Characterization of Zebrafish Models of Marinesco-Sjögren Syndrome

SIL1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone, BiP. Mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disease characterized by cerebellar ataxia, mental retardation, congenital cataracts, and myopathy. To create novel zebrafish models of MSS for therapeutic drug screening, we analyzed phenotypes in sil1 knock down fish by two different antisense oligo morpholinos. Both sil1 morphants had abnormal formation of muscle fibers and irregularity of the myosepta. Moreover, they showed smaller-sized eyes and loss of purkinje cells in cerebellar area compared to controls. Immunoblotting analysis revealed increased protein amounts of BiP, lipidated LC3, and caspase 3. These data supported that the sil1 morphants can represent mimicking phenotypes of human MSS. The sil1 morphants phenocopy the human MSS disease pathology and are a good animal model for therapeutic studies.

Zebrafish is a useful model to elucidate the pathomechanisms of a lot of human diseases including muscular dystrophies [13][14][15]. The analyses of zebrafish models for human muscle disorders have been facilitated because of rapid development and easy-to-identify muscle structural abnormalities using birefringence assay [16]. They are easily analyzed by the use of morpholinos during early development. Zebrafish models of muscle disease have also been used to rapid therapeutic drug screening for small molecules [17].
To generate a model fish of MSS for studies on the functions of zebrafish sil1 and for therapeutic drug screenings, morpholino antisense oligos that targeted zebrafish sil1 mRNA were designed and injected into zebrafish eggs. Our data indicates the reduction of sil1 expression causes the abnormal formation of muscles, small sized eyes and reduction of purkinje cells associated with increased marker proteins for ER-stress, autophagy, and apoptosis.

Materials and Methods
Fish and fish culture Zebrafish (the AB line) were cultured at 28.5°C according to standard procedures [18] and standard criteria [19]. Fertilized eggs were collected and used for injection. For anesthesia, euthanasia we used tricaine solution. Our IACUC, Tokyo Medical University animal facility, indicates that they approved this research (the approval number:S28029).

Detection of muscle phenotype of sil1 morphants by birefringence
Birefringence assay was performed to detect abnormal skeletal muscle structure by placing anesthesized fish on a polarizing filter and subsequently covering them with a second polarizing filter [17]. The filters were placed on an underlit dissection scope and the top-polarizing filter twisted until only the light refracting through the striated muscle was visible. Since the degree of birefringence is affected by the horizontal orientation of the fish, the fish were oscillated back and forth to account for differences in positioning.

Measurement of size of eyes
The diameter of the eye of 4 dpf embryos was measured under a dissection scope (Olympus, SZX10) with DP controller (Olympus) software and ImageJ (http://imagej.en.softonic.com/).

Zebrafish SIL1 antibody
A rabbit polyclonal antibody against zebrafish sil1 was produced by synthesized peptides (C+EDLEVFRPTDKWQTLRPGQ; 63-81) as an antigen (Sigma Aldrich). The synthesized peptides were also used to absorb the antibodies to confirm the specificity of this antibody.

Zebrafish sil1 morphant morphology
Two different anti-sense morpholino oligonucleotides (MO) 1 and 2 were designed to disrupt splicing pattern of sil1 mRNA during development (Fig 1A). The injected embryos were examined at 4 dpf. RT-PCR and sequence analysis confirmed that the MO1 and MO2 injection  resulted in an in-frame insertion of a whole intron 2 (91 bps) and insertion of partial intron 2 (52 bps), respectively (Fig 1B).
To confirm the knock down effect to the expression of fish sil1 by morpholino injections, endogenous zebrafish sil1 protein was analyzed with antibody against zebrafish sil1. In wild type and CMO injected fish, the sil1 antibody recognized a 52 kDa protein on western blots, a consistent size predicted by the fish sil1 sequence (Fig 1C). The expression of sil1 protein of MO1 or MO2 injected fishes was reduced to 66.5% and 46.9%, respectively compared to the wild type (Fig 1D). This result indicated that injections of MO1 or MO2 were effective to decrease the expression of sil1 protein.

