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Hes6 regulates myogenic differentiation

Judy Cossins¹, Ann E. Vernon^2,*, Yun Zhang^1,*, Anna Philpott² and Philip H. Jones^2,

¹ Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
² Cancer Research UK, Department of Oncology, University of Cambridge, Hutchinson/MRC Research Centre, Addenbrooke’s Hospital, Cambridge, UK
^* These authors contributed equally to this work

View larger version (81K): [in a new window]   Fig. 1. Hes6 transcription is detectable in developing muscle in mouse embryos and C2C12 myoblasts in culture. 35S-labelled in situ hybridisation for Hes6 mRNA on parasaggital sections of mouse embryos. (A,B) Day 13 p.c. embryo, Hes6 mRNA is present in the developing somites. (C,D) Day 16 p.c. embryo. Hes6 mRNA is expressed in the developing muscles in the forelimb; h, humerus. (E,F) Skeletal muscle (panniculus carnosus) from an adult mouse. Hes6 mRNA is undetectable. (G) Northern blot analysis of Hes6 expression in adult mouse tissues, compared with a actin control probe that also reacts with ß actin. The difference in the actin hybridisation in the heart and skeletal muscle lanes reflects tissue specific differences in the levels of and ß actin. Each lane was loaded with 2 µg of mRNA. Hes6 mRNA runs at 1.4 kb. (H) Hes6 transcription during C2C12 myoblast differentiation in vitro. C2C12 myoblasts were placed into differentiation medium for 0-4 days. RNA was isolated and analysed by northern blot. Each lane was loaded with 20 µg total RNA. The blot was also probed for myogenin, to confirm induction of differentiation and stained with Methylene Blue (Me blue) to reveal 18 S RNA. Result shown is typical of three independent experiments.

View larger version (33K): [in a new window]   Fig. 2. Hes6 protein binds to an Enhancer of Split E (ESE) box and represses transcription at an ESE box containing promoter. EMSA of Hes6. In vitro translated protein was incubated with the oligonucleotides shown, as described in the Materials and Methods. (A) In vitro translation reactions containing either no DNA (left hand lane) or cDNA encoding Hes6 protein with an N terminal His Tag (remaining lanes) were incubated with a 32P-labelled ESE box containing oligonucleotide. The binding reactions were carried out in the presence of unlabelled ESE box containing oligonucleotide present in 5-, 50- and 200-fold excess in the lanes shown. IgG indicates binding reaction carried out in the presence of control IgG; HIS, binding reaction in the presence of an anti-HIS tag antibody. (B) In vitro translations with no DNA or Hes6 cDNA were incubated with a 32P-labelled ESE box oligonucleotide, as in A. Unlabelled E box and N box competitor oligonucleotides, present in 5-, 50- and 200-fold excess were added to the reaction as shown. (C) COS cells were transiently transfected with pIRES2-EGFP plasmids with inserts encoding ß-galactosidase or mouse Hes6, with a reporter vector consisting of a collagenase promoter driving expression of firefly luciferase (pCOLluc3) or pCOLluc3 with 2 ESE box motifs cloned immediately 5' of the collagenase promoter (pCOLluc3-ESE). A renilla luciferase reporter (pRL-TK) was used as a control for transfection efficiency. The values shown represent the means of four independent experiments, each performed in triplicate or quadruplicate wells, normalised to the pIRES ß-gal + pCOLluc3-ESE control. Error bars show s.e.m. *P=0.010 using a two-tailed paired t-test, comparing Hes6 with ß-gal control, when each was co-transfected with pCOLLuc3-ESE reporter.

