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Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation

Abstract

Migrasomes are recently identified vesicular organelles that form on retraction fibres behind migrating cells. Whether migrasomes are present in vivo and, if so, the function of migrasomes in living organisms is unknown. Here, we show that migrasomes are formed during zebrafish gastrulation and signalling molecules, such as chemokines, are enriched in migrasomes. We further demonstrate that Tspan4 and Tspan7 are required for migrasome formation. Organ morphogenesis is impaired in zebrafish MZtspan4a and MZtspan7 mutants. Mechanistically, migrasomes are enriched on a cavity underneath the embryonic shield where they serve as chemoattractants to ensure the correct positioning of dorsal forerunner cells vegetally next to the embryonic shield, thereby affecting organ morphogenesis. Our study shows that migrasomes are signalling organelles that provide specific biochemical information to coordinate organ morphogenesis.

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Fig. 1: Formation of migrasomes during gastrulation.
Fig. 2: Tetraspanin 4a and 7 regulate migrasome formation in zebrafish gastrulas.
Fig. 3: tspan4a and tspan7 regulate zebrafish organ morphogenesis.
Fig. 4: Chemokines, morphogens and growth factors are enriched in migrasomes.
Fig. 5: Migrasomes are required for formation of the KV.
Fig. 6: tspan4a and tspan7 are required for the clustering of DFCs on the embryonic shield during gastrulation.
Fig. 7: Migrasomes are chemoattractants for DFCs.

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Data availability

RNA sequencing data that support the findings of this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive under the accession codes SRR9047069 and SAMN11633758.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013835. The statistical source data for Figs. 1–3,57 and Supplementary Figs. 16 have been provided as Supplementary Table 3. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Further information and requests for resources and reagents should be directed to and will be fulfilled by L. Yu.

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Acknowledgements

We are grateful to the members of the Meng and Yu groups for their helpful discussions. This research was supported by the Ministry of Science and Technology of the People’s Republic of China (grant nos 2017YFA0503404 and 2016YFA0500202 to L.Y. and 31590832 to A.M.), the National Natural Science Foundation of China (grant nos 31430053 and 31621063), the Natural Science Foundation of China International Cooperation and Exchange Program (grant no. 31561143002), and the Independent Research of Tsinghua University (grant no. 20161080135) to L.Y. We thank the State Key Laboratory of Biomembrane and Membrane Biotechnology for confocal microscopy imaging and facility support. We thank the Beijing Frontier Research Center for Biological Structure for facility support. We would like to acknowledge the assistance of the Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University for assistance with spinning disk microscopy and Imaris analysis. We thank the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University for mass-spectrometry sample analysis. We thank the Core Facility of the Center of Biomedical Analysis, Tsinghua University for assistance with light-sheet imaging. We thank the National Center for Protein Sciences at Peking University for assistance with light-sheet imaging. We thank the F. Liu lab (Institute of Zoology, Chinese Academy of Sciences) for sharing the cxcr4b mutant with us.

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Authors and Affiliations

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Contributions

L.Y. and A.M. conceived the experiments, wrote the paper and supervised the project. D.J. and Z.J. carried out the experiments. D.L., X.W. and H.L. helped to purify migrasomes and construct plasmids. J.Z. and Y.M. contributed to the mutant-zebrafish verification. D.W. and Y.H. contributed the staining of Integrin β1 in the mammalian cell line. Y.L. carried out the TEM sample preparation. Y.C. and H.D. contributed to the TMT-labelled mass spectrometry. Q.W. and J.X. provided the itgb1b mutant. All authors discussed the manuscript, commented on the project and contributed to the preparation of the paper.

Corresponding authors

Correspondence to Anming Meng or Li Yu.

