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doi: 10.1242/10.1242/dev.00557

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Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities

Department of Cell and Molecular Biology, Technical University of Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany

View larger version (20K): [in a new window]   Fig. 1. Schematic representation of Myf5 transgenes. The –58/–48 distal enhancer of the Myf5 gene (Hadchouel et al., 2000) was divided into subfragments indicated by yellow bars and the corresponding coordinates. Each fragment was linked to a SpeI fragment containing 4.8 kb of the proximal Myf5 promoter including the branchial arch enhancer (red), the Myf5 gene body (blue) and the lacZ reporter gene (purple) fused in frame. The reporter fragment was obtained from M5-YAC-95 described previously (Zweigerdt et al., 1997). Dark and light green boxes indicate homology elements H1 and H2, respectively, which are highly conserved in sequence between mouse and human. The various constructs are referred to by roman numerals.

View larger version (89K): [in a new window]   Fig. 2. Expression patterns of transgenic mouse embryos containing constructs Myf5-I (A-C) and Myf5-II (D-F). Myf5-I drives strong expression in the dorsal domain of somites at E9.5 (A), which extends more ventrally during later stages of developmental (B,C). Notice the expression in the hypaxial domain at the thoracic level, whereas the intermediate myotome appears to be free of lacZ staining (arrow in C). Myf5-I is also strongly expressed in limb muscles of E12.5 and E13.5 embryos (B,C), in the mandibular and hyoid arches (A), and later in hypoglossal cord (arrowhead in B). Ectopic expression is seen in head mesenchyme and in the notochord of the tail. The Myf5-II transgene is active in somites at E9.5, except for the cervical region (D), but somitic expression is not properly maintained at later stages (E,F). Also notice that ß-galactosidase staining is inappropriately confined to the posterior margin of somites (D). Myf5-II is never activated in limb buds.

View larger version (75K): [in a new window]   Fig. 3. Whole-mount ß-galactosidase staining of Myf5-I transgenic mouse embryos and serial transverse sections. (A) An E9.5 embryo (26 somites) illustrating that activation of the transgene occurs correctly in the epaxial domain of somites but is delayed by 5-6 somites. The red arrowhead indicates the most recently formed somite, and the black arrowhead points to the first somite expressing the transgene. Sections confirm that Myf5-I expression is properly restricted to the epaxial myotome along the entire axis but does not reach into the hypaxial domain during early stages. Expression in branchial arches occurs in the correct cells. (B) Myotomal expression of Myf5-I is maintained in an E12.5 embryo and, in addition, the transgene is now also expressed in the muscle anlagen of fore- and hindlimbs. This expression persists in all limb muscles at E13.5 (C). At this stage, the ventral myotome of tail somites shows robust transgene activity.

View larger version (85K): [in a new window]   Fig. 4. Myf5-III fails to support correct expression in myotomes and limb buds. ß-Galactosidase staining of transgenic embryos at E9.5 (A), E11.5 (B) and E13.5 (C) reveals expression in caudal but not in rostral somites at all stages of development. Transverse section of the embryo in (A) illustrates that the somitic expression is mostly limited to the ventral dermomyotome, with very few positive myotomal cells (D). At E13.5, distinct muscles at the base of the limbs weakly express the transgene in whole-mount (arrowhead, C) and in a section (arrow, E) of the hindlimb. Myf5-III is ectopically expressed in head mesenchyme and in tailbud notochord. Expression in branchial arches and later in head muscles reflects the activity of the proximal branchial arch enhancer present in Myf5-III. The incorrect activity in somites is probably due to the enhancer located within the Myf5 gene body, as demonstrated previously (Summerbell et al., 2000).

View larger version (127K): [in a new window]   Fig. 5. The transgene Myf5-IV is correctly expressed in the myotomal compartment of somites and in limb muscles. Whole-mount ß-galactosidase histochemistry and sections of transgenic mouse embryos show myotomal expression in all somites including the occipital/cervical somites at E9.5/10.0 (A-C). Somitic expression is maintained at E13.5 in the dorsal and ventral parts but not in the intermediate region of the myotomes at thoracic and lumbar levels (D,E). The transgene is also expressed normally in limb muscles (F). Few cells in neural tube (B) and dorsal root ganglia (E) exhibit ectopic expression.

View larger version (37K): [in a new window]   Fig. 6. Sequence comparison of H1 (A) and H2 (B) elements in mouse and human Myf5 enhancer. The alignment was performed by the Clustal method. Nucleotide residues in the human sequence that are identical to the mouse sequence are indicated by dots. Different nucleotides are shown in boxes. Sequence gaps are marked by dashes. Exon sequences of the overlapping ptprq gene are highlighted between lines.

View larger version (107K): [in a new window]   Fig. 7. The H1 element in the transgene Myf5-V drives correct expression in limb muscles but not in somites. Whole-mount lacZ stainings of E9.5 (A), E10.5 (B) and E13,5(C) embryos illustrate transgene activation in posterior margins of somites but no expression in cervical somites. Transverse sections at different axial levels indicate the expression in the dermomyotome at early stages (a,a'). At later stages, some expression is also seen in myotomal cells (b'). A sagittal section of the embryo in (B) shows prominent expression in the posterior part of somites (b, rostral to the left). Sections through fore- and hindlimbs of the E13.5 embryo demonstrate that all limb muscles express the transgene (c,c').

View larger version (77K): [in a new window]   Fig. 8. The limb-specific enhancer is located within the first 270 bp of the H1 element (Myf5-VI), whereas the rest (Myf5-VII) fails to support expression in limbs. lacZ staining of whole-mount embryos carrying Myf5-VI (A-D) revealed somitic expression at E9.5 (A) that is maintained until E13.5 (D). Notice, however, that expression in cervical somites is lacking at E9.5 but becomes activated at later stages (B-D). Myf5-VI is accurately activated in limb buds from E11 (B) onwards and continues in limb muscles of E12 (C) and E13.5 (D) embryos. By contrast, Myf5-VII is not expressed in limbs, and cervical somites also fail to express the transgene (E,F). Moreover, expression of Myf5-VII is not correctly maintained in most trunk somites at E13.5 (F). Both transgenes are ectopically expressed in the dermomyotome as shown on transversal sections (A',E').

View larger version (139K): [in a new window]   Fig. 9. Expression pattern of the transgene Myf5-VIII in somites of mouse embryos. lacZ staining on a whole-mount embryo at E9.5/10 indicates transgene activity in all somites including cervical somites (A). Transgene activity in the more caudal (younger) somites (C,E) is preferentially associated with the posterior half, but this is not the case in older somites (D,F). Serial transverse sections of E9.5/10 embryo show dermomyotomal expression in caudal somites (F) that gradually shifts to the myotome in rostral somites that are more mature (E,D). A sagittal section of the cranial somites confirms the expression in myotomes (C). Transgene activity in somites of E13.5 embryos is downregulated (B). Weak expression is seen in proximal muscles of fore- and hindlimbs at the dorsal side.