View larger version (74K): [in a new window]   Fig. 3. Analysis of effects of overexpression of murine Hes6 and Hes6DBM on C2C12 myoblast differentiation. C2C12 myoblasts were infected with bicistronic retroviral vectors encoding ß galactosidase (ß-gal), murine Hes6 (mHes6) or Hes6DBM (mHes6DBM) and GFP as described in the Materials and Methods. Cells were cultured for 5 days in DM and then analysed by immunofluorescence. (A) Structure of retroviral vector. A bicistonic retroviral vector was used. Transcription of RNA encoding the insert, an internal ribosome entry site (IRES) and cDNA encoding EGFP is driven by the 5' long terminal repeats (LTR). Translation of the bicistronic RNA produces two proteins, the inserted protein and EGFP. Expression of the puromycin resistance gene (puro) is driven by the SV40 viral promoter (SV40). (B-D) EGFP expression. Unstained cells were examined for EGFP fluorescence to confirm translation of the retrovirally expressed bicistronic RNA. There was no apparent difference in the level of EGFP expression, but in cells overexpressing Hes6 or Hes6DBM, the myotubes were elongated and narrower than ß-gal-expressing cells. Appearances shown are typical of five independent experiments. (E-G) Troponin-T expression. Cells were stained with an anti-Troponin-T antibody. Myotubes expressed Troponin-T, a marker of terminal differentiation. The different morphology of myotubes in murine Hes6 and Hes6DBM cultures is seen; the number of nuclei per myotube is lower in murine Hes6 and Hes6DBM transduced cells compared with ß-gal expressing cells. Appearances shown are typical of five independent experiments. (H-J) p21Cip1 expression. Cells were stained with an anti-p21Cip1 antibody, disclosed with a Cy3-conjugated secondary antibody. Nuclei were stained with DAPI. Rows of p21Cip1-positive nuclei correspond to multinucleate myotubes. Fewer p21Cip1-positive nuclei occur in the murine Hes6- and HES6DBM-expressing cells (see Fig. 4). (K-M) BrdU labelling to detect cells capable of re-entering into the cell cycle. To determine the proportion of cells in each culture that had undergone irreversible cell cycle withdrawal, cells were exposed to GM containing 50 µM BrdU for 20-22 hours after 5 days in DM. Nuclei were then stained with an anti-BrdU antibody and disclosed with a Cy3-conjugated secondary antibody. Nuclei were then stained with DAPI. More BrdU-positive cells are found in the murine Hes6- and Hes6DBM-expressing cells (see Fig. 4).

View larger version (13K): [in a new window]   Fig. 4. Overexpression of murine Hes6 (mHes6) and Hes6DBM (mHes6DBM) in C2C12 myoblasts decreases the number of cells undergoing irreversible cell cycle withdrawal. (A) C2C12 cells were cultured in DM for 120 hours, fixed and stained for immunofluorescent analysis of the proportion of nuclei in Troponin-T-positive cells, as shown in Fig. 3. Results of a typical experiment are shown. The mean of five random fields containing a total of at least 600 nuclei is shown, error bars indicate s.d. *P=0.004, **P=0.001, comparing murine Hes6 and Hes6DBM, respectively, with ß-gal control using a two-tailed unpaired t-test. There was no significant difference between murine Hes6 and Hes6DBM. (B) C2C12 cells were cultured in DM for 48 or 120 hours, fixed and stained for immunofluorescent analysis of the proportion of p21Cip1-positive nuclei as shown in Fig. 3. The mean proportion of p21Cip1-positive nuclei expressed as a percentage of the total number nuclei in three independent experiments is shown. At least 1000 nuclei for each cell type were counted from random microscope fields at each time point in each experiment. Error bars show s.e.m. *P=0.037, **P=0.040, comparing murine Hes6 with ß-gal control using a two-tailed paired t-test at 48 and 120 hours, respectively. +P=0.013, ++P=0.009, comparing murine Hes6DBM with ß-gal control using a two-tailed paired t-test at 48 and 120 hours, respectively. There was no significant difference between the percentage of p21Cip1-positive cells for murine Hes6 and Hes6DBM at either time point. (C) Cells were cultured in DM for 5 days and then placed in GM containing 50 µM BrdU for 20-22 hours. Cells were then fixed and stained for BrdU to determine the proportion of BrdU-positive nuclei as shown in Fig. 3. The mean proportion of BrdU-positive nuclei expressed as a percentage of the total number nuclei in three independent experiments is shown. At least 1000 nuclei for each cell type were counted from random microscope fields at each time point in each experiment. Error bars show s.e.m. *P=0.008, **P=0.015, comparing murine Hes6 and Hes6DBM, respectively, with ß-gal control using a two-tailed paired t-test. There was no significant difference between murine Hes6 and Hes6DBM.

