1. Knockdown of smyhc1 Expression Resulted in Paralyzed Zebrafish Embryos with Defective Thick Filament Organization in Slow Muscles
Zebrafish embryonic muscles can be divided into two major types, slow and fast, based on the expression of myosin heavy chain (MyHC). Smyhc1 represents the first and primary MyHC expressed in slow muscles of zebrafish embryos that can be labeled specifically with F59 monoclonal antibody 
. In addition, two other myhc
, are also expressed in a small subset of zebrafish slow muscles at the dorsal, ventral and myoseptum regions of the myotome in late stage embryos 
. To determine the myosin knockdown phenotype and compare it with that of hsp90α1
mutation, we knocked down smyhc1
expression using a smyhc1
-specific translational morpholino (ATG-MO) in zebrafish embryos (). The ATG-MO was targeted to the flanking sequence of the smyhc1
ATG start codon. It shares 50–70% identity with the corresponding sequences in zebrafish smyhc2
Knockdown of smyhc1 expression by smyhc1 ATG-MO.
Western blot analyses showed a significant reduction of Smyhc1 protein levels in smyhc1 ATG-MO injected zebrafish embryos (). Immunostaining of whole mount embryos confirmed that myosin expression was missing or greatly reduced in slow muscles of the knockdown zebrafish embryos (). However, expression of other MyHCs in fast muscles was not affected (). Moreover, myosin expression in a subset of slow muscles in the dorsal and myoseptum regions of the myotome that express smyhc2 and smyhc3 also appeared normal (). Together, these data indicate that the smyhc1 ATG-MO was specific in knocking down the expression of smyhc1 in slow muscles of zebrafish embryos.
To determine whether knockdown of smyhc1 affects slow muscle development, we analyzed myod expression by in situ hybridization. Compared with controls (), a similar pattern of myod expression was observed in smyhc1 knockdown embryos (). Two rows of myod-expressing adaxial cells that give rise to slow muscles were clearly detected in the smyhc1 knockdown embryos (), confirming that knockdown of smyhc1 did not alter the initial formation of slow muscle precursors in zebrafish embryos. To determine whether slow muscle differentiation was affected in smyhc1 knockdown embryos, we analyzed the expression of slow muscle-specific troponin C in the knockdown embryos. The results showed that smyhc1 knockdown did not affect the expression of slow-specific troponin C in zebrafish embryos at 24 hpf (). Together, these data confirmed that knockdown of smyhc1 did not affect the formation of slow muscles.
Effects of smyhc1 knockdown on muscle development in zebrafish embryos.
To determine whether knockdown of smyhc1
affected muscle function, the smyhc1
ATG-MO injected embryos were examined morphologically following the smyhc1
ATG-MO injection. Although the injected embryos appeared morphologically normal compared with control (), smyhc1
knockdown embryos were paralyzed at 24 hpf, a phenotype very similar to the zebrafish slotu44c
mutant carrying a nonsense mutation in the hsp90α1
Morphology of control and smyhc1 knockdown embryos at 48 hpf.
To determine whether knockdown of smyhc1 expression resulted in defective thick filament organization, we examined the organization of myosin thick filaments in smyhc1 knockdown embryos by immunostaining with the F59 and F310 antibodies that label myosin in slow and fast muscles, respectively. Compared with slotu44c mutant zebrafish embryos (), knockdown of smyhc1 expression resulted in no organized thick filaments in slow muscles of zebrafish embryos (). This was expected considering the smyhc1 is specifically expressed in slow muscles. However, knockdown of smyhc1 had no effect on thick filament organization in fast muscles (). This was in contrast to slotu44c mutant that has muscle defects in both slow and fast muscles (). Together, these data indicate that Smyhc1 is required for thick filament assembly in slow muscles.
Effects of smyhc1 knockdown or hsp90α1 mutation on myosin thick filament organization in skeletal muscles of zebrafish embryos.
To confirm the specificity of phenotype, we performed a rescue experiment by co-injecting a smyhc1 expression DNA construct with the smyhc1 ATG-MO into zebrafish embryos. The ATG-MO was not able to knock down the expression of the DNA construct because its 5′ UTR sequence targeted by the ATG-MO has been replaced with a 5′ UTR sequence from the b-globin gene. The results showed that transient expression of the smyhc1 DNA construct could rescue the thick filament defect (). The rescued myofibers appeared in a mosaic fashion, consistent with the typical mosaic pattern of gene expression from DNA injection.
2. Knockdown of smyhc1 Expression Disrupted Organization of Thin Filaments in Slow Muscles
During myofibrillogenesis, actin thin filaments align around myosin thick filaments in a hexagonal arrangement to form the highly ordered sarcomeric structure. It has been suggested that interaction with myosin is critical for α-actin thin filament organization. To test whether thin filament organization was affected in smyhc1 knockdown embryos, we examined thin filaments in smyhc1ATG-MO injected embryos by immunostaining with anti-α-actin antibody. Compared with the control-MO injected embryos (), smyhc1 knockdown embryos showed disorganized thin filaments in slow muscles (). The thin filament defects were very similar to that observed in hsp90α1 mutant zebrafish embryos (). As expected, the thin filament defects were specifically restricted to slow muscles in smyhc1 knockdown embryos. Thin filaments in fast muscles appeared normal in smyhc1 knockdown embryos (). This differs from the slotu44c mutant, which exhibited thin filament defects in both slow and fast muscles (). Together, these data suggest that disruption of myosin thick filaments could result in defective organization of thin filaments in muscle cells.
