We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct (“rescue”), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non–transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non–cell autonomous mechanisms. Of the Kiaa0319 shRNA–transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites.
Developmental dyslexia is a language–based learning disability that affects between 4% and 10% of the population, and has a strong genetic component (Fisher and Francks 2006). Post mortem examination of the brains of developmental dyslexics demonstrated the presence of neuronal migration anomalies, including molecular layer ectopias, laminar dysplasia, and occasional focal microgyria (Galaburda and Kemper 1979; Galaburda et al. 1985; Humphreys et al. 1990). More recently, an association between periventricular nodular heterotopias and developmental dyslexia has been reported (Chang et al. 2005; Sokol et al. 2006).
Recent reports proposing candidate dyslexia susceptibility genes have opened up new lines of investigation into the genetic modulation of this disorder. A number of these genes, in specific—DCDC2 and KIAA0319 on Chr 6 (Francks et al. 2004; Cope et al. 2005; Meng et al. 2005; Harold et al. 2006; Paracchini et 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 been shown to have roles in neuronal migration. Thus, we have previously demonstrated that in utero electroporation of shRNA targeted against rat homologs of DCDC2, KIAA0319, or DYX1C1 in the rat disrupts the process of neuronal migration to the cerebral cortex as assessed during the prenatal period (Meng et al. 2005; Paracchini et al. 2006; Wang et al. 2006). Further evaluation of the postnatal consequences of embryonic knockdown of Dyx1c1 and Dcdc2 function in rats revealed the presence of a variety of neocortical malformations, including molecular layer ectopias and periventricular heterotopias (PVHs) (Rosen et al. 2007; Burbridge et al. 2008). In addition to these frank neuronal migration anomalies, we found evidence of more subtle disruptions, with some transfected neurons migrating to the cortical plate (CP), albeit past their expected laminar locations (Rosen et al. 2007; Burbridge et al. 2008).
As mentioned above, embryonic knockdown of Kiaa0319 disrupted neuronal migration when assessed 4–7 days after transfection (Paracchini et al. 2006). Unknown were the long-term effects, both in terms of neuronal migration and subsequent neuronal morphology, of embryonic knockdown or overexpression of this candidate dyslexia susceptibility gene. In the current report, therefore, we examined the brains of postnatal rats that were embryonically transfected by in utero electroporation with plasmids containing 1) shRNA targeted against Kiaa0319 (knockdown), 2) a construct expressing KIAA0319 protein (overexpression), or 3) a combination of the shRNA and expression constructs (rescue). We qualitatively assessed these brains for the presence or absence of neuronal migration anomalies and the laminar disposition of transfected and non–transfected neurons. In addition, we quantified migration distances and neuronal morphology of the transfected cells.
In Situ Hybridization
In order to better interpret the knockdown and overexpression findings, we first determined the expression of Kiaa0319 in 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 and they were cut in either the horizontal, sagittal, or coronal plane on a cryostat at 18 μm, and the slides were processed for in situ hybridization of Kiaa0319 as described below.
The cDNAs prepared from frontal, parietal, and occipital lobes of human embryonic brain (20 weeks, Biochain Institute, Hayward, CA) were used as template to amplify the full-length coding sequence of Kiaa0319 using forward (5′ATGGCGCCCCCCACAGGTGTG3′) and reverse (5′ TTATCTGTCCTTTGAGCAATAACTG 3′) primers. All fragments were then cloned into pGEMT-Easy vector (Promega, Madison, WI). Rat embryonic (E14) brain cDNA was synthesized from total RNA using SuperScript III Reverse Transcriptase enzyme (Invitrogen, CA). Full-length coding sequence of Kiaa0319 was amplified from E14 cDNA using forward primer (5′ ATGGTGTCCCCACCAGGAGTAC 3′) and reverse primer (5′ TTATCTGTCCTTTGAGTAATAACCA 3′). The amplified product was cloned into pGEMT-Easy vector. All the plasmids generated by PCR were sequenced at WM Keck sequencing facility. Nonradioactive in situ hybridizations were done by UB-In Situ (Natick, MA), as previously described (Berger and Hediger 2001) using a digoxigenin-labeled cRNA probe. The antisense and sense probes were generated by using T7 and SP6 promoters flanking pGEMT-Easy-Kiaa0319 of human and rat plasmids.
