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Exp Anim
2021 Aug 06;703:378-386. doi: 10.1538/expanim.21-0001.
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Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos.
Kiyozumi D
,
Yaguchi S
,
Yaguchi J
,
Yamazaki A
,
Sekiguchi K
.
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Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.
Fig. 1. Fraser syndrome-associated proteins are conserved in sea urchin. (A) Schematic representation of human 12-CSPG proteins ECM3/FREM2, QBRICK, and FRAS1 and
their sea urchin orthologues. Human FREM3 is also shown. (B) A phylogenetic representation of human (Hs) and sea urchin (Hp) 12-CSPG proteins. (C)
Quantitative RT-PCR analyses for ecm3 and qbrick expression during embryonic development. The transcript level of
mitCO1 at any timing of development is set to 1 and relative expression levels are shown.
Fig. 2. Protein localization of 12-CSPG proteins ECM3 and QBRICK. (A–F) Co-immunofluorescence of embryos for ECM3 (magenta) and QBRICK (green) at 23 hpf. ECM3
(A, B), QBRICK (C, D), their merged view (E, F). Panels B, D, and F are magnified views of boxed area in panel A, C, and E. Closed and open arrowheads in
(A) and (C) indicate blastocoel wall localization of ECM3 and QBRICK, respectively. Asterisks in (A) and (C) indicate apically localized
immunoreactivities. Bar, 50 µm. (G–L) Immunofluorescence images of ECM3 and QBRICK in MO-injected embryos. Glycerol-injected (G, H)
ecm3-MO1-injected (I, J), and qbrick-MO1-injected (K, L) embryos. Immunofluorescent signals of ECM3 (magenta in G, I,
K), QBRICK (green in H, J, L) are shown. Closed and open arrowheads indicate ECM3 and QBRICK localizations in control embryos, respectively. Arrows
indicate fragmented ECM3 localization in QBRICK morphants.
Fig. 3. Mesh-like fibrillar ECM3 on the basal surface of ectoderm. (A–F) Immunofluorescence of ECM3 in control (A, B), ecm3-MO (C, D), and
qbrick-MO (E, F) injected embryos. B, D, and F are magnified views in the boxed areas in A, C, and E, respectively. Images are
generated by projecting several z-stack images into a single plane. Bars, 20 µm.
Fig. 4. PMC migration is abnormal in ecm3 or qbrick knockdown embryos. PMC distribution are visualized by fluorescent
in situ hybridization using SM50 RNA-probe (green). (A, B) Glycerol-injected control embryos. (C, D)
ecm3-MO-injected embryos. (E, F) qbrick-MO-injected embryos. The lateral (A, C, E) and vegetal (B, D, F) views of
embryos are shown. Filled arrowheads in (A) indicate PMCs migrating along blastocoelar wall. Open arrowheads indicated the absence of migrating PMCs
toward the anterior side in ecm3 and qbrick morphants. (G) The number of PMCs in control or MO-injected embryos at 25
hpf. Each column represents mean number ± SD (bars). Numbers in bars represent n.
Fig. 5. Antisense morpholino knockdown of ecm3 and qbrick causes skeletal patterning defects. (A–F) Glycerol or MO-injected
embryos at 72 hpf. A, B, glycerol-injected control. C, D, ecm3-MO1 injected embryos. E, F, qbrick-MO1 injected embryos.
Red asterisks, animal pole. Black asterisks, vegetal pole. In addition to side views (A, C, E), smashed views (B, D, F) are also shown to visualize
endoskeletons. Arrows indicate each skeletal rod and arrow colors correspond to rod component indicated in (I). (G, H) The length of body rod (G) and
postoral rod (H) in glycerol or MO-injected embryos. Each column represents average ± SD (bars). Numbers in bars represent n. (I–K)
Schematic representation of skeletal rod pattern in control (I), ecm3-MO (J), and qbrick-MO (K) embryos.
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