Geach TJ et al. (2015), Contractile activity is required for Z-disc sar...

XB-ART-50332

Genesis 2015 May 01;535:299-307. doi: 10.1002/dvg.22851.

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Contractile activity is required for Z-disc sarcomere maturation in vivo.

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Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto-myosin contraction in embryos of the frog X. tropicalis, and Z-disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z-disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo.

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Species referenced: Xenopus tropicalis
Genes referenced: actn1 actn2 actn3 elob myh6 tcea1 unc45b
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	FIG. 1. a-Actinin-stained Z-discs first appear in 11 somite embryos (a, d, g) Schema of somites imaged and somite numbering. Somites are sequentially added and numbered from the posterior during development (right, S0); second-to-anterior shaded black; third-youngest (SIII) gray; e5eye; cg5cement gland. a-Actinin staining in second-to-anterior somite in (b) nine somite NF23 (somite SVIII), (e) 11 somite NF24 (SX), and (h) 12 somite NF25 (SXI) embryos or third from posterior (SIII) somite at corresponding stage (c, f, i). Z-discs (arrowheads) are present in anterior somites from NF24 (e), becoming more abundant at NF25 (h). Z-bodies (arrows) are observed in the SIII somite of 11 and 12 somite embryos (f, i). Initial a-actinin staining appears in 11 somite embryos in both anterior and posterior somites Scale bar=13 um.
	FIG. 2. Z-disc maturation is delayed in the absence of contractility. a-Actinin staining in an anterior somite of (a, b) nine somite NF23, (d, e) 11 somite NF24, or (g, h) 12 somite NF25 embryos treated with vehicle control (a, d, g) or BTS (b, e, h) showing Z-discs (arrowheads) and Z-bodies (arrows). Z-disc maturation is delayed in BTS-treated embryos. (k) a-Actinin maturation is likewise delayed in genetically paralyzed dit mutant embryos at NF28 relative to (j) wild-type control. (c, f, i, l) Reverse transcription-PCR shows that actn3 mRNA is present before Z-discs are detected and remains expressed when embryos are paralyzed by BTS treatment or the dit mutation. Scale bar=18um.
	FIG. 3. Z-disc diameter and thick filament bundling are delayed in the absence of contractility. Representative fields of TEM sections from (a) DMSO vehicle control showing well-formed Z-discs (arrowheads) and (b) BTS-treated somites with an immature Z-disc (arrow) at NF24; scale bar5200 nm. Z-discs were identified as electron-dense regions crossing thick filaments (arrowheads). The number of Z-discs was reduced in BTS-treated embryos (c), as was their diameter (d). Thick filaments also appeared more disordered in BTS-treated embryos, and (e) average length of visible thick filaments per frame was reduced relative to controls.
	FIG. 4. Timely Z-disc maturation in sequentially added posterior somites also requires muscle activity. At later stages, Z-disc maturation accompanies sequential addition of posterior somites, but shows consistent delay in paralyzed embryos. NF28 a-actinin-stained posterior somites treated with (a) vehicle control, (b) BTS, or (c) tricaine; intersomitic boundaries indicated by dashed lines. Z-bodies in control embryos appear in the SII somite (a, inset), with Z-discs visible in SIII (arrowhead). Paralysis with BTS or tricaine shifts Z-disc maturation to older SV– SVI somites (b, c inset). (d–f) NF40 posterior somites stained for a-actinin, showing Z-bodies in the SIII somite in control (d, inset), in the SVI somite of BTS-treated embryos (e, inset), or SIX somite in dit mutants (f, inset). (g–i) MyHC-stained thick filaments are unaffected in NF28 BTS- and tricaine-treated embryos. (j) Quantitation of anterior shift of the last Z-disc-containing somite at NF28 and NF40 caused by pharmacological or genetic paralysis. The youngest (most posterior) Z-disc-containing somite is shifted anteriorly by pharmacological or genetic paralysis, as shown by quantitation of anterior shift at stages NF28 and NF40 in tricaine- and BTS-treated embryos and dit mutants.
	Figure 1. α-Actinin-stained Z-discs first appear in 11 somite embryos (a, d, g) Schema of somites imaged and somite numbering. Somites are sequentially added and numbered from the posterior during development (right, S0); second-to-anterior shaded black; third-youngest (SIII) gray; e = eye; cg = cement gland. α-Actinin staining in second-to-anterior somite in (b) nine somite NF23 (somite SVIII), (e) 11 somite NF24 (SX), and (h) 12 somite NF25 (SXI) embryos or third from posterior (SIII) somite at corresponding stage (c, f, i). Z-discs (arrowheads) are present in anterior somites from NF24 (e), becoming more abundant at NF25 (h). Z-bodies (arrows) are observed in the SIII somite of 11 and 12 somite embryos (f, i). Initial α-actinin staining appears in 11 somite embryos in both anterior and posterior somites Scale bar = 13 μm.
	Figure 2. Z-disc maturation is delayed in the absence of contractility. α-Actinin staining in an anterior somite of (a, b) nine somite NF23, (d, e) 11 somite NF24, or (g, h) 12 somite NF25 embryos treated with vehicle control (a, d, g) or BTS (b, e, h) showing Z-discs (arrowheads) and Z-bodies (arrows). Z-disc maturation is delayed in BTS-treated embryos. (k) α-Actinin maturation is likewise delayed in genetically paralyzed dit mutant embryos at NF28 relative to (j) wild-type control. (c, f, i, l) Reverse transcription-PCR shows that actn3 mRNA is present before Z-discs are detected and remains expressed when embryos are paralyzed by BTS treatment or the dit mutation. Scale bar = 18 μm.
	Figure 3. Z-disc diameter and thick filament bundling are delayed in the absence of contractility. Representative fields of TEM sections from (a) DMSO vehicle control showing well-formed Z-discs (arrowheads) and (b) BTS-treated somites with an immature Z-disc (arrow) at NF24; scale bar = 200 nm. Z-discs were identified as electron-dense regions crossing thick filaments (arrowheads). The number of Z-discs was reduced in BTS-treated embryos (c), as was their diameter (d). Thick filaments also appeared more disordered in BTS-treated embryos, and (e) average length of visible thick filaments per frame was reduced relative to controls.
	Figure 4. Timely Z-disc maturation in sequentially added posterior somites also requires muscle activity. At later stages, Z-disc maturation accompanies sequential addition of posterior somites, but shows consistent delay in paralyzed embryos. NF28 α-actinin-stained posterior somites treated with (a) vehicle control, (b) BTS, or (c) tricaine; intersomitic boundaries indicated by dashed lines. Z-bodies in control embryos appear in the SII somite (a, inset), with Z-discs visible in SIII (arrowhead). Paralysis with BTS or tricaine shifts Z-disc maturation to older SV–SVI somites (b, c inset). (d–f) NF40 posterior somites stained for α-actinin, showing Z-bodies in the SIII somite in control (d, inset), in the SVI somite of BTS-treated embryos (e, inset), or SIX somite in dit mutants (f, inset). (g–i) MyHC-stained thick filaments are unaffected in NF28 BTS- and tricaine-treated embryos. (j) Quantitation of anterior shift of the last Z-disc-containing somite at NF28 and NF40 caused by pharmacological or genetic paralysis. The youngest (most posterior) Z-disc-containing somite is shifted anteriorly by pharmacological or genetic paralysis, as shown by quantitation of anterior shift at stages NF28 and NF40 in tricaine- and BTS-treated embryos and dit mutants.

References [+] :

Abu-Daya, Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6. 2009, Pubmed, Xenbase