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A conserved MRF4 promoter drives transgenic expression in Xenopus embryonic somites and adult muscle - PubMed

A conserved MRF4 promoter drives transgenic expression in Xenopus embryonic somites and adult muscle

Timothy J Hinterberger. Int J Dev Biol. 2010.

Abstract

The muscle regulatory factor MRF4 is expressed in both embryonic and adult vertebrate skeletal muscle cells. In mammals the MRF4 gene has a complex cis-regulatory structure, with many kilobases (kb) of upstream sequence required for embryonic expression in transgenic mice. Here, initial functional comparison between Xenopus and mammalian MRF4 genes revealed that 610 base pairs (bp) of the XMRF4a proximal promoter drove substantial transgenic expression in X. laevis myogenic cells, from somites of neurula embryos through adult myofibers, and as little as 180 bp gave detectable expression. Over 300 bp of XMRF4a proximal promoter sequence is highly conserved among three X. laevis and X. tropicalis MRF4 genes, but only about 150 bp shows significant identity to mammalian MRF4 genes. This most-conserved XMRF4a region contains a putative MEF2 binding site essential for expression both in transgenic embryos and in transfected mouse muscle cells. A rat MRF4 minimal promoter including the conserved region also was active in transgenic X. laevis embryos, demonstrating a striking difference between the mouse and Xenopus transgenic systems. The longest XMRF4a promoter construct tested, with 9.5 kb of 5'-flanking sequence, produced significantly greater expression in transfected mouse cells than did promoters 4.3-kb or shorter, suggesting that the intervening region contains an enhancer, although no increased expression was evident when this region was included in transgenic X. laevis embryos. Further identification and analysis of Xenopus MRF4 transcriptional control elements will offer insights into the evolution of this gene and of the myogenic gene regulatory network.

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Figures

Figure 1
Figure 1. Structure of the XMRF4a genomic clone and comparison to other MRF4 gene sequences

(A) Upper portion of the figure shows the 5′-flanking region, exons, introns, and 3′-flanking sequence of the 13-kb clone. Restriction enzyme sites mentioned in the text (B, BamH I; BX, BstX I; E, EcoR V; H, Hind III; K, Kpn I; N, Nco I; R, EcoR I; S, Sac I) and the stop codon are indicated. Cross-hatching identifies the P2X7 gene homology region (see text). Lower portion shows results of VISTA alignments of 5066 bp of XMRF4a 5′-flanking sequence versus XMRF4b and X. tropicalis MRF4 loci. Shading indicates conservation of 70% or greater. For XMRF4b, only 2325 bp of sequence was available for comparison. (B) Alignment of nucleotide sequences from rat MRF4 (Ra), X. tropicalis MRF4 (Xt), XMRF4a (Xa), and XMRF4b (Xb) promoters. Numbering refers to positions 5′ from the XlMRF4a start codon. −610 indicates the EcoR I site, −360 the Nco I site, −180 the BstX I site, and −120 and −100 indicate the 5′ ends of PCR-generated deletions. The MEF2 site is in bold type and E boxes (potential MRF binding sites) are underlined. Black dots beneath nucleotides indicate conservation in all four species, while asterisks indicate conservation only among the three Xenopus genes. Shading covers the 150-bp highly conserved region described in the text.

Figure 2
Figure 2. Whole mount in situ hybridization of X. laevis embryos with the full-length XMRF4 probe or XMyf5 probe

All are oriented anterior to the right. (A) Stage 14–15 early neurula, dorsal view. XMRF4 expression is seen in presomitic mesoderm prior to segmentation, as well as in the anterior. (B) Stage 20 neurula, lateral view. XMRF4 staining is most intense in the somites but also evident in the eye primordia. (C) Stage 16 neurula, dorsolateral view. XMyf5 expression is confined to the posterior mesoderm. (D) Stage 31–32 tailbud embryo, lateral view. In addition to the myotomal staining, XMRF4 expression is evident in the eyes, brain, branchial arches, otic vesicles, and head mesoderm. (E) Stage 31–32 tailbud embryo, lateral view. XMyf5 expression is seen in tailbud mesoderm, dorsal and ventral myotomal cells, and in primordia of some cranial muscles.

