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To analyze the transcriptional regulatory mechanisms of the myoD gene, we generated transgenic mice bearing a lacZ gene driven by a 6-kb 5'-flanking sequence of the mouse myoD gene including a proximal regulatory region (PRR) and a distal regulatory region (DRR), which are sufficient for activation of muscle-specific transcription in vitro. The expression of the reporter lacZ gene was detected from 10.5 days post coitum in the myotomes, consistent with endogenous myoD mRNA. However, in limb buds and branchial arches, the appearance of the lacZ-positive cells was delayed for one day compared with the endogenous myoD mRNA, suggesting the existence of a different control mechanism among muscle cell lineages. Further, a subset of cells in the central nervous system (CNS), where endogenous myoD mRNA was not detected, expressed the transgene transiently. The same 6-kb MyoD-lacZ gene injected into Xenopus embryos was expressed in the myotomes and in CNS cells at the tailbud stage. Deletion analyses in both transgenic mice and Xenopus embryos indicated that the DRR and PRR were together sufficient for the expression pattern in skeletal muscle. In addition, analysis in Xenopus indicated that a third enhancer region between -1.3 kb and -275 bp could substitute for the DRR. The functional conservation of the regulatory region of the mouse myoD gene in Xenopus embryos suggests that the regulatory pathway that activates myoD gene transcription in axial muscles is conserved among vertebrates.
FIG. 1. Structure of MyoD-lacZ reporter constructs. (Top) The
upper line shows a scale bar. The second line represents a composite
of the genomic fragments isolated from the mouse myoD gene.
Solid boxes denote the exons of the myoD gene. Open boxes denote
the distal regulatory region (DRR) and the proximal regulatory region
(PRR). The arrow indicates the transcription start site. The
DRR contains consensus sequences for three E boxes, an MCAT
box, an MLC box, and an AT -rich sequence. The PRR contains
consensus sequences for two E boxes, two Spl sites, an MCAT box,
a CCAATbox, and a TATA-like box. (Bottom) The upstream region
of the myoD gene and the construction of lacZ reporter genes used
in this experiment. Solid lines denote the 5'-flanking region of the
myoD gene. Solid boxes denote the DRR and PRR. Shaded boxes
denote the SV40 nuclear localization signaL Open boxes denote the
bacteria /3-galactosidase gene. The MD6.0-lacZ contains a full 6-
kb upstream fragment including the DRR and PRR of the myoD
gene. The DRR/PRR-lacZ contains the PRR and the 2-kb region
including the DRR.
FIG. 5. The MyoD-lacZ expression in Xenopus embryos at late tailbud (stages 28-30). {A, B) Whole-mount lacZ staining of the Xenopus
embryo injected with the MD6.0-lacZ and cleared by BABB. {B) Higher magnification of A. Expression is restricted to a single hand in
the middle of each myotome {arrow) and to the spinal cord (arrowhead), with some staining in the brain, retina, and branchial arches. (C)
Frontal section through myotomes of the Xenopus embryo injected with the MD6.0-lacZ. (D/ Transverse section of the rostral myotome
and spinal cord of the Xenopus embryo injected with the MD6.0-lacZ. (E) LacZ staining of Xenopus embryos injected with the MD6.0-
lacZ, DRR/PRR-lacZ, MD4.0-lacZ, MD1.3-lacZ, PRR-lacZ, and MD0.04-lacZ. The left side shows the upstream region of the myoD
gene and the construction of lacZ reporter genes used in this experiment. Solid lines denote the 5'-flanking region of the myoD gene.
Solid boxes denote the DRR, PRR, and deleted PRR. Shaded boxes denote the SV40 nuclear localization signal. Open boxes denote the
bacteria .8-galactosidase gene. The MD4.0-lacZ and MD1.3-lacZ contain 4.0 and 1.3 kb of upstream sequence, respectively. The PRRlacZ
contains -275 bp upstream sequence. The MD0.04-lacZ contains 40 bp of upstream sequence (-40 to +1) including the TAT Alike
box but not the Spl site, MCAT box, or CCAAT box. Each construct was injected into more than 50 einbryos in each experiment.
Independent experiments were performed at least three times. Representative embryos from each construction are shown here. BA,
branchial arch; SC, spinal cord; MT, myotome; NC, notochord.
