The Effect of Variation in Expression of the Candidate Dyslexia Susceptibility Gene Homolog Kiaa0319 on Neuronal Migration and Dendritic Morphology in the Rat

Department of Physics, Harvard University,Cambridge, MA 02138, USAVeronica J. Peschansky, Timothy J. Burbridge, and Amy J. Volzcontributed equally to this manuscript.We investigated the postnatal effects of embryonic knockdown andoverexpression of the candidate dyslexia gene homolog Kiaa0319.We used in utero electroporation to transfect cells in E15/16 ratneocortical ventricular zone with either 1) small hairpin RNA(shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expressionconstruct, 3) Kiaa0319 shRNA along with KIAA0319 expressionconstruct (‘‘rescue’’), or 4) a scrambled version of Kiaa0319 shRNA.Knockdown, but not overexpression, of Kiaa0319 resulted inperiventricular heterotopias that contained large numbers of bothtransfected and non--transfected neurons. This suggested thatKiaa0319 shRNA disrupts neuronal migration by cell autonomous aswell as non--cell autonomous mechanisms. Of the Kiaa0319 shRNA--transfected neurons that migrated into the cortical plate, mostmigrated to their appropriate lamina. In contrast, neurons trans-fected with the KIAA0319 expression vector attained laminarpositions subjacent to their expected positions. Neurons trans-fected with Kiaa0319 shRNA exhibited apical, but not basal,dendrite hypertrophy, which was rescued by overexpression ofKIAA0319. The results provide additional supportive evidencelinking candidate dyslexia susceptibility genes to migrationaldisturbances during brain development, and extends the role ofKiaa0319 to include growth and differentiation of dendrites.Keywords: cerebral cortex, dendritic hypertrophy, heterotopias,malformation, RNAiIntroductionDevelopmental dyslexia is a language--based learning disabilitythat affects between 4% and 10% of the population, and hasa strong genetic component (Fisher and Francks 2006). Postmortem examination of the brains of developmental dyslexicsdemonstrated the presence of neuronal migration anomalies,including molecular layer ectopias, laminar dysplasia, andoccasional focal microgyria (Galaburda and Kemper 1979;Galaburda et al. 1985; Humphreys et al. 1990). More recently,an association between periventricular nodular heterotopiasand developmental dyslexia has been reported (Chang et al.2005; Sokol et al. 2006).Recent reports proposing candidate dyslexia susceptibilitygenes haveopenedupnew linesofinvestigation intothe geneticmodulation of this disorder. A number of these genes, inspeciﬁc—DCDC2 and KIAA0319 on Chr 6 (Francks et al. 2004;Cope et al. 2005; Meng et al. 2005; Harold et al. 2006; Paracchiniet al. 2006; Schumacher et al. 2006; Luciano et al. 2007;Paracchini et al. 2008), and DYX1C1 on Chr 15 (Taipale et al.2003; Brkanac et al. 2007; Marino et al. 2007)—have beenshownto have roles in neuronal migration. Thus, we have previouslydemonstrated that in utero electroporation of shRNA targetedagainst rat homologs of DCDC2, KIAA0319,orDYX1C1 in therat disrupts the process of neuronal migration to the cerebralcortex as assessed during the prenatal period (Meng et al. 2005;Paracchini et al. 2006; Wang et al. 2006). Further evaluationof the postnatal consequences of embryonic knockdown ofDyx1c1 and Dcdc2 function in rats revealed the presence ofa variety of neocortical malformations, including molecular layerectopias and periventricular heterotopias (PVHs) (Rosen et al.2007; Burbridge et al. 2008). In addition to these frankneuronal migration anomalies, we found evidence of moresubtle disruptions, with some transfected neurons migrating tothe cortical plate (CP), albeit past their expected laminarlocations (Rosen et al. 2007; Burbridge et al. 2008).As mentioned above, embryonic knockdown of Kiaa0319disrupted neuronal migration when assessed 4--7 days aftertransfection (Paracchini et al. 2006). Unknown were the long-term effects, both in terms of neuronal migration andsubsequent neuronal morphology, of embryonic knockdownor overexpression of this candidate dyslexia susceptibilitygene. In the current report, therefore, we examined the brainsof postnatal rats that were embryonically transfected by inutero electroporation with plasmids containing 1) shRNAtargeted against Kiaa0319 (knockdown), 2) a constructexpressing KIAA0319 protein (overexpression), or 3) a combi-nation of the shRNA and expression constructs (rescue). Wequalitatively assessed these brains for the presence or absenceof neuronal migration anomalies and the laminar disposition oftransfected and non--transfected neurons. In addition, wequantiﬁed migration distances and neuronal morphology ofthe transfected cells.Experimental ProceduresIn Situ HybridizationIn order to better interpret the knockdown and overexpres-sion ﬁndings, we ﬁrst determined the expression of Kiaa0319in the prenatal brain by in situ hybridization. We obtained time-mated pregnant females (Charles River Laboratory, Wilmington,MA) and sacriﬁced the litters on E14/15, E16/17, or E19/20.Three embryos from each litter were immediately frozen andthey were cut in either the horizontal, sagittal, or coronal planeon a cryostat at 18 lm, and the slides were processed for in situhybridization of Kiaa0319 as described below.The cDNAs prepared from frontal, parietal, and occipital lobesof human embryonic brain (20 weeks, Biochain Institute,