Modeling Neurodevelopmental Disorders and Epilepsy Caused by Loss of Function of kif2a in Zebrafish

A novel kif2a knock-out zebrafish model shows that kif2a is not indispensable for survival and mutations do not result in gross morphologic abnormalities. a, Linear representation of the human kinesin 2A polypeptide with highlighted four de novo MCD-associated mutations (indicated in red-pink) located in or near the ATP-binding region (indicated in green). Kinesin motor domain, coiled coil, and globular domains (UniProtKB-O00139) are highlighted. Multiple sequence alignment shows the level of conservation of the ATP binding region (indicated in dark turquoise) of human, mouse, and zebrafish. b, Localization of 4-nucleotide deletion (CCAG) in the globular region of Kif2a and electropherogram of kif2a^+/+ larvae, confirming the presence of the mutation in homozygotes. c, Kaplan–Meier survival rate curves of kif2a^+/+ (n = 5), kif2a^+/− (n = 21), and kif2a^−/− (n = 10) larvae. Larvae were obtained by the mating of heterozygous adults. Surviving larvae were counted every 24 h until 30 dpf. d, Lateral bright-field images representing normal macroscopic morphology of 3 and 5 dpf kif2a^+/+ and kif2a^−/− larvae. Scale bar, 1 mm. The design of the CRISPR/Cas9 kif2a zebrafish knock-out line is illustrated in Extended Data Figure 1-1. qPCR analysis of kif2c expression levels showed no upregulation of kif2c during development in kif2a^−/− larvae (Extended Data Fig. 1-1).

Expression and localization pattern of kif2a in the developing zebrafish. a, qPCR analysis of kif2a levels in kif2a^+/+ larvae normalized to actin and represented as the fold change expression to 0 hpf. Values are reported as the mean ± SEM of three separate experiments. Significant values are noted as ****p ≤ 0.0001 and *p ≤ 0.05. b, Relative quantification of Kif2a protein expression in kif2a^+/+ larvae of 1–7 dpf normalized to Gapdh and represented as the fold change expression to 1 dpf. Values are reported as the mean ± SEM of three separate experiments. Significant values are noted as ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. Below the graph is a representative Western blot image of Kif2a protein expression levels in kif2a^+/+ larvae of 1–7 dpf. c, Spatiotemporal expression patterns of kif2a by whole-mount RNA in situ hybridization at 8 hpf, 23 hpf, 2 dpf, and 5 dpf. B, Brain; H, hindbrain; M, midbrain; MBH, midbrain–hindbrain boundary; Som, somite; R, retina. Scale bar, 200 μm. d, Representative Western blot image of Kif2a protein expression levels comparing kif2a^+/+ with kif2a^−/− larvae at 5 and 7 dpf.

kif2a^−/− larvae exhibit locomotor abnormalities, increased locomotor behavior to PTZ and habituation learning disabilities. a, Average total movement of kif2a^+/+ and kif2a^−/− larvae from 5 to 8 dpf expressed in actinteg units. Values are reported as the mean ± SD of four separate experiments. Significant values are noted as ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. b, Average total movement of 5 dpf kif2a^+/+ and kif2a^−/− larvae after 6 mm PTZ immersion for 30 min. Values are reported as the mean ± SD of four separate experiments. Significant values are noted as ***p ≤ 0.001. c, The habituation learning assay performed on 6 dpf kif2a^+/+ and kif2a^−/− larvae was composed of different protocols: starting with a 3 h incubation period in the light (indicated in yellow), followed by a habituation training consisting of four periods with 120 DFs with a 15 s ISI (regions with gray and yellow stripes), alternated by 10 min of light, a period of 30 min pause in the light preceding the actual test, and a test consisting of 10 DFs with a 60 s ISI (region with gray and yellow stripes). d, Percentage habituation of kif2a^+/+ and kif2a^−/− larvae to DFs during the spaced training. Values are reported as the mean ± SD of three separate experiments. e, Habituation ratio of average movement of kif2a^+/+ and kif2a^−/− larvae to DFs during the test. Values are reported as the mean ± SD of three separate experiments. Significant values are noted as **p ≤ 0.01. No locomotor abnormalities and habituation learning disabilities were observed in kif2a^−/− zebrafish larvae from +/– incrosses (Extended Data Fig. 3-1).

kif2a^−/− larvae show increased susceptibility to epilepsy. a, Visual representation of the setup of local field potential recording from optic tectum (indicated in red) of 5 dpf larvae. b, Number of seizures and average seizure duration in kif2a^+/+ and kif2a^−/− larvae. c, Number of seizures and average seizure duration in kif2a^+/+ and kif2a^−/− larvae after 6 mm PTZ immersion for 15 min. d, Representative fragments of a 10 min recording (calibration: 0.1 mV, 20 s) of kif2a^+/+ and kif2a^−/− larvae after 6 mm PTZ immersion for 15 min, with enlargement of a polyspiking event (calibration: 0.02 mV, 2 s) in kif2a^−/− larvae. Data are represented as the mean ± SD. Significant values are noted as **p ≤ 0.01. No increased seizure susceptibility was observed in the brains of kif2a^−/− zebrafish larvae from +/– incrosses (Extended Data Fig. 4-1).

kif2a larvae present microcephaly that is associated with defects in neurologic development. a–d, Comparison of the individual measurements for body area (a, b) and head size (c, d) for kif2a^+/+ and kif2a^−/− larvae at 3 and 5 dpf. Data are represented as the mean ± SD. Significant values are noted as *p ≤ 0.05. e, Histologic assessment of 5 dpf kif2a^−/− and kif2a^+/+ larval brains. Six brain regions (from forebrain to hindbrain) were selected per genotype as indicated in the diagram. f, Comparison of the number of brain sections from forebrain to hindbrain (e) between kif2a^+/+ and kif2a^−/− larvae. Data are represented as the mean ± SD. Significant values are noted as ****p ≤ 0.0001. g, h, Coronal sections stained with H&E imaged at 20× and 40× (indicated by orange square) magnification. DT, Dorsal thalamus; Hi, intermediate hypothalamus; lfb, lateral forebrain bundle; PG, preglomerular complex; PrT, pretectum; Teg, midbrain tegmentum; TeO, tectum opticum. Bar graphs compare hematoxylin-positive stained nuclei between kif2a^+/+ and kif2a^−/− larvae. Data are represented as the mean ± SD. Significant values are noted as ****p ≤ 0.0001 and **p ≤ 0.01. A significant neuronal loss was observed in specific brain regions of kif2a^−/− zebrafish larvae (Extended Data Fig. 5-1).