20 μm (white bars).
Figure 4
Figure 4.Mechanical induction ofntlexpression in the zebrafish embryo mesoderm.
(a)ntl Expression in control embryos at the germ ring stage of 30% to 50% epiboly (5.7 hpf). (b)ntl labelling in blebbistatin-treated embryos at the same stage, (c) in blebbistatin-treated embryos globally compressed at the same stage (d) and after blebbistatin washing at 4.8 and 5.7 hpf, when washed embryos morphologically reach 30–50% epiboly. Scale bar, 100 μm (black bar). (e) Quantification of the number of embryos showingin situ ntl expression in controls (n=97), blebbistatin-treated (n=95), blebbistatin-treated compressed (n=73), blebbistatin-treated and washed (n=24), and blebbistatin-treated embryos with epiboly rescue by magnetic forces (n=8). Differences between control and blebbistatin-treated embryos, and between treated and rescued embryos, are statistically significant according to theχ2-test (P<0.001). (f) Quantitative reverse-transcription PCR quantification ofntl expression in controls, blebbistatin-treated, blebbistatin-treated compressed, blebbistatin-treated and washed embryos (each reaction realized in technical triplicates). Differences between control and blebbistatin-treated embryos, and between treated and rescued embryos, are statistically significant according to the Student’st-test (P<0.001). Error bars are s.d. All experiments were replicated two times.
Figure 5
Figure 5.Mechanical induction of nuclear translocation of β-cat and of Twist expression in theDrosophilaembryo mesoderm.
(a) Twi expression in the mesoderm ofDrosophila embryos at stage 8 after WT invagination (n=6). (b) Twist expression in non-invaginatingsna−/− mutants (n=7). (c) Twist expression after invagination rescue through indentation ofsna−/− mutants (n=6). (d) Twi expression in the mesoderm of TcfΔN embryos at stage 8 (n=6). (e) Quantification of Twi expression in the mesoderm of WT embryos (n=6),sna− embryos (n=7),sna− indented embryos with rescue of mesoderm invagination (n=6),sna− indented embryos without rescue of mesoderm invagination (n=6), TcfΔN embryos (n=6), Src42A-RNAi embryos (n=12) and Arm667m embryos (n=10). Data are characterized by a Mann–Whitney’s exact testP<0.001. Error bars are s.d. (f) Arm labelling at stage 5 before mesoderm invagination. (g) Arm labelling during mesoderm invagination at stage 6. (h) Arm labelling in stage-6sna−/− mutants. (i) Arm labelling in mesoderm-invaginatedsna mutants after indentation. (j) Arm labelling in Mat-Gal4*Arm667m Y667 unphosphorylable Arm mutants. Note the diffuse texture of the image associated with 1% formaldehyde procedure for which nuclear detection of Arm is possible (see Methods) but junctional resolution is lost. Higher membranar resolution is achieved with the 2% formaldehyde procedure, but characterizes membrane and cytoplasm only (Supplementary Fig. S11d). Note also that in contrast to zebrafish, signalling Arm is known to be more diffusively found in the cytoplasm with some degree of nuclear enrichment only. (k) Quantification of Arm nuclear translocation (ratio of average mesodermal co-localization signal (density of white pixels) over ectodermal co-localization signal) in the mesoderm of WT at stage 5 (n=7), at stage 6 (n=6), in thesna− at stage 6 (n=6), in thesna− indented with rescue of invagination at stage 6 (n=6), in Arm667m at stage 6 (n=8). Arbitrary units (a.u.). Data are characterized by a Mann–Whitney’s exact testP<0.01 for all relevant comparison. Error bars are s.d.sna−/− mutants were selected by phenotypea priori (no mesoderm constriction and invagination before the onset of germ band extension and a delay of 10 min in anterior gut invagination14), as well asa posteriori (profound lateral dorsal folds characteristic ofsna mutants as described in ref. 69) and by Snail labelling (see Supplementary Fig. S11a,b). All embryos are at stage 8. All experiments were replicated twice. Scale bar, 10 μm (white bars).
Figure 6
Figure 6.Mechanically induced phosphorylation of the Y667-β-cat E-cadherin interaction site in the mesoderm ofDrosophila.
