Sztal et al., 2018 - Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy. Acta neuropathologica communications   6:40 Full text @ Acta Neuropathol Commun

Fig. 1

Characterization of the neb (sa906) mutant zebrafish strain. a & b) At 4 dpf neb−/− zebrafish (aii) appear smaller in size and (bii) display a loss of birefringence compared to their wildtype siblings (ai & bi). c) RT-PCR analysis in and neb−/− mutant embryos at 2 dpf shows a reduction in neb mRNA levels compared to wildtype siblings (sibling). βAct was used as a positive control. d) ii) neb−/− mutants display a smaller eye, brain region (B) and deflated swim bladder (SB) compared to their i) wildtype siblings. e) Quantification of the maximum acceleration recorded from touch-evoked response assays of neb−/− fish compared to wildtype siblings at 2 dpf. Error bars represent mean±SEM for three independent experiments (n = 11,9,12 neb −/− , 55,25,32 neb +/− , 18,13,12 neb +/+ zebrafish per experiment), **p < 0.01. f & g) Quantification of the normalized (f) distance and (g) speed travelled by neb−/− mutants compared to wildtype siblings at 6 dpf. For f) error bars represent median±interquartile range for three independent experiments (for n = 19,23,19 neb −/− , 41,42,36 neb +/− , 31,20,21 neb +/+ zebrafish). For g) error bars represent mean±SEM range (for n = 19,23,14 neb −/− , 41,42,36 neb +/− , 30,20,21 neb +/+ zebrafish per experiment). *p < 0.5, **** p < 0.001

Fig. 2

Characterisation of skeletal muscle pathology in neb−/− fish. a Gomori trichome staining of neb−/− skeletal muscle sections reveal the presence of dark regions (arrows) throughout the muscle indicative of nemaline bodies not observed in neb +/+ fish. Nuclei (arrowhead) are evenly organized in neb +/+ , however, appear disorganized in neb−/− fish. b Quantification of normalized fiber area from Gomori trichome stained sections in neb−/− (n = 23 fibers) compared to neb+/+ fish (n = 21 fibers). Error bars represent mean±SD, *** p < 0.001. c neb−/− mutants exhibit F-actin (red) and Actinin2 (green) positive aggregates at the myosepta (arrowheads) (and zoomed inset) compared to wildtype siblings at 2 dpf

Fig. 3

Examination of neb−/− skeletal muscle by electron microscopy. a) neb −/− mutant skeletal muscles display (i, iv) thickened Z-disks (arrows), (ii) fiber breakage (asterisks), (iii) accumulations of nemaline bodies and (iv) disruption of sarcomeric structures that are not observed in b) neb +/+ wildtype siblings

Fig. 5

Quantification of the phenotypic severity of neb −/− mutants at 6 dpf. Quantification of the phenotypic severity of Tg(neb −/− ; Lifeact-eGFP) fish at 6 dpf supplemented with either L-tyrosine, taurine, L-carnitine, creatine, or water (H2O). a Phenotypes were scored as either wildtype, mild (less than five Lifeact-eGFP positive aggregates at the myosepta or a mild disruption of muscle fibres), or severe (severely disorganised fibres or an accumulation of five or more Lifeact-eGFP positive aggregates within the muscle cell). b Quantification of the phenotypic severity of Tg(neb −/− ; Lifeact-eGFP) fish supplemented with either L-tyrosine, taurine, or water. c Quantification of the phenotypic severity of Tg(neb −/− ; Lifeact-eGFP) fish supplemented with either L-carnitine, creatine, or water (H2O). b & c Error bars represent mean±SEM for three independent experiments. For b) n = 6,8,7 Tg(neb −/− ; Lifeact-eGFP) for L-tyrosine, n = 11,5,11 Tg(neb −/− ; Lifeact-eGFP) for taurine and n = 9,8,10 Tg(neb −/− ; Lifeact-eGFP) for water. For c) n = 8,10,4 Tg(neb −/− ; Lifeact-eGFP) for L-carnitine, n = 6,8,3 Tg(neb −/− ; Lifeact-eGFP) for creatine, and n = 10,9,5 Tg(neb −/− ; Lifeact-eGFP) for water per experiment). ns = not significant

Fig. S1

Characterisation of Tg(neb-/-; Lifeact-eGFP) fish.

Tg(neb-/-; Lifeact-eGFP) fish show an accumulation of Lifeact-eGFP at the myosepta (arrowheads) at both 2 dpf and 6 dpf as well as regions of disorxganized muscle fibers (arrows) at 6 dpf that are not observed in Tg(neb+/+; Lifeact-eGFP) siblings.

Fig. S8

Characterisation of facial muscles in Tg(neb-/-; Lifeact-eGFP) fish and wild type siblings at 6 dpf.

Maximum projection images of Tg(neb-/-; Lifeact-eGFP) fish supplemented with water, tyrosine, taurine, L-carnitine of creatine show no difference in the appearance of facial muscles to Tg(neb+/+; Lifeact-eGFP) siblings supplemented with water.

Acknowledgments:
ZFIN wishes to thank the journal Acta neuropathologica communications for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Acta Neuropathol Commun