Int J Dev Biol
January 1, 2010;
A conserved MRF4 promoter drives transgenic expression in Xenopus embryonic somites and adult 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
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.
Int J Dev Biol
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Fig. 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 meso- derm. (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.
Fig. 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), arrows indicate ectopic expression in cardiogenic regions. Embryos in (F,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.
Fig. 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.
Fig. 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-GFP transgenic frog. Note the neurovascular bundle in the center. (C) GFP fluorescence in the same section as in (B). Scale bar, 100 μm.
Fig. 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.
myf5 ( myogenic factor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior right, dorsal up.
myf5 (myogenic factor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31/32, lateral view, anterior left, dorsal up.
Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF4 gene expression to long-term muscle denervation.