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. 2017 Apr;222(3):1367-1384.
doi: 10.1007/s00429-016-1282-1. Epub 2016 Aug 10.

Normal radial migration and lamination are maintained in dyslexia-susceptibility candidate gene homolog Kiaa0319 knockout mice

Isabel Martinez-Garay  1   2 , Luiz G Guidi  1   3 , Zoe G Holloway  3 , Melissa A G Bailey  1 , Daniel Lyngholm  1   4 , Tomasz Schneider  5 , Timothy Donnison  3 , Simon J B Butt  1 , Anthony P Monaco  6   7 , Zoltán Molnár  8 , Antonio Velayos-Baeza  9
Affiliations

Normal radial migration and lamination are maintained in dyslexia-susceptibility candidate gene homolog Kiaa0319 knockout mice

Isabel Martinez-Garay et al. Brain Struct Funct. 2017 Apr.

Abstract

Developmental dyslexia is a common disorder with a strong genetic component, but the underlying molecular mechanisms are still unknown. Several candidate dyslexia-susceptibility genes, including KIAA0319, DYX1C1, and DCDC2, have been identified in humans. RNA interference experiments targeting these genes in rat embryos have shown impairments in neuronal migration, suggesting that defects in radial cortical migration could be involved in the disease mechanism of dyslexia. Here we present the first characterisation of a Kiaa0319 knockout mouse line. Animals lacking KIAA0319 protein do not show anatomical abnormalities in any of the layered structures of the brain. Neurogenesis and radial migration of cortical projection neurons are not altered, and the intrinsic electrophysiological properties of Kiaa0319-deficient neurons do not differ from those of wild-type neurons. Kiaa0319 overexpression in cortex delays radial migration, but does not affect final neuronal position. However, knockout animals show subtle differences suggesting possible alterations in anxiety-related behaviour and in sensorimotor gating. Our results do not reveal a migration disorder in the mouse model, adding to the body of evidence available for Dcdc2 and Dyx1c1 that, unlike in the rat in utero knockdown models, the dyslexia-susceptibility candidate mouse homolog genes do not play an evident role in neuronal migration. However, KIAA0319 protein expression seems to be restricted to the brain, not only in early developmental stages but also in adult mice, indicative of a role of this protein in brain function. The constitutive and conditional knockout lines reported here will be useful tools for further functional analyses of Kiaa0319.

Keywords: Anatomy; Cerebral cortex; Development; Dyslexia; Kiaa0319; Neuronal migration.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

This article does not contain any studies with human participants performed by any of the authors.