Figure 3
Figure 3. Relative activity of XMRF4a promoters in mouse myotubes

The graph shows averages of values ± s.e.m. from two to four independent transient transfection experiments for each XMRF4a construct, except for 4.3-kb which is a single datum. Each experimental value is the average of two separate C2C12 cell transfections, expressed as the ratio of test construct to RL-SV40 control, normalized to the value obtained for a positive control construct.

Figure 4
Figure 4. Relative activity of XMRF4a promoters in transgenic X. laevis embryos

Lengths of the promoters (described in the text) are indicated. Each panel shows lateral views of representative positive individuals from a single experiment. All specimens are oriented with dorsal sides towards the top of the figure, but because expression often was asymmetric, either the left or right side may be shown, whichever had more intense staining, and the anterior end is marked with an asterisk. In panels A–D, arrows indicate ectopic expression in cardiogenic regions. Embryos in F and G are co-transgenic for both the indicated XMRF4a-GFP construct and the gamma-crystallin-GFP construct; all are oriented with the anterior to the right side of the figure. Staining of the hindbrain (arrows) and, in some cases, the lens of the eye (arrowheads), results from gamma-crystallin-GFP expression.

Figure 5
Figure 5. Activity of the rat MRF4 minimal promoter in transgenic X. laevis embryos

In situ hybridization shows GFP expression in representative positive individuals, oriented with dorsal side towards the top of the figure, anterior towards the left.

Figure 6
Figure 6. Postmetamorphic expression of XMRF4a transgenes

(A) GFP fluorescence driven by the 610-bp promoter in skeletal muscle, ventral view, anterior at top, skin removed. This is a composite of two separate exposures of the same animal. Most of the trunk and the left forelimb and proximal hindlimb are shown, but the head is out of the frame. Inset shows reflected-light (upper) and fluorescence (lower) images of a ventral skin incision in the hindlimb of a frog carrying the 1.1-kb promoter. GFP fluorescence in muscle is clearly distinguishable by color from autofluorescence in the skin. (B) Reflected-light image of a 50-µm frozen section of hindlimb muscle from an XMRF4a-1.1-GFP transgenic frog. Note the neurovascular bundle in the center. (C) GFP fluorescence in the same section as in B. Scale bar = 100µm.

Figure 7
Figure 7. Onset of XMRF4a transgene expression

(A) In situ hybridization showing GFP expression under control of the 610-bp promoter in the early somites of an F1 individual at stage 18–20, dorsal view, anterior to the right. (B) Reflected-light image of the right hindlimb of a stage-56 F1 animal carrying the 1.1-kb promoter construct. (C) GFP fluorescence from the same limb as in B. Note that the developing muscle (arrow) in the limb shows much lower intensity of fluorescence than do the tail myotomes at the top of the figure.

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References

    1. Adams L, Carlson BM, Henderson L, Goldman D. Adaptation of nicotinic acetylcholine receptor, myogenin, and mrf4 gene expression to long-term muscle denervation. J Cell Biol. 1995;131:1341–1349. - PMC - PubMed
    1. Ataian Y, Owens J, Hinterberger T. Mrf4 gene expression in Xenopus embryos and aneural myofibers. Dev Dyn. 2003;226:551–554. - PubMed
    1. Beck CW, Slack JM. Gut specific expression using mammalian promoters in transgenic xenopus laevis. Mech Dev. 1999;88:221–227. - PubMed
    1. Berkes CA, Tapscott SJ. Myod and the transcriptional control of myogenesis. Semin Cell Dev Biol. 2005;16:585–595. - PubMed
    1. Black BL, Martin JF, Olson EN. The mouse mrf4 promoter is trans-activated directly and indirectly by muscle-specific transcription factors. J Biol Chem. 1995;270:2889–2892. - PubMed

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