Birefringence assay
Following injection of the two different morpholinos, some embryos showed abnormal shape visible upon light microscopy (Fig 2A). To better visualize the structure and organization of muscle fibers of the morphants, MO1, MO2 or CMO injected embryos and wild type embryos were analyzed by birefringence assays at 4 dpf (Fig 2A). The sil1 morphant embryos were found to have markedly reduced normal patterns of birefringence compared to wild type and control morphants (Fig 2A). Injection of 3 ng of MO1 or MO2 resulted in approximately 39.0±1.8% and 21.8±3.1% of injected embryos exhibiting reduced birefringence, 36.5±4.7% and 64.5±1.6% were normal looking, and 24.5±4.7% and 13.7±1.6% of dead, respectively ( Fig  2B). These percentages are an average of the results from three different experiments. The effects of morpholinos were dose-dependent and the ratio of abnormal embryos were increased when 6 ng of morpholinos were injected (S1 Fig). Importantly, co-injection of zebrafish sil1 mRNA with each MOs rescued the phenotypes (Fig 2C).

Immunohistochemistry of sil1 morphants with muscle structure's components
To examine the expression of muscle proteins, antibodies against beta-dystroglycan, laminin (data not shown), and myosin heavy chain (MHC, slow fibers) were used for immunohistochemistry. Beta-dystroglycan expression at the myosepta of MO1 or MO2 injected 4 dpf embryos was misshapen and had a less clear v-shaped structure as observed in wild type and CMO injected embryos (Fig 3A and 3C). Staining with anti-MHC indicated that formation of myofibers was disturbed in MO1 and 2 (in Fig 3B and 3D). This result is consistent with that observed by birefringence assay. Co-injection of zebrafish sil1 mRNA with each MO reduced the number of fishes showing marked myofibril disruptions by anti-MHC stain. dpf embryos was smaller than those of CMO injected embryos (Fig 4C, 4D and 4E). Co-injection of zebrafish sil1 mRNA with each MOs rescued the eye size (Fig 4F).
The number of purkinje cells detected with anti-parvalbumin, a purkinje cell marker [20], showed reduced number of positive cells in MO1 or MO2 injected embryos compared to those of controls (Fig 5D and 5G). Co-injection of zebrafish sil1 mRNA with each MO increased the number of positive cells in MO1 or MO2 injected embryos (MO2: 76.9%, n = 13, MO2+sil1 mRNA: 35.7%, n = 14) (Fig 5J).

Increased expression of marker proteins of ER-stress, autophagy, and apoptosis
To examine expression levels of marker proteins of ER-stress, autophagy and apoptosis, western blotting was performed. The protein amounts of BiP, lipidated form of LC3 (LC3-II), and activated caspase 3 were significantly increased in sil1 morphant embryos compared to those of CMO-injected embryos (Fig 6A and 6B).

Discussion
Here, we showed a zebrafish is a good model of MSS, an autosomal recessive multisystem disorder characterized by cerebellar ataxia, mental retardation, cataract, and myopathy [1][2][3][4][5][6]. The sil1 morphants showed altered skeletal muscle structures, judged by unshapen myosepta and disturbed muscle fibers. In addition, the sil1 morphants have smaller sized eyes and loss of purkinje cells.
Our MSS zebrafish models display an increase in the levels of markers associated with ER stress, autophagy and apoptosis, which are similar to those observed in humans and a mouse model of MSS [9][10][11]. SIL1-deficiency is expected to reduce BiP activity, an ER chaperone protein, which leads to accumulation of abnormal proteins and subsequent ER stress, increased autophagy and apoptosis. In the reduced expression of sil1, morphants might have clear phenotypes in muscle, abnormal eye size and loss of purkinje cells via activated unfolded protein response, ER stress, autophagy and apoptosis. Interestingly, sil1 morphants showed smallersized eyes instead of cataracts, which suggests that sil1 may have important roles in development of eyes in zebrafish. Recently SIL1 was reported to play a role in regulation of motor neuron subtype-selective ER stress in amyotrophic lateral sclerosis (ALS) [21]. Further analysis of the MSS model fish might reveal molecular crosstalk of ER stress, autophagy, and apoptosis in variable tissues and cells.
Further analysis of sil1 function in zebrafish should be undertaken CRISPR-Cas9 system for analysis of sil1 functions and therapeutic intervention studies for MSS.