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Integrated supplementary information

Supplementary Figure 1 Formation of migrasome-like vesicles in zebrafish gastrulas.

a, b. Tspan4-GFP-expressing L929 cells (a) or tspan4a-GFP mRNA-injected zebrafish gastrulas (b) were stained with anti-integrin β1 antibody and observed by confocal microscopy. Scale bars, 10 μm. c, d. TSPAN4-GFP-expressing L929 cells were transfected with PH-mCherry (c) and zebrafish embryos were injected with tspan4a-GFP and PH-mCherry mRNAs (d) and observed by confocal microscopy. Scale bars, 10 μm. e. Zebrafish 70% epiboly stage embryos injected with PH-GFP mRNA were stained with antibodies against GFP and endogenous Integrin β1b and observed by confocal microscopy. Scale bars, 10 μm. f. Zebrafish primary cells were isolated from PH-mCherry-expressing gastrulas, cultured in fibronectin-coated imaging chambers for 24 h and observed by confocal microscopy. Process-connected vesicles are enlarged in the inset. g. Transmission electron microscopy image of migrasome-like vesicles in zebrafish gastrulas. Scale bars, 5 μm. h. Light-sheet images of active caspase3 staining of embryos after injection of mRNAs encoding GFP, PH-GFP and Tspan4a-GFP. Scale bar, 50 μm. i. Quantification of cells positive for active caspase3 in h. Data were pooled from three independent experiment. There is no significant difference after overexpression of PH-GFP or Tspan4a-GFP. GFP, n=14 cells; PH-GFP, n=16 cells; Tspan4a-GFP, n=17 cells. ns, P>0.05. Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. j. Formation of migrasomes during embryo development revealed by light-sheet live imaging of a wild-type embryo injected with PH-GFP mRNA. k. Fluorescently labelled dextrans (10 kD) were injected into embryos at the 50% epiboly stage to label cavities in embryos. Scale bar, 20 μm. l. Embryos were injected with Tspan4a-GFP mRNA at the 8-cell stage and injected with fluorescently labelled dextrans at the 50% epiboly stage. Scale bar, 20 μm. m. Confocal image of migrasomes (arrows) generated by Tg(sox17:GFP)-positive endoderm cells. Scale bar, 5 μm. n. Confocal image of a migrasome (arrow) generated by Tg(gsc: GFP)-positive mesoderm cells. Scale bar, 5 μm. The experiments in a-g, j-l were done twice. The experiments in m-n were done once. The numerical source data for i are shown in Supplementary Table 3.

Supplementary Figure 2 Analysis of the gene expression patterns of itgb1b, tspan4a and tspan7 in zebrafish embryos by whole-mount in situ hybridization.

a. Expression pattern of itgb1b viewed laterally and from the animal pole. Scale bar, 100 μm. b. Representative images of migrasomes in WT embryos and itgb1b mutants. c. Images of gastrulas from three independent experiments were pooled and quantified for the number of migrasomes. WT, n=19 embryos; itgb1b-/-, n=26 embryos, ***P<0.001. Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. d. Quantification of the average speed of cell migration in WT embryos and itgb1b mutants. 4D images of gastrulas were acquired by light-sheet microscopy and the average migration speed was measured by Imaris software. n=3 embryos in WT, n=4 embryos in itgb1b-/-. ns, P>0.05. Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. Data were pooled from two independent experiments. e-f. Expression pattern of tspan4a (e) and tspan7 (f) viewed laterally and from the animal pole. Scale bar, 100 μm. g. The efficiency of morpholinos was tested using reporters carrying the gene-specific 5’UTR upstream of GFP. For each treatment, seven embryos were imaged simultaneously with GFP fluorescence and bright field. Scale bar, 1 mm. h, i. Quantification of the number of migrasomes after morpholino injection. Control and gene-specific MOs were injected at the one-cell stage (10 ng per embryo). Migrasomes were counted at the shield stage. (h) Ctrl MO, n=10; tspan4a MO, n=10 embryos; *P<0.05. (i) Ctrl MO, n=11 embryos; tspan7 MO, n=13 embryos; ***P<0.001. Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. j. Whole-mount in situ hybridization of 1-cell-stage wild-type, MZtspan4a and MZtspan7 embryos. 19 embryos were imaged simultaneously in each group. Scale bar, 1 mm. The experiments in a, e, f were done twice. The experiments in g-j were done once. The numerical source data for c,d,h and i are shown in Supplementary Table 3.

