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First published online 15 August 2007
doi: 10.1242/dev.003905

Development 134, 3371-3382 (2007)
Published by The Company of Biologists 2007
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Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation

Lisa Maves1,*, Andrew Jan Waskiewicz2, Biswajit Paul1, Yi Cao1, Ashlee Tyler1, Cecilia B. Moens3 and Stephen J. Tapscott1

1 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G2E9, Canada.
3 Howard Hughes Medical Institute and Division of Basic Science, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.


Figure 1
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  		Fig. 1. Pbx and Myod are required for the proper initiation of myog expression. (A-R) RNA in situ expression of (A-C,G-I) myod and krox-20, (D-F,J-L) myog and krox-20, (M-O) myog (blue) and myod (red), or (P-R) desm and krox-20 in (A,D,G,J,M,P) wild-type control, (B,E,H,K,N,Q) pbx2-MO; pbx4-MO, (C,F,I,L,R) myod-splMO, or (O) myod-MO embryos. Similar phenotypes were observed for both myod splice-blocking MOs (myod-splMOs). Somite (s) staging was confirmed by counting somites using Nomarski optics. All embryos are shown in dorsal view, anterior towards the left. (A) Arrowheads point to adaxial cells; arrows point to lateral somite cells; and r3 and r5 indicate krox-20 expression in hindbrain rhombomeres. (S-U) Graphs of real-time reverse-transcriptase (RT)-PCR quantities for (S) myod, (T) myog and (U) desm. (T, bottom) Enlarged view of 6s stage myog expression and the key for S-U. x-axes are developmental stage and y-axes are relative mRNA expression level. Quantities were normalized to odc1 expression. Error bars represent standard deviation. myodE2I2-splMO targets the exon 2-intron 2 boundary (see Fig. S3 in the supplementary material). The myod real-time primers measure only mRNA with properly spliced intron 2. Quantitative (q)RT-PCR shows that less than about 10-15% of normally spliced myod transcripts remain in myod-splMO embryos (Fig. 1S and see Fig. S3 in the supplementary material; and data not shown). Similar quantities for myog and desmin were observed for both myod-splMOs.

 

Figure 2
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  		Fig. 2. Validation of microarray-identified Pbx-dependent genes. (A-Z) RNA in situ expression of genes from Table 1 in (A,C,E,G,I,K,M,O,Q,S,U,X) wild-type control, (B,D,F,H,J,L,N,P,R,T,V,Y) pbx2-MO; pbx4-MO, or (W,Z) myod-MO embryos. (S,T) Control gene, desm; (X-Z) slow-muscle gene, smyhc1. Although pbx2-MO; pbx4-MO embryos appear delayed relative to controls, all embryos are stage-matched at (A-T) 18-somite stage (18s), (U-W) 16s or (X-Z) 14s. krox-20 was included in all in situs to control for the pbx MOs. (A) r3 and r5 indicate krox-20 expression in hindbrain rhombomeres. (Q) Arrow marks vmhc expression in the heart primordium. Embryos are shown in (A-P,S-T) left-side view or (Q-R,U-Z) dorsal view, anterior towards the left.

 

Figure 3
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  		Fig. 3. Pbx is required for the proper initiation of fast-muscle differentiation. (A-D) Antibody staining of slow muscle with F59 antibody (green; A,B), or fast muscle with F310 antibody (green; C,D), in (A,C) wild-type control or (B,D) pbx2-MO; pbx4-MO embryos. Antibody staining of muscle with MF20 antibody (red; A-D) is included to control for somite staging. Embryos are shown in left-side view, anterior towards the left. (E,F) Quantification of (E) F59/slow- and (F) F310/fast-muscle marker expression. MF20 expression is included to control for somite (s) staging. Graphs show number of somites expressing a muscle marker at different developmental stages. Similar phenotypes were observed for both myod splice-blocking MOs (myod-splMOs). Error bars represent standard deviation. P values (Mann-Whitney test; normalized to somite number): *P<0.0001 compared to same-stage control; except for pbx2-MO; pbx4-MO F310 compared to control at 24 hpf, P<0.03.

 

Figure 4
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  		Fig. 4. Pbx functions with Myod in fast-muscle, but not slow-muscle, differentiation. RNA in situ expression at the 10-somite (10s) stage of (A-G) myog and krox-20, (H-N) desm and krox-20 or (O-U) smyhc1 and krox-20, or at 16s of (V-BB) mylz2 and krox-20 in (A,H,O,V) wild-type control, (B,I,P,W) pbx2-MO; pbx4-MO, (C,J,Q,X) myod-MO, (D,K,R,Y) myf5-MO, (E,L,S,Z) myf5-MO; myod-MO, (F,M,T,AA) myf5-MO; pbx2-MO; pbx4-MO, or (G,N,U,BB) myod-MO; pbx2-MO; pbx4-MO embryos. Embryos are shown in dorsal view, anterior towards the left. Somite staging was confirmed by counting somites using Nomarski optics. (A) r3 and r5 indicate krox-20 expression in hindbrain rhombomeres.

 

Figure 5
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  		Fig. 5. Pbx is required downstream of Shh signaling to induce fast-muscle, but not slow-muscle, gene expression. (A-L) RNA in situ expression of (A-F) smyhc1 (blue) and myod (red) at the 14-somite (14s) stage or (G-L) mylz2 (blue) and myod (red) at 16s in embryos injected with (A,G) pbx2-MO mismatch control and gfp mRNA, (B,H) pbx2-MO mismatch control and shh mRNA, (C,I) myod-MO and gfp mRNA, (D,J) myod-MO and shh mRNA, (E,K) pbx2-MO; pbx4-MO and gfp mRNA, or (F,L) pbx2-MO; pbx4-MO and shh mRNA. The most-anterior 8-10 somites of each embryo are shown, in dorsal view, anterior towards the left. Control embryos injected with shh mRNA (control+shh) show increased expression of smyhc1 (B, 106/135 embryos; 29/135 show normal smyhc1 expression) or increased expression of mylz2 (H, 88/121; 33/121 show normal mylz2 expression). myod-MO+shh embryos show less-frequent and less-extensive induction of smyhc1 compared with control+shh (46/90 embryos resemble D; 44/90 show normal smyhc1 expression) and fail to upregulate mylz2 expression beyond that seen in myod-MO+gfp embryos (84/88 embryos resemble J; 4/88 show slightly increased mylz2 expression). pbx2-MO; pbx4-MO+shh embryos show strongly increased expression of smyhc1 (77/79 embryos resemble F; 2/79 have normal smyhc1 expression). Approximately half (43/102; L) of pbx2-MO; pbx4-MO+shh embryos show expansion of weak levels of mylz2 across the somites; the other half (59/102) resemble pbx2-MO; pbx4-MO+gfp embryos. Similar results were observed in multiple experiments.

 

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© The Company of Biologists Ltd 2007