View larger version (83K): [in a new window]   Fig. 5. XHes6 is expressed in the presomitic mesoderm and somitic chevrons. Embryos were analysed by whole-mount in situ hybridisation at early gastrula stage (stage 10.5) (A,C) and tailbud stage (stage 22) (B,D) for expression of MyoD (A,B) and Hes6 (C,D). Both MyoD and Hes6 are found in a ring of prospective mesoderm around the dorsal pore at stage 10.5 (A,C, respectively). At stage 22, MyoD is restricted to myotome but is expressed uniformly throughout (B). Hes6 is found in the eye, brain (asterisk), neural tube, tailbud domain (TBD) and in two to three chevrons anterior to the TBD (arrows, D).

View larger version (80K): [in a new window]   Fig. 6. XHes6 and XHes6DBM increase myotome size. Embryos were injected with 2 ng of XHes6 (A,D,G), XHes6DBM (B,E,H) or XHes6WRPW (C,F,I) along with ß-gal (light blue, injected side to left) and analysed at the neural plate stage for NßTub (A-C), MyoD (D-F) or -sarcomeric actin (MA) (G-I) expression by whole-mount in situ hybridisation (purple). Overexpression of all three constructs upregulated NßTub expression (A-C), while only XHes6 (D,G) and XHes6DBM (E,H) upregulated the muscle markers MyoD and MA.

View larger version (60K): [in a new window]   Fig. 7. XHes6 and XHes6DBM upregulate myogenesis and disrupt somitogenesis. Embryos were injected in one of two cells with 2 ng of (A,D,E) XHes6, (B,F,G) XHes6DBM or (C,H,I) XHes6WRPW along with nuclear ß-gal. At stage 22, the embryos were fixed and transversely (A-C) or longitudinally (D-I) sectioned and analysed for expression of MA (red); nuclei are stained with Hoechst (blue). Transverse sections are oriented with injected side to the left. Longitudinal sections are arranged with anterior towards the left and the injected side upwards. Broken white lines indicate the outlines of the embryo, the neural tube and the notochord. Quantitative image analysis was performed and the ratio of MA-expressing areas on injected and uninjected sides analysed as described in the Materials and Methods; results are shown in J. The error bars indicate the s.e.m. P values with a two-tailed t test, comparing mean ratios on injected and uninjected sides, were 0.017 for XHes6, 0.004 for XHes6DBM and 0.33 (not significant) for XHes6WRPW. Both XHes6 and XHes6DBM cause complete disruption of somitogenesis (100% of embryos disrupted, n=26, 25 respectively), while XHes6WRPW has a minimal effect (10% of embryos disrupted, n=20). In each embryo, the ß-gal tracer was distributed both in the mesoderm and the ectoderm (data not shown)

View larger version (103K): [in a new window]   Fig. 8. XHes6 and XHes6DBM inhibit terminal myogenic differentiation. Embryos were injected in one of two cells with 2 ng of (A,B) XHes6, (C,D) XHes6DBM or (E,F) XHes6WRPW with ß-gal (light blue) and analysed at stage 22 for 12/101 expression (purple) by whole-mount antibody staining. XHes6 and XHes6DBM both decrease the area, intensity and pattern of 12/101 expression (compare A,C with B,D). However, XHes6WRPW has little effect on the expression of this terminal myogenic differentiation marker (E,F).