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The early epaxial enhancer is essential for the initial expression of the skeletal muscle determination gene Myf5 but not for subsequent, multiple phases of somitic myogenesis

Lydia Teboul1,2,{dagger}, Juliette Hadchouel3,*,{dagger}, Philippe Daubas3, Dennis Summerbell1,2, Margaret Buckingham3 and Peter W. J. Rigby1,2,{ddagger}

1 Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Section of Gene Function and Regulation, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK
3 Département de Biologie du Développement, CNRS URA 1947, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France
* Present address: Molecular Physiology, The University of Edinburgh, Wilkie Building, Medical School, Teviot Place, Edinburgh EH8 9AG, UK
{dagger} These authors contributed equally



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  		Fig. 1. Time-course of pEEBZ transgenic embryos stained for ß-galactosidase. (A) pEEBZ construct map. (B) Transgene expression starts before 8.5 dpc. At 9.5 dpc (C), epaxial somitic expression is evident in all somites and expression is also seen in the branchial arches (star) and in the head. At 10.5 dpc (D), dermomyotomal expression can be seen in the youngest somites. Transgene expression can be observed in the myotome in older somites where the dermomyotomal structure is breaking down. Additional ectopic expression can be seen in limbs (higher magnification inset). At 12.5 dpc (E), continued epaxial somitic expression and ectopic limb and head expression are seen.

 


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  		Fig. 2. Transverse sections of a 9.5 dpc (26 somite) pEEBZ transgenic embryo following X-gal staining. When using the criteria of Spörle and Schughart (Spörle and Schughart, 1997Go) this embryo is at the 27 somite stage. (PSM) No transgene expression can be seen in the presomitic mesoderm adjacent to the first somite. As soon as the somite is born (So Ip), ß-galactosidase-positive cells are detected adjacent to the dorsal neural tube. (So IIa-IIIa) Stronger epaxial expression is seen in the dermomyotome of the subsequent, older somites. (So V-XIX) The expression of ß-galactosidase is observed more hypaxially throughout the dermomyotome and the myotome as the somite matures (p, posterior; a, anterior).

 


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  		Fig. 3. Characterisation of the head expression in pEEBZ transgenic embryos. (A) Reporter gene expression is seen in the head at 9.5 dpc. (B) In transverse sections (plane of section shown in A) strong expression is detected in the cephalic mesoderm together with a few stained cells in the brain (arrows). (C) Dissection of the head of a 12.5 dpc y200-Myf5-nlacZ embryo shows reporter expression in prosomere p1 and in the mamillary body as previously described (Tajbakhsh and Buckingham, 1995Go; Daubas et al., 2000Go). (D) Dissection of the head of a 12.5 dpc pEEBZ embryo reveals that the reporter expression is scattered but centred on the posterior mesencephalon extending into the pons. ß-galactosidase expression takes place predominantly outside of the previously described territories (red arrow indicates the boundary between the mesencephalon and the metencephalon; blue arrow indicates mamillary body; mes, mesencephalon; met, metencephalon).

 


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  		Fig. 4. Deletion analysis of the early epaxial enhancer in the y200-Myf5-nlacZ context. The expression patterns of 9.5 (A,B), 10.5 (C,D) and 11.5 (E,F) dpc embryos bearing y200-Myf5-nlacZ (A,C,E) and y200{Delta}E-Myf5-nlacZ (B,D,F) and stained for ß-galactosidase. The deletion abolishes dermomyotomal transgene expression (arrows in A and B) although some epaxial myotomal expression remains. The only difference between the two series of embryos is that the early epaxial transgene expression is missing in the y200{Delta}E-Myf5-nlacZ line.

 


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  		Fig. 5. Transverse sections of a 9.5 dpc (26 somite) y200{Delta}E-Myf5-nlacZ transgenic embryo stained for ß-galactosidase. At this stage no transgene expression can be seen in the dermomyotome. Transgene expression starts in the epaxial half of the myotome of somite VII (arrow in So VII). The staining extends further epaxially and hypaxially in the consecutive somites until it occupies the entire length of the myotome (So IX-XII).

 


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  		Fig. 6. Analysis of reporter gene transcription. In situ hybridisation was performed on 9.5 dpc embryos bearing (A) pEEBZ, (B) y200{Delta}E-Myf5-nlacZ and (C) y200-Myf5-nlacZ constructs. (A) Transverse sections of a 9.5 dpc (26 somite) pEEBZ transgenic embryo stained for ß-galactosidase. When using the criteria of Spörle and Schughart (Spörle and Schughart, 1997Go), this is a is 27 somite stage embryo. (PSM) No transgene expression can be seen in the presomitic mesoderm adjacent to the first somite. (So O) As soon as the somite is born, nlacZ transcripts are detected in cells in the dorsal half of the somite. (So I-IIa) Stronger epaxial expression is seen in the dermomyotome of the subsequent older somites. (So X-Brachial) Intensity of dorsal dermomyotomal expression decreases thereafter. (Brachial-Cervical) The expression of nlacZ transcripts is observed more hypaxially through the myotome as the somite matures. (B) The y200{Delta}E-Myf5-nlacZ reporter transcripts are detected in the myotomes of all somites (red arrowhead) and in the hypaxial dermomyotome at the interlimb level. (C) The y200-Myf5-nlacZ transgene transcript pattern is the sum of those of pEEBZ and y200{Delta}E-Myf5-nlacZ. The higher magnification picture of the youngest somites illustrates the switch from the transcriptional output of the EEE (white arrowhead) to that of the enhancers that operate in the myotome (red arrowhead). Dashed line indicates the level at which this switch is seen.

 


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  		Fig. 7. Localisation of ß-galactosidase-positive cells within the myotomes of y200-Myf5-nlacZ and y200{Delta}E-Myf5-nlacZ 9.5 dpc embryos. (A-D) Transverse sections of y200-Myf5-nlacZ, (E-H) transverse sections of y200{Delta}E-Myf5-nlacZ. A and E are stained with Hoechst 33258 to show the anatomy. Desmin (red), used as a marker of myogenic cytodifferentiation, is detected throughout the myotome except in cells immediately adjacent to the DML (dorsomedial lip) (B,F). ß-galactosidase-positive cells (green) extend up to and within the epaxial DML in the y200-Myf5-nlacZ control sections (C) but are restricted to relatively ventral regions of the myotome in y200{Delta}E-Myf5-nlacZ (G). D and H show overlays (merged images) of B,C and F,G, respectively. White arrows mark the boundary of DML, red arrows mark dorsal desmin boundary, green arrows dorsal ß-galactosidase boundary.

 




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