In Utero Electroporation
In utero electroporations of litters designated for postnatal analysis were performed at the Beth Israel Deaconess Medical Center. One litter that was analyzed in the prenatal period was transfected at the University of Connecticut. The Institutional Animal Care and Use Committees of these institutions approved all procedures.
A total of 11 pregnant Wistar rats were obtained (Charles River Laboratory) and each litter was assigned to 1 of 3 groups: Kiaa0319 shRNA, KIAA0319 Overexpression, and Rescue. Within each litter, pups were randomly assigned to receive 1 of 2 treatments (see Table 1). This balanced design was essential for the analysis of migrational distance as it controlled for between–litter variation in gestational age. In utero electroporation of plasmid DNA was performed at E15/16 as described previously (Bai et al. 2003; Rosen et al. 2007; Burbridge et al. 2008). The concentration of enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein (mRFP) plasmids was 0.75 μg/μL, the shRNA was 1.5 μg/μL, and expression plasmids were 1.5 μg/μL.
|Group||Treatment 1||Treatment 2|
|Kiaa0319 shRNA||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (12)||pU6shRNA-Kiaa0319 scram + pCAGGS-mRFP + pCAGGS-eGFP (11)|
|KIAA0319, overexpression||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (8)||pCAG-KIAA0319-IRES-eGFP + pCAGGS-mRFP (7)|
|Rescue||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (9)||pU6shRNA-Kiaa0319 + pCAG-KIAA0319-IRES-eGFP + pCAGGS-mRFP (10)|
|Group||Treatment 1||Treatment 2|
|Kiaa0319 shRNA||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (12)||pU6shRNA-Kiaa0319 scram + pCAGGS-mRFP + pCAGGS-eGFP (11)|
|KIAA0319, overexpression||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (8)||pCAG-KIAA0319-IRES-eGFP + pCAGGS-mRFP (7)|
|Rescue||pU6shRNA-Kiaa0319 + pCAGGS-eGFP (9)||pU6shRNA-Kiaa0319 + pCAG-KIAA0319-IRES-eGFP + pCAGGS-mRFP (10)|
For the Kiaa0319 shRNA condition, plasmids encoding shRNA (pU6shRNA-Kiaa0319) and plasmids encoding eGFP (pCAGGS-eGFP) were cotransfected into the ventricular zone (VZ). We have previously demonstrated that cotransfection is highly efficient, as virtually all neurons cotransfected with eGFP and mRFP were colabeled when sacrificed 4 days post-transfection (Rosen et al. 2007). Littermates were cotransfected with plasmids encoding a scrambled version of the shRNA (pU6shRNA-Kiaa0319 scram) along with plasmids encoding mRFP (pCAGGS-mRFP) and plasmid encoding eGFP. Pups in the KIAA0319 overexpression group were cotransfected with an IRES construct coding both for the human KIAA0319 protein and eGFP (pCAG-KIAA0319-IRES-eGFP) and pCAGGS-mRFP, whereas their littermates were transfected with pU6shRNA-Kiaa0319 + pCAGGS-eGFP. In the Rescue condition, subjects were cotransfected with pU6shRNA-Kiaa0319, pCAGGS-KIAA0319-eGFP, and pCAGGS-mRFP. Littermates were transfected with pU6shRNA-Kiaa0319 + pCAGGS-eGFP. The effectiveness of these plasmids in knocking down exogenous Kiaa0319 function was validated by Western blot (Supplemental Fig. 1).
Pregnant dams at E18/19 were anesthetized with isoflurane (5%) and intraperitoneally injected with 50 mg/kg of 5-bromo-2′-deoxyuridine (Sigma Aldrich, St. Louis, MO, 10 mg/mL solution).
One litter transfected with Kiaa0319 shRNA was sacrificed 4 hours after BrdU, whereas the remaining litters were sacrificed at P21. Animals were deeply anesthetized (Ketamine/Xylazine 10:1, 100 mg/mL) and sacrificed by transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde. The brains were removed from the skull and postfixed for 24 h before being cryoprotected in 10% and then 30% sucrose phosphate buffer. The brains were sectioned coronally at 40 μm on a freezing microtome. Sections were then mounted and coverslipped with VECTASHIELD Mounting Medium (Vector Labs, Burlingame, CA) and visualized under fluorescence for the presence of eGFP and/or mRFP. One series of every tenth section was stained for Nissl substance using Thionin. One adjacent series of free-floating sections was processed for immunohistochemical detection of eGFP (Chemicon, 1:200) using ABC protocols.
