Fig 1.
RT-PCR and protein expression of sil1 morpholino-injected embryos.
A: Morpholino oligo-nucleotides target for zebrafish sil1. Two different anti-sense morpholino oligo-nucleotides targeted to disrupt splicing of sil1 mRNA are designed by Gene Tools LLC. B: A single RT-PCR product of 355 bp is seen in 4 dpf embryos injected 3 ng of control morpholino (CMO), whereas 4 dpf embryos injected 3 ng of sil1 morpholino 1 (MO1) had two different sized products of 355 and 446 bp. Two RT-PCR products of 355 bp and 407 bp were detected in 4 dpf embryos injected morphant 2 (MO2). C: Western blotting analysis shows reduced amounts of sil1 protein extracted from 20 each of MO1- or MO2-injected morphants compared to 4 dpf wild-type embryos (WT) and CMO. D: Injections of MO1 or MO2 are effective to reduce the expression of sil1 protein to 66.5% (MO1, light gray) and 46.9% (MO2, gray) compared to CMO-injected fish (black), respectively.
Fig 2.
Altered skeletal muscle in sil1-morpholino injected fish.
A: Left panels are pictures under bright field, and left panels are birefringence assay (BR). Abnormal structure of muscle is obvious in sil1 morpholino injected fish (MO1 and MO2) compared to control (CMO) under birefringence assay. B: Histogram of the percentage of normal, affected, and dead fish by morpholino-injection. Injection of 3 ng of MO1 or MO2 resulted in approximately 39.0% and 21.8% of injected embryos exhibiting reduced birefringence, respectively. C: Restoration of sil1 morphant with co-injection of fish sil1 mRNA (50 pg). Histogram of the percentage of dead, affected fish of morphants and recovered fish. Co-injection of zebrafish sil1 mRNA with each MO (3 ng) rescued the phenotypes. White bar shows normal %, gray shows affected % and black shows dead fish %. Non: non injected fish, MO1: MO1 injected fish, MO2: MO2 injected fish.
Fig 3.
Immunohistochemistry of skeletal muscle tissue of morpholino-injected fish.
Immunostaining of CMO injected fish (A, B) and sil1 morphant (MO2) injected fish (C, D) with antibodies against beta-dystroglycan (beta-DG) (A, C) and myosin heavy chain (MHC) (B, D). Beta-dystroglycan expression at the myosepta of MO2 injected 4 dpf embryos is misshapen and has a less clear v-shaped structure. Staining with anti-MHC indicated that formation of myofibers is disturbed in MO2. Arrowheads indicate the disturbance of myosepta in MO2-injected fish. Bar: 100 μm. Arrows indicate the abnormal structure of myofibers.
Fig 4.
Smaller sized eyes in sil1-morpholino injected fish (4 dpf).
The diameter of eyes in MO1 or MO2 injected 4 dpf embryos are smaller than those of CMO injected embryos. Co-injection of zebrafish sil1 mRNA with each MOs rescues the eye size. Eye size is measured under a dissection scope with DP controller software and ImageJ. (A) wild type, (B) CMO injected fish, and (C) MO2 injected fish, showing white lines. Measurement of the diameter of eyes (D: MO1, E: MO2). (F): Restoration of eye diameter of sil1 morphant with co-injection of fish sil1 mRNA. Single asterisk indicates Student’s t-test p = 1.25E-07 (MO1; n = 19, CMO; n = 14), double asterisks indicate p = 7.36E-08 (MO2; n = 16, CMO; n = 28) and triple asterisks indicate p = 0.00441 (MO2; n = 19, MO2+sil1 mRNA 50 pg; n = 14, CMO; n = 14).
Fig 5.
Staining of purkinje cells with anti-purvalbumin in cerebellar area.
The number of purkinje cells detected with anti-parvalbumin shows reduced number of positive cells in MO1 or MO2 injected embryos. (A, B and C): CMO injected fish, (D, E and F): MO1 injected fish, (G, H and I): MO2 injected fish. (A, D and G: Staining Purkinje cell. (B, E and H): DAPI, nuclei. (C, F and I): Merged images. (J): Increased number of Purkinje cells in sil1 morphants with co-injection of fish sil1 mRNA (50 pg) from 23.1% (MO2, n = 13) to 64.3% (MO2+sil1 mRNA, n = 14).
Fig 6.
Protein expression analysis of sil1-morpholino injected fish.
Western blot analysis of control morpholino (CMO) and sil1-morpholino2 injected fish (MO). The protein amounts of BiP, lipidated form of LC3 (LC3-II), and activated caspase 3 are significantly increased in sil1 morphant embryos compared to those of CMO-injected embryos. A: Immunoblot with anti-BiP, LC3-II, activated-Caspase 3, and beta-actin. B: Relative expression level is analyzed via immunoblot (*p = 0.0226, ** p = 0.000102 and *** p = 0.000705, versus CMO, Student’s t-test, n = 3).