Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

Abstract

Background: Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.

Results: We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines.

Conclusions: From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species.

Figure 1

The rat MLC1/3 locus and lentiviral construct. Top: The rat MLC locus consists of two separate promoter elements which generate two alternatively spliced transcripts. A downstream enhancer augments expression from both promoters in skeletal muscle. Stippled exons are specific for MLC3. The internal MLC3 promoter and the downstream enhancer were cloned upstream of a lacZ reporter construct in an EIAV replication defective lentiviral vector to generate pONY-MLZ. Restriction sites for HindIII (red arrows) and Sph1 (black arrows) are indicated. Bottom: Sequence comparison of homologous downstream regions of the MLC1/3 locus of eutherans and chickens. Grey boxes highlight core enhancer elements identified in human, mouse, and rat. The internal basepairs of the E box of Site A are changed to the sequence of site B in the chicken. Site C is in reverse orientation in the chicken to that in mammals.

Figure 2

MLC3 regulatory elements are transcriptionally active in chicken skeletal muscle but not cardiac muscle primary cultures. Cardiac (A, A') and skeletal (B, B') muscle chick primary cultures were co-transfected with the MLC3 lentiviral construct and a CAG-GFP reporter construct. Three independent experiments were carried out and a representative field is shown. A, B: GFP fluorescence A'B': X-gal staining of primary cultures. Large myotubes are stained in B'.

Figure 3

Southern blot analysis of transgenic G₁ chickens. Genomic DNA from birds containing the MLZ proviral sequences were analysed for integration events by digestion with HindIII (left) or Sph1 (right) to generate junction fragments. The number of G₁ birds generating identical junction fragments is indicated.

Figure 4

Transgene expression is limited to skeletal muscle. Top: Protein samples from various tissues were analysed by ELISA for the presence of β-galactosidase. Each data point is the average of two independent assays on tissues from one transgenic bird for each line. Birds were a minimum of four months of age when assayed. Line 76 is not visible on the graph due to the low expression levels of this line. Bottom: Sections from various tissues of the same birds were stained for β-galactosidase activity. A) Transgenic breast muscle. B) Transgenic leg muscle. C) Immunofluorescence of the endogenous MLC1 and 3 protein D) Control breast muscle. E) Transgenic atrium. F) Transgenic ventricle. Staining in skin appears to be in striated muscles associated with feather tracts. Scale bars, 0.5 mm.

Figure 5

Transgene expression is restricted to fast skeletal muscle fibres and is expressed in a similar fibre type as the endogenous locus. A) A section from an interior leg muscle of a transgenic chicken was stained for β-galactosidase activity. The animal was at least four months old when assayed. B) A serial section to the one above was immuno-stained for fast (red) and slow (green) muscle isoforms. Fibres that are negative for β-galactosidase are labelled with an antibody to slow muscle. C) A section from an interior leg muscle of a second line of transgenic chicken was stained for β-galactosidase activity. D) A serial section to the one above was immuno-stained for MLC1 and 3 muscle isoforms (green). Fibres that are negative for β-galactosidase do not express MLC1 and 3. White arrows indicate corresponding myofibres. Scale bars, 0.1 mm.

Figure 6

Transgene expression in the forming chicken embryo. Transgenic and control embryos were stained for of β-galactosidase activity. A) Transgenic and (B) control embryos at day 3 of development. C) Higher magnification of the embryo in (A) to show staining in the forming myotome. D) Transcripts from the endogenous locus at day 3 are revealed by in situ hybridisation for both MLC1 and 3. E) Transgenic and (F) control embryos at day 10 of development. Arrow, myotome.