Adjacent series of sections were processed for immunofluorescence detection of laminar markers. These included Cux1 (CDP (M-222), Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000) and FoxP2 (FoxP2 (N–16), Santa Cruz Biotechnology, 1:50). Cux1 is a transcription factor that predominantly labels layer 2–4 neurons, especially in the in parietal cortex (Nieto et al. 2004), whereas FoxP2 labels neurons in layer 6 throughout the cortex (e.g., Keays et al. 2007). An antibody for the connective tissue growth factor Ctgf (Heuer et al. 2003), (L-20 Santa Cruz Biotechnology, 1:50) was used to label neurons in layer 6b (Molyneaux et al. 2007), and cells that contained BrdU were labeled with Anti-BrdU (BD Bioscences, San Jose, CA, 1:100). Primary antibodies were detected with one of the following secondary antibodies: Alexa Fluor 555, Alexa Fluor 594 (Invitrogen, Carlsbad, CA, 1:200), or Cy5 (Jackson ImmunoResearch, West Grove, PA, 1:50).
Additional sections were stained for gamma-aminobutyric acid-ergic (GABAergic) antibodies Calretinin (MAB1468, Milipore Corp., Billerica, MA, 1:1000) and Parvalbumin (MAB353, Millipore, 1:200). The presence of progenitor cells was assessed by staining for Nestin (MAB1572, Millipore, 1:1000).
In Situ Quantification
Individual sense and antisense sections from horizontally prepared brains (1 each from E14/15, E16/17, and E19/20 rats) were imaged with monochrome digital camera (Insight, Diagnostic Instruments, Sterling Heights, MI) on a light box (Aristo Grid Lamp Products, Waterbury, CT) and interfaced via firewire to Macintosh G4 computer (Apple Computer, Cupertino, CA). Each antisense section image and its corresponding sense section were captured using SPOT software (Diagnostic Instruments) with common exposure settings. Using ImageJ <http://rsb.info.nih.gov/ij/>;, optical density values were measured for the combined CP and VZ. A total of 9–13 sections were measured for each brain. The average difference in optical density between sense and antisense images were computed and expressed as a percent of sense density.
Postnatal Assessment of Pathology
All analyses of postnatal brains were performed blind with respect to condition. Nissl-stained sections were surveyed for the presence of neocortical and/or hippocampal malformations, and their location noted.
Quantitative analysis of migrational distance was conducted using a custom Matlab (Mathworks, Natick, MA) program. The location of eGFP+ cells was charted in 4 randomly chosen immunohistochemically stained series using Neurolucida (MBF Biosciences, Williston, VT). The program then determined the location of each cell in a user–defined region of interest as the percentage of cortical depth, with 0% being the white matter/subplate border and 100% being the pial surface. Frequency distributions were determined for each animal, and the mean value across all animals within each condition was determined (Supplemental Fig. 2). Differences in the distribution of migrated neurons were assessed using ANOVA. Initial analysis determined that there were no differences among the Kiaa0319 shRNA-transfected groups (F2,24 = 1.4, NS), and so their data were pooled for all analyses.
Five neurons from each brain were randomly selected from all laminar locations for morphological analysis. The cell body and the extent of each apical and basal dendrite were traced with Neurolucida (MBF Biosciences). Using Neurolucida Explorer (MBF Biosciences), Sholl analysis and branch analysis (dendritic length and numbers of nodes, ends, and dendrites) were then performed, and a single measure derived for each animal (mean value from 5 neurons for each of the dependent measures). Statistical differences were determined by single factor and repeated measures ANOVA. Initial analysis determined that there were no differences among the Kiaa0319 shRNA–transfected groups (F2,27 < 1, NS) and so their data were pooled for all analyses.
Fluorescent images were obtained on a confocal microscope (Zeiss LSM 510 Meta, Carl Zeiss, Inc., Thornwood, NY). Photomicrographs were adjusted for exposure and sharpened (unsharp mask filter) using Adobe Photoshop (Adobe Inc., San Jose, CA). Some brightfield images were acquired using the Virtual Slice Module of Neurolucida. Image montages were created in Canvas X (ACD Systems, Miami, FL).
