Generation of Kiaa0319-targeted mice
To study the putative role in cortical development postulated for human and rat KIAA0319 genes, we generated mice targeted at the homologous D130043K22Rik locus. All animals used in this work were derived from a single male chimera. Although the original ES clones had passed KOMP quality control tests, we analysed the Kiaa0319-KO1 allele in the obtained mice to confirm all important elements were as expected. This was carried out by amplification of four overlapping fragments (KO1-1 to KO1-4), specific to the Kiaa0319-KO1 allele, covering the whole region included in the targeting vector plus short flanking regions to both homology arms (Figs. S2a, S2b, S3, S4). PCR amplification of fragments with combinations of a KO1-allele specific primer and a flanking sequence specific primer (PCRs KO1-1 and KO1-4) confirmed the correct targeting into the Kiaa0319 locus. After sequencing, only minor discrepancies were found (Fig. S3).
Homozygous Kiaa0319-KO1 (tm1a allele) and Kiaa0319-NZ (tm1b allele) mice are viable, show no obvious differences with heterozygous and wild-type littermates, and breed normally. Western blotting analysis of brain lysates from E15 and E18 embryos showed that the specific band corresponding to the KIAA0319 protein was absent in homozygous samples (Fig. 1b). We additionally obtained Kiaa0319-Flx mice (tm1c allele), with conditional KO potential and in which the KIAA0319 protein could now be detected (Fig. 1c), and Kiaa0319-Null mice (tm1d allele) in which exon 6 is removed. As with KO1 and NZ mice, Kiaa0319-Null mice do not show any obvious phenotype and do not have detectable KIAA0319 protein (Fig. S6c).
Western blotting analysis of lysates from different mouse tissues shows that the KIAA0319 protein is not only detected at early developmental stages (Fig. 1b) but also in adult brain (Fig. 1c). Interestingly, the same pattern of the presence/absence of signal found for the ~170 kDa band, corresponding to the glycosylated full-length protein, can be detected for a small, under 25-kDa band (Fig. 1c, bottom panel) probably corresponding to a C-terminal cleavage fragment (Velayos-Baeza et al. 2010). Specific KIAA0319 signal was not detected in other tissues (liver, kidney, spleen, lung, and heart) except perhaps weekly in testis (Fig. S2d), suggesting that the function of this protein is probably restricted to the nervous system.
We selected Kiaa0319-NZ mice as the ko line for characterisation, and used Kiaa0319-Flx mice to occasionally knockout the Kiaa0319 gene at specific locations.
Neurogenesis is not affected in Kiaa0319 ko animals
Kiaa0319 has been reported to be expressed in the VZ of the developing cortex both in mice and humans by RNA in situ hybridization experiments (Paracchini et al. 2006; Peschansky et al. 2010). This expression pattern in progenitor cells suggests that Kiaa0319 might play a role during neurogenesis. To investigate this possibility, we analysed embryonic brains of wild-type, heterozygous, and homozygous Kiaa0319 ko animals at E15.5 and E18.5. Brain sections were stained for markers Ki67 and pH3 to label cells in the cell cycle and in mitosis, respectively (Fig. 2). Quantification of the number of positive cells for these two markers revealed no significant differences between the three genotypes (Fig. 2). To check whether the proportion of RG cells and intermediate progenitors, the two main progenitor types in mice, was altered in Kiaa0319 mutant animals, we stained E15.5 and E18.5 sections with antibodies against Pax6 and Tbr2, respectively. Again, quantification of the Pax6- or Tbr2-positive cells did not show any significant differences between genotypes (Fig. 2). In addition, the thickness of the cortical wall in E18.5 brain slices did not differ between the three conditions (data not shown). Together, these results suggest that Kiaa0319 is not playing a major role in neurogenesis during cortical development or that, alternatively, its absence may be functionally compensated by other proteins.
