Kuriyama S and Tanaka M (2023), Characteristic tetraspanin expression patterns ...

XB-ART-59649

Dev Growth Differ 2023 Feb 01;652:109-119. doi: 10.1111/dgd.12836.

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Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development.

Kuriyama S , Tanaka M .

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The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.

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Species referenced: Xenopus laevis
Genes referenced: cd151 cd63 cd81 cd82 cd9 myc rpe tspan14 tspan15 tspan16 tspan17 tspan18 tspan31 tspan33 tspan36 tspan4 tspan9
GO keywords: tetraspanin-enriched microdomain

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	Figure 1. Various tetraspanins in the notochord group. (a) Evolutionary tree of tetraspanins (Tspans) described in this study. The expression patterns of Tspans indicated by red dots were analyzed. The expression domains of each Tspan are indicated. The pale color indicates that the border between detected and not detected is obscure. (b) The primers used for cloning Xenopus cDNAs. Uppercase letters indicate artificial sequences. (c) Xlcd9.S expression (st.27). (d) Xlcd151.S expression. (e) XlTspan14.L expression. (f) XlTspan16.S. expression. (g–j) The transverse thick slices show expression inside. (g) Xlcd9.S expression was seen in the notochord (nc), somites, and the roof plate. (h) Xlcd151.S notochordal expression. (i) XlTspan14.L was expressed only in nc. (j) XlTspan16.S was expressed in nc. (k) Expression of Xlcd9.S in the tail tip (arrowhead). (l) Expression of Xlcd81.L was not observed in the notochord (arrow, nc). The dorsal and ventral edges of the tail tip are positive (open arrowheads). Br; brain, Ey; eye, Pl; placodes, Cn; cranial neural crest cells, Tn; trunk neural crest cells, So; somites, Nc/nc; notochord, Ga; gastrula, Kd; kidney.
	Figure 2. Brain and eye group of tetraspanins. Expression patterns of (a–d) Xltspan9.S, (e–h) Xltspan14.L, and (i–m) Xlcd82.L. The stages are indicated in the figures. (d, h, m, p, r, u) Transverse thick slice. (d) Eye/diencephalon-level section from (c). (h) Eye/mesencephalon-level section from (g). (k) Expression observed in the dorsal top. (m) Eye/diencephalon-level section of (l). Expression was observed in the inner layer of the retina (rt). (n–p) Highly bleached embryo with Xltspan17.S probe at stage 32. (o) Eye/mesencephalon-level expression. (p) Ear-level expression. (q, r) Highly bleached embryo with Xltspan31.L (SAS). (r) Eye/diencephalon-level expression of SAS. (s–u) Xltspan15.L expression patterns. (u) Xltspan15.L was observed in the lens equatorial zone (ls, arrowheads). (v) Xlotx2 expression. (w) Xlpax2 expression. rt, retina; HB, hindbrain; MHB, mid-hindbrain boundary; eb, epibranchial placode; pa, pharyngeal arch; ov, otic vesicle; vrt, ventral retina; di, diencephalon; ls, lens; me, mesencephalon.
	Figure 3. Tetraspanins in neural crest cells and placodes. The expression patterns of (a, b) Xlcd81.L, (c, d) Xltspan31.L (SAS), and (e, f) Xltspan17.S. The stages are indicated in the figures. (g–i) Xltspan4.S is expressed in the mandibular cranial mesenchyme and diencephalon at stage 32. (g) Lateral view. (h) Anterior view. (i) Transverse slice of (g). Expression was not seen in the retina, and it was only seen in the peripheral area of the eye. Expression patterns of (j–m) Xlcd63.L and (n–p) Xltspan36.L. (j) Transient cranial expression of Xlcd63.L. (l) The whole eye was dissected from the tadpole, and RPE was stained. (o) Focus was adjusted from two pictures of the same field. (q) Xltspan18.S is observed in the hindbrain at stage 27. (r) Xltspan18.S is expressed in trigeminal (tg), lateral line (ll), and epibranchial (eb) placodes. (s) Xlcd9.S expression is observed in trigeminal, lateral line, and epibranchial placodes at stage 32. (t) Xlfoxic1 is an epibranchial placode marker at stage 32.