Supplementary Figure 3 Phenotypic analysis of MZtspan4a and MZtspan7 gastrulas.

a. Morphology of wild-type, MZtspan4a and MZtspan7 embryos from the sphere stage to the bud stage. MZtspan4a and MZtspan7 mutants showed no obvious defects or development delay. Scale bar, 100 μm. b. Quantification of epiboly speed measured from 6.5 hpf to 9 hpf in WT, MZtspan4a and MZtspan7 embryos. Embryos were incubated at 28 °C. WT, n=22 embryos; MZtspan4a, n=24 embryos; MZtspan7, n=23 embryos; ns, P>0.05 (MZtspan4a vs. WT; MZtspan7 vs. WT). Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. c. Examples of embryos showing how the epiboly speed was calculated. D6.5 (μm) is the distance from the animal pole to the margin at 6.5 hpf. D9 (μm) is the distance from the animal pole to the margin at 9 hpf. Epiboly speed (μm/h)= (D9-D6.5)/2.5h. d. ISH of dlx3b shows convergence movement; ISH of ntl shows extension movement. WT, n=25 embryos; MZtspan4a, n=24 embryos; MZtspan7, n=25 embryos. ns, P>0.05 (MZtspan4a vs. WT; MZtspan7 vs. WT). Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. Scale bar, 100 μm. e. ISH of bmp4 and eve1 marks ventral fate; ISH of chd and gsc marks dorsal fate. The ratio shown in each image represents ‘the number of embryos with the indicated abnormality/the number of embryos analysed’. The experiment was done once. Scale bar, 100 μm. f. cp, pdx1 and nanos3 riboprobes were fluorescein-labelled and the embryos were incubated with POD-conjugated anti-fluorescein antibodies and then dyed with TSA Plus fluorescein solution to reveal green fluorescence. myl7, slc20a1a and foxa3 riboprobes were labelled with digoxygenin and the embryos were incubated with POD-conjugated anti-digoxygenin antibodies and then dyed with TSA Plus Cy3 solution to reveal red fluorescence. Z-stack images were acquired by light-sheet microscopy. Three embryos are shown for each group. Scale bar, 200 μm. The experiments in a-e were done once. The experiments in f were performed three times independently showing similar results. The numerical source data for b and d are shown in Supplementary Table 3.

Supplementary Figure 4 Samples from lysed cells do not rescue the phenotype.

a. in situ hybridization of spaw in 20-somite stage embryos (dorsal view) of WT, MZtspan4a and MZtspan7. The ratio shown in each image represents ‘the number of embryos with the indicated abnormality/the number of embryos analysed’. Scale bar, 100 μm. The experiment was done once. b. Transmission electron microscopy image of purified samples from lysed cells prepared similar to the migrasome purification. Scale bar, 1 μm. The experiment was done once. c. Categorization of MZtspan4a phenotypes as ‘normal’ or ‘abnormal’. All deformed embryos (including left–right defects and impaired induction or development of tissue) are classed as ‘abnormal’. This statistic describes the general impairment of organ morphogenesis. Embryos pooled from three independent experiments were used for quantification. WT, n=107 embryos; MZtspan4a, n=119 embryos. ns, P>0.05. d. Categorization of MZtspan4a phenotypes as ‘normal’ or ‘laterality defects’. Only embryos with a clear left–right phenotype are classified as ‘laterality defects’. Samples are same as c. WT, n=107; MZtspan4a, n=119 embryos. ns, P>0.05. In c-d, data represent mean ± s.d., P values were calculated using a two-tailed, unpaired t-test. The experiments in c and d were performed three times independently showing similar results. e. Verification of Integrin β1b antibody by itgb1b morphant. Note that the lower band does not reduce in itgb1b morphant, thus, is unspecific band. The experiment was done once. The numerical source data for c and d are shown in Supplementary Table 3. Unprocessed blots for e are shown in Supplementary Figure 8.

