QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 10, 2003

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, C. Articles by Du, S.-J. Search for Related Content PubMed PubMed Citation Articles by Xu, C. Articles by Du, S.-J. Social Bookmarking                         What's this?

Analysis of myostatin gene structure, expression and function in zebrafish

Cheng Xu^*, Gang Wu^*, Yonathan Zohar and Shao-Jun Du

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA

View larger version (54K): [in a new window]   Fig. 1. The genomic structure and promoter sequence of zebrafish myostatin gene. (A) The zebrafish myostatin gene contains three exons and two introns with conserved exon and intron boundaries. Sequence analysis identified seven putative E box sites in the 5' flanking sequence. Some of the E boxes are conserved compared with that in the bovine myostatin promoter. (B) The DNA sequence of zebrafish myostatin gene 5' flanking region. The putative TATA box and the seven putative E box sites are underlined in the DNA sequence. The GenBank accession number is AY323521.

View larger version (51K): [in a new window]   Fig. 2. Expression of myostatin in zebrafish embryos, larvae, juvenile and adult. (A,B) In situ hybridization showing little or no myostatin expression in developing somites of zebrafish embryos at 24 h.p.f. (hours post-fertilization). (C) RT-PCR results showing myostatin (MSTN) expression in early stage zebrafish embryos, 1-4 d (days post-fertilization), swimming larvae (16 d), juvenile (1 month) and adult skeletal muscles (3 months). Elongation factor 1 (Ef-1) was used as control for RT-PCR. 16d, 1m and 3m are duplicated.

View larger version (45K): [in a new window]   Fig. 3. Structure of myostatin-GFP (green fluorescent protein) construct and activity of myostatin promoter in zebrafish embryos. (Top) The myostatin-GFP construct contains a 1.2 kb zebrafish myostatin 5' flanking sequence including the 5'UTR (5'-untranslated region). To ensure proper processing of the RNA transcripts, the rabbit ß-globin intron-2 sequence (in) and the SV40 polyadenylation and transcription termination signals are included in the myogenin-GFP construct. (A) GFP expression in myofibers and forebrain region of zebrafish injected with the myostatin-GFP construct at 24 h.p.f. (hours post-fertilization). Forebrain expression is indicated by the white arrow. (B) Higher magnification view showing GFP expression in muscle fibers. (C) GFP expression in the floor plate (white arrows) of zebrafish embryos injected with the myostatin-GFP construct at 24 h.p.f. (D) Whole-mount anti-GFP staining confirming GFP expression in myofibers and brain (arrow) of injected zebrafish embryos (24 h.p.f.). (E) Higher magnification view of anti-GFP staining in muscle fibers. (F) Expression of GFP in the forebrain region (arrow) of a myostatin-GFP injected embryo, revealed by whole-mount antibody staining. Scale bars, 500 µm (A,D); 200 µm (B,C,E,F).

View larger version (47K): [in a new window]   Fig. 4. Transient expression analysis of the rat myosin light chain (mylc) promoter in zebrafish embryos and structure of the mylc-GFP (A) and mylc-MSTNpro (B) transgenes. The mylc-GFP gene construct (A) contains a 1.3 kb rat myosin light chain promoter (mylc) at the 5', the 0.8 kb GFP coding region, a 0.8 kb SV40 poly(A) signal, and a mylc 1/3 enhancer from the rat mylc gene (Donoghue et al., 1991). The mylc-MSTNpro gene construct (B) contains the 1.3 kb rat myosin light chain promoter (mylc) at the 5', the 0.8 kb prodomain coding region of zebrafish myostatin (MSTNpro), the 0.8 kb SV40 polyA signal, and the mylc 1/3 enhancer from the rat mylc gene. The ATG start codon, RIRR proteolytic site and the TAA stop codon are indicated. (C,D) Muscle-specific expression of GFP reporter gene in 24 h.p.f. zebrafish embryos injected with the mylc-GFP: (C) fluorescence, (D) immunostaining. Scale bar, 100 µm. The mosaic pattern of expression is typical for transient expression studies in zebrafish embryos (Westerfield et al., 1992; Du and Dienhart, 2001).

View larger version (62K): [in a new window]   Fig. 5. Expression analysis of the mylc-MSTNpro transgene in F1 transgenic embryos by in situ hybridization. The expression of the transgene was visualized by in situ hybridization with a probe that could hybridize with transcripts of both the endogenous myostatin gene and the prodomain transgene. (A,C,E,G-I) Expression of Myostatin prodomain transcripts in transgenic fish embryos of line 33. Strong expression of Myostatin prodomain was found in transgenic fish embryos. (B,D,F,J) In situ results of non-transgenic zebrafish embryos using the same probe. All embryos were analyzed at 24 h.p.f. except G (16 h.p.f.). (H-J) Cross sections showing the fast muscle-specific expression of the transgene directed by the rat mylc promoter. Note the absence of staining in the slow muscle pioneer region at the myoseptum region of the somite (arrow). Scale bars, 500 µm (A-D); 200 µm (E,F); 150 µm (G-J).

View larger version (10K): [in a new window]   Fig. 6. Expression of the prodomain transgene and the endogenous myostatin gene in skeletal muscles of adult transgenic and non-transgenic zebrafish. For each sample, RT-PCR was performed using three different sets of primers that are specific for the transgene (mylc-MSTNpro), the endogenous myostatin gene and the transgene (Total MSTN), and the elongation factor 1- (Ef-1) mRNA transcripts, respectively. Lane 1, RT-PCR results using mylc-myostatinpro plasmid DNA as positive control. Lanes 2 and 3, RTPCR results showing the expression of the endogenous gene (total MSTN) and EF-1 in the skeletal muscle of two individual non-transgenic fish (2 and 3). Lanes 4 and 5, RT-PCR results showing the expression of the Myostatin prodomain transgene (mylc-MSTNpro), the endogenous myostatin gene and the transgene (total MSTN) and the EF-1. Lane 6, RT minus control of non-transgenic fish. Lane 7, RT minus control of transgenic fish.

View larger version (54K): [in a new window]   Fig. 7. In situ hybridizations comparing myogenic gene expression in transgenic and non-transgenic fish embryos. (A,B,G,H) Myf-5 expression in transgenic (TG; B,H) and wild-type (WT; A,G) zebrafish embryos. (C,D,I,J) MyoD expression in transgenic (D,J) and wild-type (C,I) zebrafish embryos. (E,F,K,L) myogenin expression in transgenic (F,L) and wild-type (E,K) zebrafish embryos. (A-F) 16 h.p.f. embryos; scale bar, 200 µm. (G-L) 24 h.p.f. embryos; scale bar, 500 µm.

View larger version (65K): [in a new window]   Fig. 8. Profile of real-time PCR showing MyoD, Myf-5, myogenin (Myog) and Ef-1 expression in transgenic (MSTN) fish and non-transgenic (WT) control at day 7.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter