Snapshots of zebrafish embryos of both control and strong SMF-exposed groups at 4, 8 hpf and 1–6 dpf. (a,c,e,g,i,k,m,o) Morphology of control embryos; (b,d,f,h,j,l,n,p) morphology of SMF-exposed embryos. Note that before 3 dpf, zebrafish curled up in the chorion and looked spherical under a stereoscope. At 3 dpf, zebrafish hatched and stretched their bodies. Scale bar, 1 mm.

Developmental indices of zebrafish. (a) Survival rate of embryos with or without 24 h SMF exposure, calculated when embryos were fetched out from SMF. Data came from three samples with 50 embryos each (p = 0.77). (b) Hatching rate from 2 to 4.5 dpf, calculated every 0.5 day. Control data came from three samples with 91 embryos each, and SMF-exposed data were from three samples with 75 embryos each. From left to right, the p-value is 0.41, less than 0.001, 0.009, 0.46, 0.22 and 0.93. (c) Deformation rate calculated after most embryos had hatching. Data source was the same as b. From left to right, the p-value is 0.57, 0.10 and 0.03. (d) Body length measured with ImageJ. Data were collected from 30 samples. From left to right, the p-value is less than 0.001, less than 0.001, 0.43 and 0.99. (e) Heart rate from 1 to 6 dpf. Each data point came from 12 embryos. From left to right, the p-value is 0.06, 0.79, 0.52, 0.11, greater than 0.99, 0.54. Data are shown as mean with s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance.

Alcian blue staining of the seven pharyngeal arches of zebrafish embryos from 4 to 6 dpf. (a,a′) Control embryo formed five ceratobranchial (CB) bones at 4 dpf. (b,b′) SMF-exposed embryo developed only three CB bones. (c,c′,d,d′) Pharyngeal arches of 5 dpf control (c,c′) and SMF-exposed (d,d′) embryos displayed no significant difference. (e,e′,f,f′) Pharyngeal arches of 6 dpf control (e,e′) and SMF-exposed (f,f′) embryos displayed no significant difference. The seven pharyngeal arches are Meckel's cartilage (M), ceratohyal (CH) bone and the five pairs of ceratobranchial (CB1–5) bones as illustrated in e′. (a–f) use the same scale bar, 1 mm. (a′–f′) are the magnifications, respectively, and use the same scale bar, 0.5 mm.

qRT–PCR analysis of eight indicator genes from 1 to 6 dpf. Each data point came from three samples, with 30 embryos each at 1 and 2 dpf, and 15 embryos each at 3–6 dpf. Results were analysed with multiple t-tests and the p-values in each figure, from left to right, are (a) 0.008, 0.90, 0.71, 0.82, 0.10, 0.35; (b) 0.002, 0.04, 0.68, 0.36, 0.004, 0.19; (c) 0.63, 0.74, 0.05, 0.08, 0.69, 0.82; (d) 0.49, 0.04, 0.45, 0.36, 0.95, 0.79; (e) 0.04, 0.20, 0.13, 0.90, 0.24, 0.08; (f) 0.88, 0.30, 0.70, 0.52, 0.78, 0.63; (g) 0.49, 0.62, 0.04, 0.03, 0.29, 0.57; and (h) 0.33, 0.93, 0.87, 0.94, 0.66, 0.11. Data were normalized and shown as mean with s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance.

Behavioural tests of the effects of strong SMF. (a) The magenta line portrayed the swimming path of zebrafish larvae in 1 min. Experiments were performed in a 24-hole dish, with one larva to each hole. (b) Moving time of larvae in free swimming tests. (c) Average speed of larvae in free swimming tests. (d) Schematic depiction of OKR tests. (e,f) Performance of larvae in OKR tests at 5 and 6 dpf, respectively. Each data point came from 24 larvae in (b,c). Data were analysed with multiple t-tests and the p-values are (b) 0.96, 0.90, 0.67, 0.10; (c) 0.91, 0.79, 0.57, 0.96. (e) and (f) used 10 larvae in each group. Data were analysed with t-test and the p-values are (e) 0.002 and (f) 0.22. All data are shown as mean with s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance.

Immunohistochemistry of zebrafish embryos during early cleavage in control condition. (a) Metaphase of the second cleavage, (b) magnification of the spindle in a, (c) embryo of 4-cell stage after the second cleavage, (d) telophase of the third cleavage, (e) magnification of the departing spindle in (d) and (f) embryo of 8-cell stage after the third cleavage and before the telophase of the fourth cleavage. Green indicates microtubules and red indicates chromosomes. Scale bar in a, c, d and f is 50 µm. Scale bar in b and e is 10 µm.

Model of the delaying effect of strong SMF on early development. (a) Dissociative tubulin dimers are randomly distributed in cytoplasm in control condition. (b) Tubulin dimers tend to orient with α–β axis parallel to SMF direction, altering the polymerization rate at astral microtubule ends. The thickest arrow means the largest polymerization rate. (c) Microtubules are rotated under magnetic torque from SMF. The thickest arrow means the largest rotating speed. (d) Microtubules bend to the direction of SMF. The largest deflection happens at the microtubule ends. (e,f) Schematic diagram shows the positioning and orienting of spindle by microtubules (e) without or (f) with an external SMF. (g) Computational simulations of the positioning process of spindle with and without SMF. t* = t/τ_d, where τ_d is the relaxation time for positioning process without SMF [25]. (h) Computational simulations of the orientation process of spindle with and without SMF. t* = t/τ_α, where τ_α is the relaxation time for orientation process without SMF [25].

Exposing zebrafish embryos to strong SMF. (a) Zebrafish eggs were put in a Petri dish, which was placed in a stainless iron bore. (b) The stainless iron bore. (c) The iron bore was put into the centre of the superconducting magnet.