Figures

Fig. 1
Fig. 1
Generation and characterisation of Kiaa0319-targeted mice. a Schematic representation of strategy followed for targeting exon 6 of the Kiaa0319 gene to obtain a “knockout first” (tm1a or KO1) allele. Alleles “Null-lacZ” (tm1b or NZ) and “floxed” (tm1c or Flx) are obtained after Cre and Flp recombination, respectively. Further Cre recombination from Flx allele generates a “Null” (tm1d or Null) allele where exon 6 is deleted. b Western blotting analysis of brain lysates from E15 and E18 embryos confirms the presence at these stages of the KIAA0319 protein (arrow) in wild type mice and its absence in homozygous KO1 animals. Non-specific bands are detected due to cross-reactivity of R7 antiserum. 30 µg total protein per lane. c Western blotting analysis with R7 antibody of brain lysates from 6-week old mice shows the presence of the KIAA0319 protein (arrow, top panel) in adult wild type and Flx mice and its absence in homozygous KO1 and NZ animals. The same pattern is detected for a < 25 kDa band (arrow, bottom panel), probably corresponding to a C-terminal cleavage fragment. 50 µg total protein per lane. M male, F female
Fig. 2
Fig. 2
Neurogenesis is not altered in Kiaa0319 mutants. a Immunostaining of Kiaa0319 +/+, +/− and −/− embryos at E15.5. There are no differences between the three genotypes in the numbers or distribution of dividing (pH3 + , green) or cycling (Ki67 + , red) cells along the cortical wall (upper panels). Staining for Pax6 (red, middle panels) and Tbr2 (green, lower panels) also shows no changes in Kiaa0319 deficient animals. Nuclei were stained with DAPI (blue). Quantifications of number of cells per 100-μm wide sections in the ventricular zone and rest of cortical wall (upper panels) or per bin (middle and lower panels) are shown on the right. b Immunostaining of +/+, +/− and −/− embryos at E18.5. The number of dividing (pH3 + , green) or cycling (Ki67 + , red) cells and their distribution, quantified as number of cells per 100-μm wide section in the ventricular zone and the rest of the cortical wall, shows no variation between +/+, +/− and −/− animals (upper panels). Immunostaining against Pax6 (red, middle panels) and Tbr2 (green, lower panels) shows similar numbers and distribution of positive cells in all three conditions. Nuclei were stained with DAPI (blue). Quantifications are shown on the right. VZ ventricular zone, SVZ subventricular zone, IZ intermediate zone, CP cortical plate. Scale bars 75 μm
Fig. 3
Fig. 3
Normal lamination in layered brain regions of Kiaa0319-deficient mice. a Immunostaining with anti-Ctip2 (red) and anti-Cux1 (green) antibodies in the P2 somatosensory cortex of Kiaa0319 +/+, +/− and −/− animals, revealing normal lamination of deep and upper layers. b P2 hippocampi stained for Ctip2 (red, left panels) and Calbindin (green, right panels), showing no differences between the three Kiaa0319 genotypes. Nuclei were stained with DAPI (blue). c Upper panels: immunostaining against Ctip2 (red) and Cux1 (green) in P10 somatosensory cortex shows normal lamination in Kiaa0319 deficient mice. Lower panels further staining against NF-H (red) and Calbindin (green) confirms normal neuronal distribution in the mutants. d No lamination defects are apparent in P10 hippocampi stained for Ctip2 (red, left panels) or Calbindin (red) and NF-H (green) (right panels). Nuclei were stained with DAPI (blue). e Immunostaining against NeuN (green) and Calbindin (red) shows normal foliation and lamination of P10 cerebella in +/+, +/− and −/− animals. Nuclei were stained with DAPI (blue). Cortical layers are labelled with roman numbers (II to VI); WM white matter, DG dentate gyrus, CA1–CA3 Cornu Ammonis areas 1–3. Scale bars 100 μm
Fig. 4
Fig. 4
Effects on radial migration upon Kiaa0319 depletion or overexpression. a Illustration of the strategy to study the effects of altered Kiaa0319 levels on neuronal migration at embryonic stages. To reduce KIAA0319 levels, embryos from wild-type (+/+), heterozygous (F/+), and homozygous (F/F) floxed animals were electroporated in utero at E14.5 with pCIG-Cre. For overexpression, C57Bl/6J embryos were electroporated at E14.5 with either pCIG or pCIG-mKiaa0319. Neuronal position was analysed at E18.5. b Strategy to assess the effect of Kiaa0319 overexpression on migration at postnatal stages. C57Bl/6J embryos were electroporated at E13.5 with either pCIG or pCIG-mKiaa0319 and analysed at postnatal day 12. c Representative images of coronal sections of embryos electroporated with pCIG-Cre. Electroporated neurons are shown in green and nuclei (DAPI stained) in blue. KIAA0319 depletion at E14.5 does not alter the radial migration of cortical projection neurons. d Quantification of the percentage of electroporated neurons in the VZ/SVZ, IZ, LCP, and UCP shown in c. Neurons were counted in three slices of 7 (F/F), 8 (F/+) or 5 (+/+) electroporated brains originating from three separate experiments. The data represent mean ± SEM. No statistically significant differences were found by one-way ANOVA analysis. e Representative images of coronal sections of embryos electroporated with pCIG or pCIG-mKiaa0319. Electroporated neurons are shown in green and nuclei (DAPI stained) in blue. Kiaa0319 overexpression slightly impairs migration. f Immunohistochemistry to detect the overexpressed KIAA0319 protein. Neurons electroporated with pCIG-mKiaa0319 express EGFP (green) and stain with anti-KIAA0319 R5 antibody (red). g Quantification of the percentage of electroporated neurons in the VZ/SVZ, IZ, LCP, and UCP shown in e. The data represent mean ± SEM. *p < 0.05 by Student’s t test. Three slices from five brains coming from three separate electroporations were counted for each condition. h Representative images of coronal sections of embryos electroporated at E13.5 with pCIG or pCIG-mKiaa0319 and analysed at P13. Electroporated neurons are shown in green. Cux1 staining to label upper cortical layers is shown in red. No difference in the distribution of targeted neurons is apparent. i Quantification of the distribution of electroporated neurons shown in h. The data represent mean ± SEM. No statistically significant differences were found for any of the bins by Student’s t test. Three slices from four brains coming from three separate electroporations were counted for each condition. MZ marginal zone, LCP lower half of the cortical plate, UCP upper half of the cortical plate, other abbreviations as in Figs. 2, 3. Scale bars 100 μm
Fig. 5
Fig. 5
No changes in the intrinsic properties of cortical neurons upon partial or total elimination of Kiaa0319. a–c Superimposed responses of pyramidal cells to suprathreshold depolarising and hyperpolarising current injection for electroporated cells in wild-type (a; +/+), heterozygous (b; F/+) and homozygous (c; F/F) floxed animals. d–f Action potential phase (dV/dT) plots for 10 action potentials shown in panels a–c. g–o Active (g–l) and passive membrane properties (m–o) for electroporated cells recorded from +/+ (n = 6), F/+ (n = 5) and F/F (n = 5) animals. No significant differences were observed. The data represent mean ± SEM. Significance was assessed by Kruskal–Wallis test with Dunn’s correction for multiple comparisons
Fig. 6
Fig. 6
Subtle changes detected in light-induced anxiety and prepulse inhibition in Kiaa0319 mutant mice. a–c Anxiety related behaviour measured in the light/dark box over the full 10 min duration of the test (a), in the initial 5 min of the light/dark box test (b) and in the elevated plus maze (EPM) (c) of Kiaa0319 +/+ (n = 16; 17 for EPM), +/− (n = 17) and −/− (n = 14) mice. Kiaa0319 −/− mice display reduced anxiety in the first 5 min of the light/dark box test when compared with +/+ and +/− animals. No significant difference in anxiety is observed between genotypes in the EPM. Light–dark ratio ratio of time spent in the dark vs time spent in the light. Post hoc statistically significant Bonferroni LSD results, *p ≤ 0.01, **p ≤ 0.001. d–e Prepulse inhibition/facilitation of the acoustic startle response in male (d) and female (e) Kiaa0319 +/+ (male = 7, female = 7), +/− (male = 8, female = 4) and −/− (male = 7, female = 6) mice. Homozygous mutant (−/−) males show a deficit in prepulse facilitation at 400- and 800-ms gaps between prepulse and pulse compared with the other two genotypes. Post hoc statistically significant Bonferroni LSD results: homozygous (−/−) vs heterozygous (+/−) or wild-type (+/+), **p ≤ 0.001. The data represent mean ± SEM
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