Normal lamination of cortex, hippocampus and cerebellum in Kiaa0319 ko mice
Functional experiments using in utero electroporation of shRNA against Kiaa0319 in rat embryos have shown a defect in radial migration following Kiaa0319 knockdown: groups of electroporated neurons fail to migrate and form heterotopias in the white matter (Paracchini et al. 2006; Peschansky et al. 2010; Szalkowski et al. 2012; Adler et al. 2013). These experiments suggest that Kiaa0319 plays an important role in the migration of newborn neurons into the CP, which could impact on the correct formation of the cortical layers. However, because in utero electroporation only targets a small percentage of neurons, effects on cortical lamination could not be analysed by this method. To check if a partial or total absence of Kiaa0319 affects the final position of projection neurons, we assessed cortical lamination at P2, when deep layer neurons have finished migrating but upper layer neurons are still on their way, and at P10, when all projections neurons have reached their final position. Staining of brain sections with antibodies against Ctip2 and Cux1, to label layer V neurons and upper layer neurons, respectively, showed no aberrant distribution of cells in the Kiaa0319 heterozygous or homozygous ko animals. Ctip2 positive cells were located in a band below Cux1 positive cells that occupied the upper part of the cortical plate at P2 (Fig. 3a). A similar distribution could be seen at P10, where layer IV could also be readily identified by the presence of the barrel cortex in all three conditions (Fig. 3c). Further staining for NF-H at P10 also showed no differences in the location and shape of labelled neurons and Calbindin staining revealed similar interneuron distribution across the three genotypes (Fig. 3c). To analyse whether other layered structures could be affected by a lack of KIAA0319 protein, we performed immunohistochemistry with anti-Ctip2 and anti-Calbindin antibodies to stain CA1 and the Dentate Gyrus of the hippocampus at P2 and P10 (Fig. 3b,d). Again, no migration defects were apparent, and no differences in the appearance or distribution of cells could be identified between wild-type, heterozygous, and homozygous Kiaa0319 ko animals. Finally, we looked at the cerebellum of P10 animals, using antibodies against Calbindin and NeuN (Fig. 3e). Calbindin-positive Purkinje cells were correctly positioned, and no lamination defects could be seen in the heterozygous or homozygous ko animals. To account for the possibility that any migration defects could be transitory and not visible at postnatal stages, we repeated the cortical stainings on sections of E15.5 (Ctip2) and E18.5 (Ctip2 and Cux1) brains, but the distribution of the labelled cells was the same in all three conditions (Fig. S5). Together, these results demonstrate that a partial or complete reduction in the levels of KIAA0319 protein in the mouse does not affect migration in any of the layered structures of the brain.
Acute elimination of KIAA0319 protein by Cre recombination does not affect radial migration of projection neurons
The lack of lamination defects in the Kiaa0319 ko mouse is in contradiction with the shRNA electroporation data obtained in rat by other laboratories (Paracchini et al. 2006; Peschansky et al. 2010; Szalkowski et al. 2012; Adler et al. 2013). One possible explanation for this discrepancy could be the timing of Kiaa0319 knockdown. Because in the ko animals the protein is missing from the beginning, compensatory mechanisms might be operating to counteract its absence by the time neuronal migration takes place. An acute knockdown of the protein right before migration commences, as achieved by shRNA electroporation, might overcome this putative compensation. To acutely eliminate KIAA0319 in migrating neurons, we carried out in utero electroporation experiments to deliver Cre-recombinase encoding plasmids into Kiaa0319-Flx embryos (Fig. 4a). Plasmid pCIG-Cre, expressing Cre-recombinase and EGFP under the control of a general promoter (Chicken Beta Actin), was electroporated at E14.5 into Kiaa0319
F/+, and Kiaa0319
F/F embryos, and brains were harvested four days later, at E18.5 (Fig. 4c). The number of neurons in four different regions of the developing cortex (VZ/SVZ, IZ, lower CP, and upper CP) was quantified (Fig. 4d), but no significant differences could be found between the three conditions. Consistent with these results, there were no misplaced neurons in the white matter of Kiaa0319
F/+ and Kiaa0319
F/F postnatal animals electroporated with pCIG-Cre at E13.5 (data not shown). Cre expression from the pCIG-Cre plasmid was verified by immunohistochemistry, confirming the presence of Cre recombinase in the electroporated cells (Fig. S6a). The lack of a suitable antibody to exclusively detect the endogenous KIAA0319 protein precluded us from confirming that the protein was effectively absent following Cre electroporation in brain slices. However, we tested that the Kiaa0319-Null allele can indeed be obtained from Kiaa0319-Flx allele after Cre recombination in primary cortical cultures transfected with the same plasmid (Fig. S6b), and Western blotting analysis of protein lysates obtained from these same cultures shows a reduced KIAA0319 protein signal (Fig. S6c, left).
Kiaa0319 overexpression delays radial migration, but does not affect final neuronal position
Overexpression of Kiaa0319 has been reported to alter the final location of migrating neurons in rats, where cells electroporated at E15/16 with an overexpression construct occupied lower positions than control cells in the cortical plate at P21 (Peschansky et al. 2010). To check if we could detect a similar effect in mice, we electroporated pCIG-mKiaa0319 into wild-type embryos at E14.5 (Fig. 4a) and analysed the brains 4 days later (Fig. 4e). We confirmed overexpression of KIAA0319 protein using immunohistochemistry (Fig. 4f) and quantified the percentage of electroporated cells in the different zones of the cortical wall (Fig. 4g). We could see a tendency of Kiaa0319 overexpressing cells to lag behind control electroporated cells, with an increase in the percentage of targeted cells both in the IZ (24.04 % for pCIG vs 32.75 % for pCIG-mKiaa0319) and the LCP (14.14 % vs 18.6 %), and a concomitant reduction in the neurons that had reached the UCP (47.15 % vs 33.83 %). Due to the high variability in the distribution of the neurons, only the differences in the LCP were significant. To assess how this delay might affect the final position of excitatory projection neurons within the cortex, we electroporated pCIG-mKiaa0319 into E13.5 embryos and checked the position of the targeted cells in somatosensory cortex at P12 (Fig. 4b, h). Electroporation at E13.5 targets neurons from different layers, allowing us to check the effect of Kiaa0319 overexpression in different neuronal populations. The distribution of Kiaa0319 overexpressing neurons across the cortex, analysed as the percentage of targeted cells in each of 10 equal bins, did not differ significantly from the distribution of control electroporated cells (Fig. 4i). These results suggest that Kiaa0319-overexpressing neurons are delayed in their migration to the cortical plate, but manage to reach their final position within the cortex postnatally.
