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doi: 10.1242/10.1242/dev.00552

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Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus

Juliette Hadchouel^1,*,, Jaime J. Carvajal^2,*, Philippe Daubas¹, Lola Bajard¹, Ted Chang¹, Didier Rocancourt¹, David Cox², Dennis Summerbell², Shahragim Tajbakhsh¹, Peter W. J. Rigby² and Margaret Buckingham^1,

¹ Département de Biologie du Développement, CNRS URA 2578, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France
² Section of Gene Function and Regulation, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK

View larger version (21K): [in a new window]   Fig. 1. The regulatory region at -58/-48 kb upstream of Myf5. (A) Schematic representation of the different fragments of the -58/-48 kb region and its upstream extension to -63 kb, which are subcloned in front of the Myf5-nlacZ cassette (2.6 kb, including a branchial arch element, neural tube element and the Myf5 promoter) (Hadchouel et al., 2000; Summerbell et al., 2000). For each construct, the cloned region is indicated by a black line. Distances in kb are from the Myf5 Cap site. (B) The nlacZ expression pattern, revealed by X-gal staining, is shown for an embryo at E11.5 (40-45 somites) with a -58/-48 tk-nlacZ transgene, where the region between -58 kb and -48 kb has been placed in front of a heterologous thymidine kinase promoter.

View larger version (100K): [in a new window]   Fig. 2. The -53.3/-48 region drives Myf5-nlacZ expression mainly in cervical somites and the hindlimb bud. X-gal staining at E12, with a -53.3/-48 Myf5-nlacZ transgene (A), and at E11.0, with a -56.4/-48 Myf5-nlacZ transgene (B). Black arrows indicate X-gal staining in fore- and hind-limbs. ß-galactosidase-positive cells are detected in the brain in prosomeres p1 and p4 (white arrowheads). The embryo shown in B was sectioned at the level of the cervical somites (white line) and the corresponding transverse cryostat section is shown under phase contrast (C) or fluorescence (D), after immunoreaction with an antibody against muscle myosin heavy chain (red labelling) to show differentiated muscle cells in the myotome, the extent of which is indicated by white arrows. Black arrowhead in C indicates ß-galactosidase-positive neurones within the ventral neural tube (NT). DRG, dorsal root ganglia.

View larger version (68K): [in a new window]   Fig. 3. The -56.6/-53.7 region drives Myf5-nlacZ expression only in the central nervous system. (A) X-gal staining at E11.5 of an embryo, with a -56.6/-53.7 Myf5-nlacZ transgene, showing nlacZ expression in the embryonic brain (prosomeres p1 and p4) and in the neural tube. The embryo in A was sectioned at levels indicated by the white lines, and sections at these levels are shown in B-D. ß-galactosidase-positive cells are detected in the ventral neural tube (B) and in the neuroepithelium at the levels of prosomeres p1 (C) and p4 (D). In these brain sections, telencephalic vesicles are on the right.

View larger version (122K): [in a new window]   Fig. 4. The -58/-54 region drives Myf5-nlacZ expression to several sites of myogenesis in the somites, limb buds and hypoglossal cord. (A,C,D) X-gal staining of an embryo with a -58/-54 Myf5-nlacZ transgene at E9.75 (A, 26 somites), E10.5 (C, 30 somites) and E11.5 (D, 36 somites). At E9.75, ß-galactosidase-positive cells are detected in the developing myotome in the central epaxial region, as shown in a transverse cryostat section (stained with Eosin) (B) of the same embryo shown in A (white line indicates level of sectioning). At E10.5 (C), Myf5-nlacZ expression is still restricted to the same area of the myotome. It is detected in the extremities of the more mature somites at E11.5 (D). (E) Phase contrast microscope view of a section in the interlimb region of the embryo shown in D (white line indicates level of sectioning) where both the epaxial lip of the epithelial dermomyotome and the hypaxial somitic bud contain cells with blue nuclei (black arrowheads). At this stage, there is strong X-gal staining in the forelimb buds and labelling is also detectable in the less mature hindlimb buds. Cells in the hypoglossal cord are also positive for ß-galactosidase. (F) X-gal staining of an embryo at E11.5 (40 somites), with a -63/-54 Myf5-nlacZ transgene showing a pattern which is essentially similar to that shown in D. With these two constructs, Myf5-nlacZ activity is also detected in the brain and neural tube, owing to the central nervous system element (-56.6/-53.7), and in the branchial arches, owing to the control branchial arch sequence. There is also ectopic labelling of head mesenchyme, which is particularly notable in C. NT, neural tube; HC, hypoglossal cord.

