Maguire RJ et al. (2012), Early transcriptional targets of MyoD link myog...

XB-ART-45868

Dev Biol 2012 Nov 15;3712:256-68. doi: 10.1016/j.ydbio.2012.08.027.

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Early transcriptional targets of MyoD link myogenesis and somitogenesis.

Maguire RJ , Isaacs HV , Pownall ME .

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In order to identify early transcriptional targets of MyoD prior to skeletal muscle differentiation, we have undertaken a transcriptomic analysis on gastrula stage Xenopus embryos in which MyoD has been knocked-down. Our validated list of genes transcriptionally regulated by MyoD includes Esr1 and Esr2, which are known targets of Notch signalling, and Tbx6, mesogenin, and FoxC1; these genes are all are known to be essential for normal somitogenesis but are expressed surprisingly early in the mesoderm. In addition we found that MyoD is required for the expression of myf5 in the early mesoderm, in contrast to the reverse relationship of these two regulators in amniote somites. These data highlight a role for MyoD in the early mesoderm in regulating a set of genes that are essential for both myogenesis and somitogenesis.

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Species referenced: Xenopus laevis
Genes referenced: dlc esr1 esr2 foxc1 hes3 hes5 hes5.2 hes5.3 lama1 msgn1 myf5 myod1 notch1 nucb1 rbm24 tbx6 tbxt tcea1
???displayArticle.antibodies??? Lama1 Ab1 Somite Ab1
???displayArticle.morpholinos??? myod1 MO1 myod1 MO2

Phenotypes: Xla Wt + myod1 (fig.4.a) [+]