No changes in the intrinsic electrophysiological properties of cortical neurons are detected upon partial or total elimination of KIAA0319
The absence of KIAA0319 does not seem to affect radial migration of cortical projection neurons, but it might impact their intrinsic electrophysiological properties. To lower the chances of having normal physiological maturation as a result of network compensation due to global knock out of Kiaa0319, we did not perform the analysis in ko animals. Instead, we electroporated pCIG-Cre, together with pCAG-RFP to enable the identification of electroporated neurons, into Kiaa0319
F/+, and Kiaa0319
F/F embryos at E13.5 and harvested the electroporated animals between P12 and P16. Recordings were performed from acute in vitro brain slices. The response of electroporated cells to either depolarising or hyperpolarising current steps did not differ between genotypes (Fig. 5a–c). There was no significant difference in either action potential dynamics (Fig. 5d–l) or passive membrane properties (Fig. 5m–o). These results suggest that the basic intrinsic electrophysiological properties of the cohort of E13.5 cortical pyramidal neurons are unaltered following removal of KIAA0319.
Kiaa0319 mutants exhibit subtle behavioural anomalies in sensorimotor gating and anxiety measures
Despite the absence of overt anatomical or electrophysiological defects in Kiaa0319 ko mice, underlying subtle circuit-level changes affecting their behaviour may exist. This hypothesis is supported by previous studies on mice mutated in other candidate dyslexia susceptibility homologous genes, such as Dcdc2 and Dyx1c1. These animals do not show the expected migration deficits, and lack lamination defects or clear anomalies in the cortex; however, changes in learning, memory or auditory processing have been reported in both cases (Gabel et al. 2011; Truong et al. 2014; Rendall et al. 2015). Similarly, in utero knockdown of Kiaa0319 in the embryonic rat cortex leads to spatial learning deficits and affect responses to complex acoustic stimuli (Szalkowski et al. 2012; Centanni et al. 2014a, b). We, therefore, decided to perform a general behavioural characterisation of the Kiaa0319 mutant animals to elucidate functional effects of Kiaa0319 knockout and conducted a standard series of mouse behavioural tests on a cohort of control and mutant mice.
There was no difference between genotypes in locomotor activity in the 60-min open field test (Fig. S7a), sociability and social novelty preference measured in the 3-chamber apparatus (Fig. S7b), locomotor habituation (Fig. S7c), or stress and motor coordination (inverted screen, weight lifting or accelerating rotarod) (Fig. S7d–f). There was also no difference in spatial and non-spatial learning and memory (spontaneous alternations using T maze, spatial novelty in the Y maze, and object recognition tests) (Fig. S7g–i); however, subtle differences were found in anxiety and sensorimotor gating (Fig. 6).
Anxiety was measured in the light/dark box and the elevated plus maze. In the light/dark box, significant interaction between genotype x sex x within-session period was found for the ratio of time spent in the dark vs light, a classic measure of anxiety, [F(2,417) = 2.969, p ≤ 0.05] (Fig. 6a). The difference was significant for the initial 5 min of the session [F(2,41) = 3.561, p ≤ 0.05] (Fig. 6b) with decreased anxiety in homozygous animals compared to both wild type (p < 0.001) and heterozygous animals (p < 0.01). Similarly, homozygous ko mice tended to spend more time in the open arms of the elevated plus maze, suggesting decreased anxiety, but this effect did not reach statistical significance (Fig. 6c).
We also assessed sensorimotor gating, i.e., an ability to filter out redundant stimuli, in the Kiaa0319 mutant mice using the prepulse inhibition/facilitation (PPI/PPF) of the acoustic startle response. We used different latencies to the startle-eliciting stimulus (SES) to measure both prepulse facilitative and inhibitory effects on the reactivity to the SES. No differences in prepulse inhibition across genotypes and sex were detected, although there was a significant genotype x sex x stimulus type interaction [F(16,3423) = 1.92, p ≤ 0.05]. Further post hoc analysis revealed increased prepulse inhibition in homozygous males for stimuli with long gaps (400 and 800 ms) between prepulse and SES, compared both with wild-type and heterozygous mice (p ≤ 0.001) (Fig. 6d). There was no difference between females (Fig. 6e) or for any other stimuli combination in males.
Taken together, those data suggest the possibility of subtle alterations in anxiety-related behaviour and in sensorimotor gating resulting from Kiaa0319-deletion, although further analyses with larger cohorts and/or different tests may be necessary to replicate and extend these initial results.