(a) Phospho-Y667-β-cat labelling in stage-5Drosophila embryos. (b) Phospho-β-cat labelling in stage-6 invaginating embryos. (c) Phospho-β-cat labelling in Src42A-RNAi invaginating embryos. (d) Phospho-β-cat labelling in Arm667m stage-6 invaginating embryos. (e) Phospho-β-cat labelling in the non-invaginating mesoderm of stage 6sna−/−Drosophila embryos. (f) Phospho-β-cat labelling in the indented invaginatingsna−/− mutants rescued in mesoderm invagination (note mesoderm invagination rescue can also be shallower than WT, see Supplementary Fig. S11b). Scale bar, 20 μm (white bars). (g) Twist expression in the WT at stage 8. (h) Twist expression in Src42A-RNAi at stage 8. (i) Twist expression in Arm667m embryos at stage 8. See Fig. 5e for quantification. Scale bar, 10 μm (white bars). (j) Quantification of Arm Y667 phosphorylation in the ventral furrow ofDrosophila WT embryos (n=18), in Src42A-RNAi (n=13), in Arm667m (n=8), insna−/− embryos (n=7) and in indented invaginatedsna−/− embryos (n=6).P<0.001 according to Mann–Whitney’s exact test. Error bars are s.d. All experiments were replicated two times.
Figure 7
Figure 7.Mechanically induced phosphorylation of the Y667-β-cat conserved E-cadherin interaction site in the mesoderm ofDanio.
(a) Phospho-β-cat labelling in zebrafish marginal cells before epiboly (sphere stage). (b) Phospho-β-cat labelling at the start of epiboly (dome stage). (c) Phospho-β-cat labelling after blebbistatin treatment that suppressed movements. (d) Phospho-β-cat labelling after blebbistatin treatment with epiboly movements rescued by global compression. (e) Phospho-β-cat labelling after blebbistatin treatment with epiboly movements rescued by drug washing. (f) Phospho-β-cat labelling after blebbistatin treatment rescued by magnetic manipulation of UML-injected embryos leading to epiboly movement resumption. (g) Phospho-β-cat labelling in the presence of PP2 Src-family inhibitor treatment at dome (associated β-cat nuclear translocation tests in Supplementary Fig. S15b). (h) β-cat labelling in blebbistatin globally compressed embryos treated with PP2. (i) β-cat labelling in blebbistatin magnetically deformed UML-injected embryos treated with PP2. Associated quantitative results in Supplementary Fig. S15b. (j) Levels of pY667 β-cat in the margin relative to the blastoderm centre and in the margin relative to the background in sphere stage (n=6), dome stage (n=9), PP2 treated (n=7), blebbistatin-treated (n=22), blebbistatin-treated and washed (n=7), and blebbistatin-treated UML-injected magnetically rescued embryos (n=9). Differences between control dome, blebbistatin-washed or blebbistatin-compressed embryos and all other conditions are statistically significant (P<0.05 according to Mann–Whitney’s exact test. Error bars are s.d. All experiments were replicated two times. (k)ntl expression at 30% epiboly. (l)ntl expression at 30% epiboly upon PP2 treatment in blebbistatin-treated embryos. (m)ntl expression at 30% epiboly upon PP2 treatment in blebbistatin-treated embryos with margin cell epiboly deformation rescued by global compression. (n)ntl expression at 30% epiboly upon PP2 treatment in blebbistatin-treated embryos with margin cell epiboly deformation rescued by magnetic forces. (o) Proportion of embryos showingntl expression in controls (n=49), PP2-treated embryos (n=8), PP2- and 1-azakenpaullone-treated embryos (n=9), PP2- and blebbistatin-treated embryos (n=15), PP2 and blebbistatin globally compressed embryos (n=18), PP2 and blebbistatin UML-injected embryos submitted to the ring magnet (n=5). All data are characterized byP<0.05 byχ2-test. All experiments were replicated two times. Scale bar, 20 μm (white bars) and 100 μm (black bars),
Figure 8
Figure 8.Gastrulation induced pY667β-cat leads to early mesoderm specification in zebrafish andDrosophilathat have diverged at the time of mesoderm emergence.
(a) In bothDanio andDrosophila, early maternally defined polarities allow the first morphogenetic movements of gatrulation that lead to specific deformation of mesodermal cells. (b) This deformation causes the mechanically induced Src-family-kinase-mediated phosphorylation of Y667-β-cat, its junctional release, nuclear translocation and transcriptional activity. (c) This results into either induction (ntl in zebrafish) or maintenance (twi inDrosophila) of early panmesodermal transcription factors. These strikingly parallel situations probably date back to Urbilateria, the last common ancestor of bilaterians, in which the mesoderm has been proposed to have arisen from the margin of the balstopore, which is by defiinition a strongly deformed zone.
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