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	Fig. 3. Expression patterns of shortlisted targets. In situ hybridisation analysis of Esr1, Esr2, FoxC1, mesogenin (MSGN1), Myf5, SEB4 and Tbx6 expression patterns at gastrula, neurula and tailbud stages. All the putative target genes are expressed circumblastoporally at gastrula stages. After gastrula stages, many of the targets are still expressed in the posterior mesoderm in the tailbud region of the embryo, which is the source of myogenic progenitors at this stage. On the right, a temporal expression profile, based on microarray data, is shown for each putative target genes depicting relative expression levels from NF stage 8 to stage 15. The expression of MyoD is shown in the three bottom panels for comparison.
	Fig. 4. Validation of the effect knocking down MyoD on shortlisted targets. Embryos were injected unilaterally with 10 ng of Morpholino A and 30 ng of Morpholino B (knock-down), 2.5 ng XtMyoD mRNA (overexpression) or both (rescue). Control embryos were injected with the equivalent amount of control MO. The injected side is shown by an asterisk. Numbers and percentages indicate the number of embryos with the phenotype shown. Numbers are from several experiments. The knock-down of MyoD protein causes the loss of expression of all shortlisted targets. Overexpression of MyoD up-regulates the expression of most targets, with the exception of Myf5. Target expression is rescued effectively for all targets when XtMyoD mRNA is coinjected with morpholino. Xbra is expressed in all mesoderm and is included as a control. Embryos have been analysed at mid-gastrula NF stage 11.5 and are viewed vegetally. For numbers and statistical analysis see Supplementary data Table S1.
	Fig. 6. MyoD is required for somite morphogenesis. (A) and (B) each depict a plane of focus from a Z-stacked confocal image of an embryos in which MyoD has been knocked down unilaterally and immunostained with anti-laminin. (A) is the control side of the embryo and (B) is the morpholino injected side. (C and C show a cryosection of an embryo unilaterally injected with MyoD AMO and immunostained with 12/101 and anti-laminin; the nuclei have been labeled with DAPI. The top is the control side and the arrow heads mark out normal tight somite boundaries. (C shows a magnification of the disrupted somite boundary on the myoD knock-down side. (D and D show a vibratome section of an embryo unilaterally injected with MyoD AMO and mRNA coding for NLS-beta-galactosidase immunostained with 12/101. The top side is the control side and the arrow heads mark out the normal somite boundaries. The MyoD knock-down side is at the bottom of the panel and the blue nuclei indicate it is the injected side. (D shows a magnification of the disrupted somite boundary on the myoD knock-down side. (E and E show a paraffin wax section of an embryo unilaterally injected with MyoD AMO (bottom side) and stained with borax carmine and picro-blue-black. The normal somite boundaries are indicated by arrow heads on the control side. All specimens are at NF stage 35 and the somites shown are anterior to mid-way along the axis. The confocal images in (A and B) were taken sagitally through the embryo. The sections shown in (C) are frontal sections. (C, (D, and (E are each a magnified field of the panel to its right showing the myofibres crossing the weak somite boundaries. Scale bars are 500 mm. Scale bar in (A) is for (A and B); scale bar in (C) is for (C, D and E); scale bar in (C is for (C D E.
	Fig. 7. A transient requirement for MyoD. (A) Embryos were unilaterally co-injected with mRNA coding for nucLacZ together with either MyoD AMO or the equivalent amount of control MO. The injected side is marked with an asterix. (B and E) are injected with control MO, (A, C, D, and F) are injected with MyoD AMO. In situ hybridisation shows Tbx6 (A) and mesogenin (D) expression. (A and D) are gastrula stage 11, viewed vegetally. (B, C, E, and F) are neurula stage 19 and viewed dorsally. (G) qPCR shows levels of target gene expression in sibling embryos at gastrula stage 11(blue bars) and neurula stage 14 (red bars). All qPCR data were normalized to ODC TM and the Ct values from three technical replicates and primer efficiency were then exported into REST confidence interval of the mean fold change. (H) In situ hybridisation shows Delta2 expression in embryos co-injected with mRNA coding for nucLacZ together with either MyoD AMO or control MO. (H, K, and N) are control MO injected, while (I, J, L, M, and O) are injected with MyoD AMO. (H) are open neural plate stage 13, viewed dorsally, and Delta expression shows the first segmentation of somites. (K) are stage 19, viewed dorsally. (N and O) are stage 30 viewed laterally and injected side is shown. Arrows indicate forming somites. (P) A frontal section through a Stage 21 (7 somites) embryo that has been unilaterally co-injected with membrane-RFP and MyoD AMOs (asterix). The structure of the somite cells is revealed by staining with anti-beta-catenin (green) and nuclei are marked with DAPI (blue). The forming somites are numbered on the control side for reference. On the control side, we have highlighted a single cell in the PSM/somitomere, S0, SI and SII to draw attention to the rotation that occurs. On the MyoD knock down side we also highlight cells at progressive points in their rotation; we were unable to highlight a cell in the PSM as the cells were disorganized (arrow heads).
	tbx6 (T-box 6) gene expression in bisected Xenopus laevis/ tropicalis embryo, assayed via in situ hybridization, NF stage 10-10.25, vegetal view, dorsal up.
	tbx6 (T-box 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	tbx6 (T-box 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	hes5.1 (hes family bHLH transcription factor 5 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5-11, vegetal view, dorsal up.
	hes5.1 (hes family bHLH transcription factor 5 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	hes5.1 (hes family bHLH transcription factor 5 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	hes3.3 (hes family bHLH transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
	hes3.3 (hes family bHLH transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	hes3.3 (hes family bHLH transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	foxc1 (forkhead box C1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
	foxc1 (forkhead box C1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	foxc1 (forkhead box C1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	msgn1 (mesogenin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
	msgn1 (mesogenin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	msgn1 (mesogenin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	myf5 (myogenic factor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
	myf5 (myogenic factor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	myf5 (myogenic factor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27/28, lateral view, anterior left, dorsal up.
	rbm24 (RNA binding motif protein 24 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
	rbm24 (RNA binding motif protein 24 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	rbm24 (RNA binding motif protein 24 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	Figure 5 Anterior somite disruption. Embryos were unilaterally injected with mRNA coding for nuc-LacZ together with control morpholino oligo (CMO) or antisense morpholino oligos targeted against MyoD (MyoD AMO). The myocyte nuclei align down the center of each somite in normal embryos (100%, n=14). Disrupted somites are evident by the failure of these nuclei to align (arrows). This disruption is most evident in somites 4-7 in MyoD knock-down embryos (58%, n=12) while more posterior somites in these same embryos form normally. Somites form in an anterior-to-posterior direction, so this suggests that the effects of MyoD knockdown on somite morphogenesis is transient; this is consistent with the recovery of segmentation genes like Tbx6 and mesogenin at neurula stages.