	Figure 4. Changing properties of EVs by overexpressing several Tspans. (a) HA-hCD81mCher+ overexpression in MX-1 breast cancer cells. Exogenous CD81 was detected in EVs and Exo. (b) HA-rTspan31mCher+ overexpression. Tspan31 was seen in both EVs and Exo, and enriched in Exo. (c) Myc-rTspan17mCher+ overexpression. Tspan17 was detected in Exo. (d–f) Time-lapse imaging of CNCs with overexpression of mCherry-labeled Tspans. (d) Tspan31mCher+ expressing CNCs. The small EVs were seen from early captures (~1 h). Many EVs were observed to be secreted from the budding of membrane. (e) Tspan17mCher+ red fluorescence (above) and membrane EGFP fluorescence (below). The large EVs were seen from 6 h after the start of capture. Arrows indicate that large fragments of the cells stayed longer than those of other Tspans. (f) CD81mCher+ CNCs. The overexpressing CNCs moved as a cohort. The single cells around the cluster were also connected to each other with a long tail. (g) Quantification of small EVs (<1 μm diameter). (h) Quantification of large EVs (1–10 μm diameter). (i–i′) Confocal images of sections immunostained with anti-Myc or anti-HA. (i) Transverse section of the craniofacial region of a Myc-rTspan17mCher+ RNA injected embryo stained with mouse monoclonal anti-Myc and an Alexa647-labeled secondary antibody (gray). The sections were not permeabilized. (i′) There were Tspan17mCher+ (red) cells without gray dots. Myc staining was observed on the skin side. (j) Section of an HA-rTspan31mCher+ embryo stained with anti-HA. (j′) Gray dots overlapped with Tspan31mCher+ cells. (k, l) Section of an HA-rTspan31mCher+ with Myc-Tspan17 (non-fluorescent) injected embryo. (k, k′) Anti-Myc gray dots appeared more spread out than in (i′) along Tspan31mCher+. (l, l′) Anti-HA staining. Gray dots looked similar to (j′). ins, inside; out, outside.
	Figure S1. Early expression and expression in somites. (A, A′) Xlcd9.S expression. Faint expression was seen in the dorsal region at st.10.5. No expression was observed in the posterior end of the axial mesoderm. (B, B′) Xlcd151.S expression. The early gastrula expression domain was not clear. Axial expression was highest at st.12. (C, C′) XlTspan14.L expression was seen in the neural ectoderm. (D, D′) Xlcd81.L expression. Expression was observed in invaginating edges at st.10.5. Axial expression was absent at st.12. (E) Xlcd63 expression was observed in the entire ectoderm. (F) Xlcd82.L expression was seen at the dorsal lip. (G) XlTspan17.S expression was seen in the early neural plate. (G′) The image in (G) was cut in half along the midline. (H, I) XlTspan9.L and XlTspan4.S were negative at st.11. (J) XlTspan4.S expression. Expression was broadly observed in the dorsal ectoderm. (K) Xlcd151.S expression was seen in the notochord. (L) Xlcd81.L expression was absent from the axial mesoderm and present in the neuroectoderm at st.16. (m) XlTspan31.L expression was seen in the cranial neural crest and the neural plate. (N) XlTspan9.L expression was seen in the distal tip of the eye field. (O) Xlcd82.L expression was seen in the eye and forebrain. (P) XlTspan9.L expression. A similar embryo as in Figure 2a. (Q) XlTspan16.S expression at st.24. Notochord expression was not seen at this stage. (R) XlTspan18.S expression. The expression in somites started and extended posteriorly from the position where somite expression of XlTspan9.L finished. (S) Dorsal view of an embryo similar to the one in (P). XlTspan9.L expression was missing from the midline. (T) High-bleached embryo with XlTspan17.S staining. Expression was observed in each somite and presegmented mesoderm. (U) XlTspan9.S expression was slightly shifted posteriorly. Expression was seen at the center of each segment. (V) XlTspan17.S was seen in every somite. Expression was also seen in the pronephric duct (pn). (W) Xlcd81.L expression. The arrows indicate the same position as the arrows in (V). The ventral halves of the expression area were missing. (X) XlTspan31.L was also seen in every somite. (Y) Even in later-stage embryos, expression was observed in every somite. (Z) XlTspan18.S expression remained in the posterior somites. (AA) Expression of Xlcd82.L in the whole tadpole. (BB) The XlTspan4.S expression pattern was almost the same as in a previous publication (Kashef et al., 2013). (CC) XlTspan33.L expression. Expression was observed in the retina. (DD) XlTspan16.S expression was seen in the forebrain and pronephore (pn).