Kiaa0319 is Highly Expressed in a Regionally Distinct Manner
In situ hybridization revealed that Kiaa0319 was expressed in a regionally distinct manner (Fig. 1). At all ages examined, there was increased expression in the VZ, intermediate zone (IZ), CP, striatum, hippocampus, and brain stem. Expression in the CP increased over time, and there was evidence of Kiaa0319 expression in migrating neurons (Fig. 1). Quantitatively, differences in optical density between the antisense and sense probes ranged from 0.5-fold (brain stem) to 1.2-fold (striatum). In addition, there was increased expression in the mitral cell layer of the olfactory bulb at E18/19. In situ hybridizations performed on E14.5 mouse embryos by GenePaint (www.genepaint.org, GeneID = D130043K22Rik) and in adult mice by the Allen Brain Atlas (www.brain-map.org, GeneID = D130043K22Rik) confirmed this, although expression in the adult striatum was diminished compared that of the embryo.
There are PVHs in Kiaa0319 shRNA–Transfected Subjects
Nissl- and eGFP-stained sections from each of the 57 animals were examined for the presence of neuronal migration anomalies, including molecular layer ectopias, hippocampal dysplasias, and PVHs. There were molecular layer ectopias associated with the embryonic injection site in half of the animals in all conditions, but there was no evidence of separate molecular layer malformations that suggested a migrational disorder. There were hippocampal dysplasias in 3 of the 29 animals transfected with Kiaa0319 shRNA (Treatment 1 in Table 1), which were identical to those previously reported (Rosen et al. 2007; Burbridge et al. 2008).
All of the Kiaa0319 shRNA–transfected animals had evidence of disruption of neuronal migration to the neocortex (Fig. 2A). Transfected neurons were seen throughout the neocortex, with the highest concentrations at the border with the white matter and in layer 2/3. In approximately 75% of the cases, there were PVHs (Fig. 2E–H) that were visible for much of the rostral–caudal extent of the brain. In contrast, there were few unmigrated neurons in the Kiaa0319 scram and KIAA0319 overexpression conditions (Fig. 2B, C). One animal in the scram condition did have a small collection of neurons at the white matter border that resembled a PVH, but the frequency and severity of these malformations were significantly greater in the Kiaa0319 shRNA condition (χ2 = 91.2, df =1, P < 0.001). There were no malformations in the brains of any animals in the KIAA0319 overexpression condition.
In order to determine the specificity of the shRNA for Kiaa0319 we cotransfected animals with a plasmid encoding human KIAA0319 along with the Kiaa0319 shRNA plasmid. Human KIAA0319 nucleotide sequence does not match rat Kiaa0319 sequence in the region targeted by the Kiaa0319 shRNA, and therefore it is not susceptible to RNAi. Of the rats simultaneously transfected with Kiaa0319 shRNA and the human KIAA0319 expression construct, 4 out of 10 had small, focal regions of PVHs, which were not as extensive as those seen in the Kiaa0319 shRNA treatment condition (Fig. 2D). The remainder had no obvious malformations (Fig. 2C, D). There was a significant difference in the number of PVHs (χ2 = 4.3, df = 1, P < 0.05), which suggests that overexpressing the human KIAA0319 protein in Kiaa0319 shRNA-treated rats at least partially rescued this phenotype, and indicates that the effects of the RNAi are not due to off-target effects.
We assessed the PVH for the presence of Nestin-positive progenitor cells. There was no evidence of progenitor cells within the PVH, but there was a marked increase of Nestin-positive fibers (Fig. 3). Thus, although there were sparse Nestin-positive fibers contained within homologous regions of the nontransfected hemisphere (Fig. 3E), there were dense collections of these fibers within the PVH (Fig. 3D) that resembled radial glial fibers. Some of fibers could be seen to invade the upper layers of the cerebral cortex. These results suggest that there is an undue preservation of radial glial-like morphology within the PVH.
There are Non–Cell Autonomous Effects of Embryonic Kiaa0319 Knockdown
In order to assess whether embryonic transfection with Kiaa0319 shRNA affects the normal laminar position of neurons, we stained Kiaa0319 shRNA-transfected brains with laminar markers. Staining with Cux1, a marker of layer 2–4 neurons in parietal and other medial cortices, revealed a number of Kiaa0319 shRNA–transfected neurons in both the PVHs and layer 2/3 that were colabeled with Cux1. In contrast, staining for Ctgf, a marker of layer 6b neurons, did not disclose any colabeled neurons (Fig. 4). Similarly, staining with the layer 6 marker Foxp2 did not reveal any transfected cells that were colabeled (Fig. 5). These results suggest that PVHs and some of the deeper cortical areas contain neurons that are normally destined for supragranular layers.