Knockdown of smyhc1 expression or hsp90α1 mutation resulted in defective thin filament organization in skeletal muscles of zebrafish embryos.
3. Inhibition of Myosin Function by BTS Resulted in Defective Thick and Thin Filaments in Skeletal Muscles of Zebrafish Embryos
It has been shown that inhibition of myosin ATPase activity by BTS (N-benzyl-p-toluene sulphonamide) blocks thick and thin filament assembly in cultured cells in vitro 
. Moreover, treating zebrafish embryos with BTS induce paralysis in fish embryos 
. To determine whether inhibiting myosin function by BTS affects myofibril assembly and muscle contraction in zebrafish embryos in vivo
, we incubated zebrafish embryos with BTS starting at 12 hpf, a developmental stage correlating with myofibrillogenesis. A clear dose-dependent effect was observed on inhibition of muscle contraction in BTS-treated zebrafish embryos (). BTS could effectively block muscle contraction at a dose of 20 µM. BTS-treated embryos appeared morphologically normal except the lack of muscle contraction (). A clear edema and weak cardiac muscle contraction were also detected in BTS treated embryos at 120 hpf ().
Dose-dependent effects of BTS on muscle contraction in zebrafish embryos.
BTS inhibits skeletal muscle contraction and suppresses thick and thin filament assembly in skeletal muscles of zebrafish embryos.
The myofibril organization of thick and thin filament was analyzed in BTS-treated zebrafish embryos by immunostaining with anti-MyHC (F59), and anti-α-actin antibodies. Unlike the ATG-MO injection, BTS treatment did not significantly reduced the levels of myosin and actin expression in muscle cells (). However, thick and thin filament organization was significantly disrupted in both slow and fast muscles of BTS-treated embryos (). In contrast, incubation with DMSO, used in making BTS solution, had no effect on thick and thin filament organization (), confirming that the muscle defects were BTS-specific. Together, these data indicate a critical role for myosin ATPase activity in myosin thick filament assembly and organization. In addition, the myosin-actin interaction is required for thin filament assembly in skeletal muscles.
4. Blocking Myosin Function and hsp90α1 Mutation Had Different Effects on Organization of Z- and M-Lines in Skeletal Muscles of Zebrafish Embryos
To determine whether disruption of myosin thick filaments could affect the organization of other sarcomeric structures in skeletal muscles, we analyzed the M- and Z-line structures in smyhc1 knockdown and BTS-treated zebrafish embryos, and compared them with that from the hsp90α1 mutation. Immunostaining was performed with anti-myomesin and anti-α-actinin antibodies that specifically label the M- or Z-lines, respectively. Compared with control (), Z-line organization was clearly altered in slow muscles of smyhc1 knockdown embryos (). Although Z line-like structures were clearly detected in the smyhc1 knockdown embryos, they failed to align correctly to form the straight Z-line (). As expected, the Z-line defect was restricted to slow muscles. The Z-lines appeared normal in fast muscles (). However, compared with smyhc1 knockdown, the Z-line organization appeared less affected in BTS-treated embryos, although the myofibers appeared to be twisted (). In contrast, the Z-line organization was significantly affected in hsp90α1 mutant embryos (). Very few organized Z-lines could be observed, although Z-bodies could be clearly detected at a higher magnification (). Together, these data indicate that compared with the loss of myosin function, hsp90α1 mutation has a strong effect on Z-line organization.
The effect of smyhc1 knockdown, BTS treatment or hsp90α1 mutation on Z body formation, and Z-line organization in skeletal muscles of zebrafish embryos.
To determine whether M-lines were affected by thick filament disruption in smyhc1 knockdown, BTS-treated or slotu44c mutant embryos, we analyzed M-line organization by immunostaining using an anti-myomesin antibody. The data showed a striking difference in myomesin staining among the three different groups of embryos. Although the organization of M-lines appeared normal in smyhc1 knockdown and BTS-treated embryos (), the M-line localization of myomesin was completely abolished in hsp90α1 mutant embryos (). Very little myomesin staining could be detected in the hsp90α1 mutant embryos (), suggesting that the hsp90a1 mutation could result in a dramatic disruption of M-line organization in skeletal muscles. Collectively, these data indicate that defective thick filament assembly could not account for all myofibril defects in hsp90a1 mutant embryos, suggesting that Hsp90α1 may play additional roles in the assembly and organization of other sarcomeric structures, such as M-lines in skeletal muscles.
The effect of smyhc1 knockdown, BTS treatment or hsp90α1 mutation on M-line organization in skeletal muscles of zebrafish embryos.