TY - JOUR
T1 - Visualization of Sox10-positive chromatoblasts by GFP fluorescence in flounder larvae and juveniles using electroporation
AU - Miyake, Minato
AU - Sekine, Michiharu
AU - Suzuki, Toru
AU - Yokoi, Hayato
N1 - Funding Information:
We are grateful to Drs. S. Higashijima and S. Ansai, and NBRP Medaka ( https://shigen.nig.ac.jp/medaka/ ) for providing the pBS‐Tbait‐:loxP‐‐loxP‐ plasmid and Drs. M. Kinoshita and K. Kishimoto for providing the Cre plasmid. This study was supported by JSPS KAKENHI Grant Numbers 17H03867, 15KK0272, and 18K05813. olhs RFP GFP
Funding Information:
We are grateful to Drs. S. Higashijima and S. Ansai, and NBRP Medaka (https://shigen.nig.ac.jp/medaka/) for providing the pBS-Tbait-olhs:loxP-RFP-loxP-GFP plasmid and Drs. M. Kinoshita and K. Kishimoto for providing the Cre plasmid. This study was supported by JSPS KAKENHI Grant Numbers 17H03867, 15KK0272, and 18K05813.
Publisher Copyright:
© 2021 Wiley Periodicals LLC
PY - 2021/7
Y1 - 2021/7
N2 - Japanese flounder are left–right asymmetrical, with features, such as dark, ocular-side specific pigmentation. This pigmentation arises during metamorphic stages, along with the asymmetric differentiation of adult-type chromatophores. Additionally, among juveniles, tank-reared specimens commonly show ectopic pigmentation on their blind sides. In both cases, neural crest-derived Sox10-positive progenitor cells at the dorsal fin base are hypothesized to contribute to chromatophore development. Here, we developed a method to visualize Sox10-positive cells via green fluorescent protein (GFP) fluorescence to directly monitor their migration and differentiation into chromatophores in vivo. Electroporation was applied to introduce GFP reporter vectors into the dorsal fin base of larvae and juveniles. Cre-loxP system vectors were also tested to enable cell labeling even after a decrease in sox10 expression levels. In larvae, undifferentiated Sox10-positive progenitor cells were labeled in the dorsal fin base, whereas newly differentiated adult-type chromatophores were seen dispersed on the ocular side. In juveniles, Sox10-positive cells were identified in the connective tissue of the dorsal fin base and observed prominently in areas of ectopic pigmentation, including several labeled melanophores. Thus, it was suggested that during metamorphic stages, Sox10-positive cells at the dorsal fin base contribute to adult-type chromatophore development, whereas in juveniles, they persist as precursors in the connective tissue, which in response to stimuli migrate to generate ectopic pigmentation. These findings contribute to elucidating pigmentation mechanisms, as well as abnormalities seen in hatchery-reared flounders. The electroporation method may be adapted to diverse animals as an accessible gene transfer method in various research fields, including developmental and biomedical studies.
AB - Japanese flounder are left–right asymmetrical, with features, such as dark, ocular-side specific pigmentation. This pigmentation arises during metamorphic stages, along with the asymmetric differentiation of adult-type chromatophores. Additionally, among juveniles, tank-reared specimens commonly show ectopic pigmentation on their blind sides. In both cases, neural crest-derived Sox10-positive progenitor cells at the dorsal fin base are hypothesized to contribute to chromatophore development. Here, we developed a method to visualize Sox10-positive cells via green fluorescent protein (GFP) fluorescence to directly monitor their migration and differentiation into chromatophores in vivo. Electroporation was applied to introduce GFP reporter vectors into the dorsal fin base of larvae and juveniles. Cre-loxP system vectors were also tested to enable cell labeling even after a decrease in sox10 expression levels. In larvae, undifferentiated Sox10-positive progenitor cells were labeled in the dorsal fin base, whereas newly differentiated adult-type chromatophores were seen dispersed on the ocular side. In juveniles, Sox10-positive cells were identified in the connective tissue of the dorsal fin base and observed prominently in areas of ectopic pigmentation, including several labeled melanophores. Thus, it was suggested that during metamorphic stages, Sox10-positive cells at the dorsal fin base contribute to adult-type chromatophore development, whereas in juveniles, they persist as precursors in the connective tissue, which in response to stimuli migrate to generate ectopic pigmentation. These findings contribute to elucidating pigmentation mechanisms, as well as abnormalities seen in hatchery-reared flounders. The electroporation method may be adapted to diverse animals as an accessible gene transfer method in various research fields, including developmental and biomedical studies.
KW - Cre-loxP
KW - Sox10
KW - asymmetry
KW - chromatophore
KW - electroporation
KW - flounder
UR - http://www.scopus.com/inward/record.url?scp=85104823753&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85104823753&partnerID=8YFLogxK
U2 - 10.1002/jez.b.23045
DO - 10.1002/jez.b.23045
M3 - Article
C2 - 33900043
AN - SCOPUS:85104823753
VL - 336
SP - 393
EP - 403
JO - Journal of Experimental Zoology Part B: Molecular and Developmental Evolution
JF - Journal of Experimental Zoology Part B: Molecular and Developmental Evolution
SN - 1552-5007
IS - 5
ER -