Intriguingly, there were large numbers of Cux1+ neurons in the PVHs that were not colabeled with eGFP (indicating that they were not transfected with Kiaa0319 shRNA) that failed to migrate (Fig. 4). This suggested that there were non–cell autonomous effects of transfection with Kiaa0319 shRNA. Alternatively, it could be that these Cux1+ neurons were originally transfected with Kiaa0319 shRNA + eGFP, and that the fluorescent protein had been subsequently lost from the cells. In order to test this alternative, we examined the brains of animals transfected with Kiaa0319 shRNA + eGFP on E15/16 and subsequently injected with BrdU at E18/19. We first assessed the distribution of BrdU+ and eGFP+ cells following a 4-h post-BrdU injection interval (Fig. 6A–F). We found that there were very few colabeled cells, indicating that cells labeled by BrdU at E18/19 were largely nontransfected cells (Fig. 6C,F; Supplemental Movies 1 and 2), and that the BrdU+ and eGFP+ populations belong to distinct populations. We then examined similarly treated animals in the postnatal period, and examined for the colocalization of BrdU+, Cux1+, and eGFP+ neurons in layer 2/3 and the PVH (Fig. 6G–N).
As expected, there were large numbers of neurons colabeled for BrdU and Cux1 as well as Cux1 and eGFP colabeled cells in layer 2. However, there were no neurons colabeled with eGFP and BrdU. In the PVH, the results were identical to those described in Figure 5. Specifically, there were large numbers of Cux1+ neurons that were not colabeled with eGFP. Moreover, there were large numbers of E18/19 BrdU+ neurons in the PVH and none of these were colabeled with eGFP (although some coexpressed Cux1). Taken together, these results indicate that the nontransfected neurons in the PVH did not lose the eGFP label, but rather represent non–cell autonomous effects of Kiaa0319 knockdown in the developing rat brain.
We sought further confirmation of the non–cell autonomous effects of embryonic knockdown of Kiaa0319 by staining for GABAergic interneurons in the PVH using antibodies against Calretinin and Parvalbumin. GABAergic interneurons are generated in the medial ganglionic eminence, and are therefore not likely to have been transfected following in utero electroporation. Thus, the presence of positive immunoreactive cells in the PVH would argue strongly for non–cell autonomous effects. We found both Calretinin- and Parvalbumin-positive interneurons within the PVH (Fig. 7), which supports the notion that embryonic transfection with shRNA targeted against Kiaa0319 has non–cell autonomous effects on both radially migrating and tangentially migrating neurons.
Altered Expression of KIAA0319 Disrupts the Laminar Position of Pyramidal Neurons
Previously, we demonstrated a distinctive pattern of migrational disturbance following transfections with shRNA targeted against Dyx1c1 or Dcdc2. Specifically, we found a bimodal pattern of migration, with approximately 7–20% of the transfected neurons not migrating past the cortical–white matter border, and the remaining neurons “overmigrating” past their expected laminar locations (Rosen et al. 2007; Burbridge et al. 2008). In the current experiment, we sought to determine whether this overmigration phenotype occurred following either knockdown of Kiaa0319 or overexpression of its protein.
Of the 57 animals, 4 were excluded from this analysis because the transfections were outside the main region of analysis (somatosensory cortex). The results of the migration distance analysis for all groups are summarized in Figure 8. Virtually, all (98.4%) of the neurons in the Kiaa0319 scram (control) transfected group migrated into the neocortex, with the majority peaking in layer 2/3, at approximately 75% of the cortical depth, with an average of 69.8 ± 1.0%. In comparison, 10% of the Kiaa0319 shRNA—transfected cells remained at the cortical/white matter border, which is significantly different from the Kiaa0319 scram group (F1,36 = 9.1, P < 0.01). Interestingly, the distribution of those neurons that did migrate is virtually identical to that of the Kiaa0319 scram group, with an average migration distance of 68.8 ± 1.0% (F1,36 < 1, NS). Thus, although the bimodal migration distance phenotype is consistent among all 3 candidate dyslexia susceptibility genes examined, unlike Dcdc2 and Dyx1c1 (Rosen et al. 2007; Burbridge et al. 2008) there is no overmigration phenotype following knockdown of Kiaa0319.