	Figure S2. Subcellular localization of Tspans. (A) The fold increase of the signal intensity of HA-CD81mCher+ bands detected by anti-HA (16B2) and an HRP-conjugated anti-mouse IgG in Figure 4a. (B) The fold increase of the signal intensity of HA-Tspan31mCher+ bands detected by anti-HA and an HRP-conjugated anti-mouse IgG in Figure 4b. (C) The fold increase of the signal intensity of Myc-Tspan17mCher+ bands detected by anti-Myc (9E10) and an HRP-conjugated anti-mouse IgG in Figure 4c. (D–G″) Co-electroporation of membrane EGFP and Tspan-mCherry fusion constructs or membrane mCherry in MX-1 human breast cancer cell cells. (D–D″) The localization of membrane mCherry was seen in membrane edges around the cell with membrane EGFP. (D) Green and red (magenta) channel image. (D′) Blue indicates cell shape and yellow indicates overlap of EGFP and mCherry. (D″) High magnification. No secretion was seen around the cell. (E–E″) CD81-mCherry fusion protein in MX-1 cells. (E) The intracellular vesicles were not associated with membrane tips. (E′) The co-localization of CD81mCher+ and membrane EGFP was seen in EVs far away from the cell (arrows). (E″) High magnification of EVs. Arrowheads indicate co-localization of CD81mCher+. (F) Tspan17mCher+-overexpressing cell. Arrows indicate Tspan17mCher+ protein in EVs. The number of EVs was increased by overexpression; however, not many EVs were co-localized with Tspan17mCher+. (F″) Membrane ruffles with co-localization of Tspan17mCher+ and membrane EGFP. (G–G″) Tspan31mCher+-overexpressing MX-1 cells. (G) An increase in the number of EVs was seen. (G′) Co-localization was seen in intracellular vesicles, EVs, and membrane edges (arrows). (G″) Arrow indicates co-localization of Tspan31mCher+ with membrane EGFP. Arrowhead indicates EV localization of Tspan31mCher+. (H, I) Statistical analysis of EV numbers around 7 h. (H) The number of small EVs of each sample per 250 NC cells. Statistical differences between samples were analyzed by one-way ANOVA (p = .0003), followed by analyses of individual differences. p = .001, **p < .0001. (I) The number of large EVs. The results were similar to the results for small EVs.
	Figure S3. In vivo Tspan localization. (A) Co-injection of membrane EGFP and membrane mCherry mRNA. Cranial neural crest cells were seen underneath the skin. EGFP fluorescence (left) and mCherry fluorescence overlapped, even in small cell protrusions. (B) The immunofluorescence of the section from Myc-tagged Tspan17mCher+ with HA-tagged hCD81 RNA injected embryos. Anti-HA staining (CD81 distribution) was dispersed, and very weakly associated with Tspan17mCher+ fluorescence. (C) Anti-Myc staining was strongly associated with Tspan17mCher+ fluorescence. (D) High magnification images revealed that Myc-positive particles were spread over mCherry fluorescence as multiple tiny particles (arrows). (E) anti-HA staining of an HA-CD81mCher+ RNA injected embryo. HA staining and mCherry fluorescence overlapped. (F) Anti-Myc staining of Myc-Tspan17mCher+ RNA injected embryo. Myc staining and mCherry fluorescence overlapped; however, Myc-positive particles were often associated with the epidermis.
	Figure S4. Cross-hybridization of Xenopus laevis probes to Xenopus tropicalis embryos. (A–F) Xenopus tropicalis embryos hybridized with X. laevis Tspan probes. (A) CD9 probe. Expression in the notochord was detected similar to that of Xlcd9. (B) Tspan9 expression. Eye-specific expression was conserved. Expression in somites seemed slightly wider in X. tropicalis. (C) Tspan14 expression. The expression pattern was similar; however, analysis of transverse slices showed that expression was observed in the neural tube (nt) and notochord (nc), and in X. laevis only notochord expression was observed (Figure 1i). (D) CD81 expression. Neurula expression was quite similar to that of Xlcd81. However, in the tailbud or tadpole stage, expression in the pronephric duct was strong, and expression in the ventral vein was observed in X. tropicalis. (E) Tspan17 expression at the tadpole stage was very similar to that in X. laevis. (F) CD63 expression at the tadpole stage was similar to that of Xlcd63 (Figure 3j). However, expression in melanocytes was weak. (G) X. laevis embryos and X. tropicalis embryos were hybridized in the same vial. Staining and bleaching were also done in the same vial.