Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10.
AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan.
Chih-Hao
CH
Tang
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan.
Yun-Ren
YR
Lai
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.
Yi-Chung
YC
Chen
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan.
Chen-Hsiu
CH
Li
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.
Yu-Fen
YF
Lu
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan.
Hung-Yen
HY
Chen
Institute of Fisheries Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan.
Huang-Wei
HW
Lien
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Chung-Hsiang
CH
Yang
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Chang-Jen
CJ
Huang
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.
Chen-Yi
CY
Wang
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.
Cheng-Fu
CF
Kao
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan. Electronic address: zoslh@gate.sinica.edu.tw.
Sheng-Ping L
SP
Hwang
D000818:Animals; D030801:Animals, Genetically Modified; D004423:Ear / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055757:SOXE Transcription Factors / Q000235:genetics / Q000378:metabolism*; D012665:Semicircular Canals / Q000166:cytology / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
C519664:AGR2 protein, zebrafish; D011993:Recombinant Fusion Proteins; D055757:SOXE Transcription Factors; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; C442229:sox10 protein, zebrafish; D049452:Green Fluorescent Proteins
Agr2 gene regulation; Otic vesicle; Zebrafish; sox10