Supplementary Figure 5 Tspan4a is enriched in migrasomes, and the Cxcl12a/Cxcr4b signalling axis is required for organ morphogenesis.

a. Upper panel, the isotopic peaks of Tspan4a peptide DLYAQN labelled by TMT reagents. Lower panel, the MS/MS spectrum of the peptide DLYAQN. The standard peptide DLYAQN was labelled by TMT-126.1284, the peptides in migrasomes were labelled by TMT-127.1249, and the peptides in cells were labelled by TMT-129.1314. b. Immunostaining of Cxcl12a in WT embryos and cxcl12a/b morphants (75%-epiboly stage). Scale bar, 20 μm. c. Verification of the Cxcl12a antibody by western blot analysis of proteins isolated from cxcl12a-/- mutants and cxcl12a/b morphants. d. tspan4a-GFP mRNA-injected zebrafish gastrulas were stained with antibodies to detect GFP and endogenous Cxcl12a, then observed. Scale bar, 10 μm. The experiments were performed three times independently with similar results. e. ISH of cxcl12a and cxcr4b. Scale bar, 100 μm. f, g. Phenotypic categorization of cxcr4b and cxcl12a mutants. In f, the phenotype was characterized as ‘normal’ or ‘abnormal’. All deformed embryos (including those with left–right defects, and impaired induction or development of tissue) were classed as ‘abnormal’. This statistic describes the general impairment of organ morphogenesis. In g, the phenotype was characterized as ‘normal’ or ‘laterality defects’. Only embryos with a clear left–right phenotype were classed as ‘laterality defects’. Embryos pooled from three independent experiments were used for quantification, WT, n=84 embryos; MZcxcr4b, n=80; cxcl12a-/-, n=143 embryos. *P<0.05; **P<0.01; ***P<0.001 (MZcxcr4b vs. WT; cxcl12a-/- vs. WT). h, i. Phenotypic categorization, as described in f and g, of cxcl12a morphants and cxcl12a morphants injected with purified migrasomes. Embryos pooled from three independent experiments were used for quantification, cxcl12a MO, n=91 embryos; cxcl12a MO + Migrasomes, n=79 embryos. **P<0.01; ***P<0.001. In f-i, Data represent mean ± s.d., P values were calculated using a two-tailed, unpaired t-test. The experiments in a-c were done once. The experiment in e were performed twice independently showing similar results. The experiments in f-i were performed three times independently showing similar results. The numerical source data for f-i are shown in Supplementary Table 3. Unprocessed blots for c are shown in Supplementary Figure 8.

Supplementary Figure 6 Migrasomes are largely absent from embryonic shield cavity in both MZtspan4a and MZtspan7 mutants.

a. Representative images of the embryonic shield cavity from different Z-axis slices in WT, MZtspan4a and MZtspan7. Scale bar, 20 μm. b. Quantification of migrasomes in WT, MZtspan4a and MZtspan7 embryonic shield cavities. Data from three independent experiments were pooled. WT, n=35 embryos; MZtspan4a, n=20 embryos; MZtspan7, n=16 embryos. ***P<0.001. Data represent mean ± s.e.m, P values were calculated using a two-tailed, unpaired t-test. The numerical source data for b are shown in Supplementary Table 3.

Supplementary Figure 7 Cxcr4 is expressed in DFCs and required for chemoattraction of DFCs.

a. ISH of cxcr4a. cxcr4a expressed in DFCs (arrow). Scale bar, 100 μm. b. ISH of sox17. cxcr4b mutants show obvious defects in DFC positioning at the 70% epiboly stage. The ratio shown in each image represents ‘the number of embryos with the indicated abnormality/the number of embryos analysed’. The experiments in a-b were done once. Scale bar, 100 μm.

Supplementary Figure 8 Unprocessed images of all gels and blots.

a-b. Unprocessed data for Fig. 4c. Verification of purified migrasomes by western blot analysis using an anti-Integrin β1b antibody. c-f. Unprocessed data for Fig. 4f. Enrichment of Cxcl12a in isolated migrasome. Lysates of cell bodies and migrasomes were normalized by total protein concentration. Integrin β1b is the positive control, while Actin and Gapdh were used as the negative controls. Some blots were cut into several pieces and incubated with different antibodies.

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Jiang, D., Jiang, Z., Lu, D. et al. Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation. Nat Cell Biol 21, 966–977 (2019). https://doi.org/10.1038/s41556-019-0358-6

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