Gbx2 functions as a transcriptional repressor to regulate the specification and morphogenesis of the mid-hindbrain junction in a dosage- and stage-dependent manner.
The Gbx subfamily of homeodomain transcription factors is involved in the positioning of the isthmus, which patterns the midbrain and cerebellum in vertebrates. To uncover the details of Gbx functions, we first examined the dose dependency of its effects on brain formation in zebrafish and found that high-dose gbx2 mRNA injection affected the entire forebrain and midbrain, whereas low-dose mRNA specifically disrupted the isthmic folding at the midbrain-hindbrain boundary (MHB) but only weakly affected the expression of genes involved in MHB specification. Thus, isthmus morphogenesis, and not its early specification, is highly sensitive to gbx2. Transient induction of heat-inducible gbx2 using transgenic fish showed that MHB specification is most sensitive to gbx2 at the end of epiboly and further suggested that otx2 is the direct target gene. These together demonstrate that gbx2 regulates both specification and morphogenesis of the MHB/isthmus region. Deletion analyses showed that both the N- and C-terminal regions contribute to the suppressive activity of Gbx2 against the anterior brain and that the N-terminal core region, including the Eh1 and proline-rich sequences, is required for this Gbx2 activity. Comparison of the effects of activated and repressive forms with wild-type Gbx2 suggested that Gbx2 functions as a transcriptional repressor, which was further evidenced by a luciferase assay in which gbx2 repressed the MHB enhancer of fgf8a in mouse P19 cells.
Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan.
Yukiko
Y
Nakayama
Hiroshi
H
Kikuta
Maiko
M
Kanai
Kimihito
K
Yoshikawa
Akinori
A
Kawamura
Kana
K
Kobayashi
Zhe
Z
Wang
Alam
A
Khan
Koichi
K
Kawakami
Kyo
K
Yamasu
D000818:Animals; D030801:Animals, Genetically Modified; D019521:Body Patterning / Q000235:genetics*; D045744:Cell Line, Tumor; D004625:Embryo, Nonmammalian; D051524:Fibroblast Growth Factor 8 / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007276:Injections, Intraventricular; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008636:Mesencephalon / Q000033:anatomy & histology / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D016548:Prosencephalon / Q000033:anatomy & histology / Q000196:embryology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D012249:Rhombencephalon / Q000033:anatomy & histology / Q000196:embryology / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic; D015027:Zebrafish / Q000033:anatomy & histology / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
C496457:Fgf8 protein, mouse; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D029961:Zebrafish Proteins; C513181:gbx2 protein, zebrafish; D051524:Fibroblast Growth Factor 8; D008156:Luciferases
Brain formation; Domain structure; Gbx2; Midbrain–hindbrain boundary; Transcriptional regulation; Zebrafish