View larger version (112K): [in a new window]   Fig. 5. The -58/-56.6 region contains elements that direct transgene expression to distinct regions of the somite, as well as to the limb buds and hypoglossal cord. X-gal staining of embryos at E11.5 (38-40 somites) in which the Myf5-nlacZ transgene contains a -58/-56.6 kb fragment (A) or subfragments of it: -58/-57 (B), -57.5/-56.6 (F) or -57.5/-57 (H). In A, expression is no longer seen in the brain (compare with Fig. 4D), but in somites, limb buds and hypoglossal cord. In B, the -58/-57 subfragment directs expression to the limb buds and hypoglossal cord (black arrow) and to the epaxial and hypaxial extremities of the somite. The cryostat section in C (stained with Eosin), corresponds to a transverse section (white line in B) at the thoracic level of embryo (B). Higher magnifications (D,E) show ß-galactosidase-positive cells in the epithelial structures of the epaxial lip (D) and hypaxial somitic bud (E). In F, somitic expression is mainly seen in the epaxial intercalated region of the myotome, shown on a transverse cryostat section at the thoracic level (white line) in G. The outline of the myotome, which is marked by immune staining with a myosin heavy chain antibody (not shown) is presented as a broken line. In H, the 500 bp (-57.5/-57) fragment directs expression to the limb buds, hypoglossal cord, and epaxial and hypaxial extremities of the somite.

View larger version (50K): [in a new window]   Fig. 6. The -63kb to -48kb region is necessary for Myf5 expression in limb buds, central nervous system and domains of the somite. Comparison of X-gal staining profiles between embryos of BAC195APZ (A,C,E) and BAC195PZ63-48 (B,D,F) transgenic lines at different stages of embryonic development. (A,B) At E9.0, the transgene is activated in the epaxial somitic lip as directed by the early epaxial enhancer (Teboul et al., 2002). (C,D) At E11.5, limb and brain expression are missing from the deleted BAC line. Expression in the thoracic somites at this stage does not comprise the full epaxial-hypaxial myotome extension, whereas staining shows a clear caudal bias as shown on higher magnification of these regions of E11.5 embryos (E,F). Expression in the thoracic somites at this stage does not comprise the full epaxial-hypaxial extent of the myotome (indicated by the red lines in E,F). (G,H) Transverse cryostat sections, stained with Eosin and X-gal from the thoracic level (white lines in C,D) of the embryos shown in C,D. Arrows in H indicate the full extent of the myotome (see G).

View larger version (64K): [in a new window]   Fig. 7. Multiple deletions within the -63 kb to -48 kb interval reveal interactions between different regulatory regions. Comparison of Myf5-nlacZ expression profiles between BAC195APZ63-48 (A,E), 59-54 (B,F), 54-49 (C,G) and p8.8 (D,H) constructs. At E9.5 (A-D), deletion of the entire -63 kb to -48 kb region (A) or the -59 kb to -54 kb region (B) results in a marked caudal somitic expression in the dermomyotome, reminiscent of the expression pattern obtained with plasmid p8.8 (D), and driven by the intragenic enhancer element (Summerbell et al., 2000). Deletion of the -54 kb to -49 kb region does not seem to affect the expression pattern at this stage, as it results in a similar pattern to the wild type. This suggests that the -59 kb to -54 kb interval contains the predicted suppressor of the dermomyotome element. At E11.5 (E-H), limb expression is missing in the -63 kb to -48 kb deletion (E), whereas expression in both limbs is seen with both the half-deletion constructs (F,G). In the -59 kb to -54 kb deletion (F), expression in the hindlimb is stronger, whereas in the -54 kb to -48 kb deletion, the relative intensity of expression between fore- and hindlimbs is reversed. This suggests that both regions are required for the full recapitulation of limb expression at this stage. (H) The intragenic region in p8.8 directs a pronounced band of labeling in the myotome of more mature somites.

View larger version (75K): [in a new window]   Fig. 8. Deletion of the -63 kb to -48 kb region reveals an additional element(s) directing a second phase of limb expression. Transgenic embryos at E13.5 where nlacZ expression is directed by BAC195APZ (A) or by BAC195APZ63-48 (B). In C, an embryo with the -58/-48 Myf5-nlacZ transgene is shown. Comparison of the expression patterns of the limbs between embryos of BAC195APZ (D,F,H,J) and BAC195APZ63-48 (E,G,I,K) transgenic lines. (E) At E12.5, all pre-muscle masses show weak X-gal staining, not seen in the hindlimb at this stage (not shown). (F,G) By E13.5, the forelimb expression pattern driven by the two constructs is similar. (I) Very weak expression is detected in hindlimb pre-muscle masses. (J,K) By E14.5, the hindlimb expression pattern driven by the two BAC constructs is indistinguishable.