Virtually all (98.7%) of the neurons transfected with the KIAA0319 expression construct migrated into the neocortex, with the major peak at lower layer 2/3 at a cortical depth of 65%, (average = 63.6 ± 2.5%). This average migration distance differs from the Kiaa0319 scram group (F1,16 = 5.1, P < 0.05), suggesting that KIAA0319 overexpression disrupts the normal migration of neurons. Of the neurons that were co–transfected with Kiaa0319 shRNA and KIAA0319 expression construct (the rescue condition), 5.5% did not migrate in the CP. This percentage of unmigrated cells is significantly smaller than that in the Kiaa0319 shRNA group (F1,33 = 5.6, P < 0.05), which indicates that the presence of the KIAA0319 protein rescues the “nonmigration” phenotype. Interestingly, the migration distance for the rescue condition peaks at the same point as the KIAA0319 expression vector alone group, at around 65% of the cortical depth, with an average of 63.3% ± 1.0. This differs significantly from the Kiaa0319 scram group (F1,17 = 12.6, P < 0.01), and does not significantly differ from the KIAA0319 overexpression group (F1,13 < 1, NS). Taken together, the evidence suggests that neurons that are transfected with the KIAA0319 expression construct (both the overexpression and rescue groups) tend to migrate to lower laminar positions than would be expected for normal cohort of migrating cells. As the KIAA0319 expression construct used to rescue is insensitive to complete knockdown by the Kiaa0319 shRNA this would suggest that overexpression of KIAA0319 alters the position of cells within cortical lamina. It is not yet clear whether this shift in position by overexpression of KIAA0319 results from impairment of migration or from impairment of laminar sorting, which could result from changes in cell adhesion.
Knockdown of Kiaa0319 Causes Increased Arborization of Apical Dendrites
The results from the quantitative analysis of dendritic arborization are summarized in Figure 9. We analyzed the data from the Sholl analysis (Fig. 9A,B) using a repeated measures ANOVA, with dendrite length within each 10-μm concentric circle as a dependent measure, treatment groups as the independent measure, and the concentric circles (“bins”) as the repeated measure. For apical dendrites, there were significant main effects for both treatment group (F3,52 = 5.7, P < 0.01) and bins (F332,4316 = 171.2, P < 0.001), as well as a significant bins × treatment group interaction (F249,4316 = 6.33, P < 0.001). We further analyzed the treatment group main effect, and found that the Kiaa0319 shRNA group significantly differed from each of the other 3 groups (shRNA vs. Overexpression: F1,35 = 9.3, P < 0.01; shRNA vs. Rescue: F1,37 = 8.9, P < 0.01; shRNA vs. Scram: F1,38 = 4.3, P < 0.05). This indicated that there was significant dendritic hypertrophy in neurons embryonically transfected with Kiaa0319 shRNA. In contrast, there was no significant difference among the 4 treatment conditions for basal dendrite length (F3,52 = 2.0, NS), although there were significant effects for bins (F48,2496 = 351.2, P < 0.001) and bin × treatment group interaction (F144,2496 = 2.5, P < 0.001). These results suggest a trophic effect on apical, but not basal, dendritic arborization caused by Kiaa0319 interference.
In order to dissect the components of the dendritic tree that differed among the treatment groups, we analyzed the number of dendrites (for basal only), nodes, ends, and total length (Fig. 9C). For apical dendrites, we found that the Kiaa0319 shRNA–transfected neurons had more ends, nodes, and longer dendritic length than neurons in the Rescue group (F1,35 = 7.5, P < 0.01; F1,35 = 8.0, P < 0.01; F1,35 = 7.1, P < 0.05, respectively). In addition, we found that there were more ends (F1,37 = 7.6, P < 0.01) and nodes (F1,37 = 7.8, P < 0.01) in the Kiaa0319 shRNA group when compared with the Kiaa0319 scram group. There were no significant differences in any of these apical dendrite measures between neurons embryonically transfected with Kiaa0319 shRNA neurons and those transfected with the KIAA0319 expression construct. Moreover, there were no significant differences among any of the basal dendrite measures. Taken together, these results support the Sholl analysis, and indicate that knockdown of Kiaa0319 results in apical, but not basal, dendritic hypertrophy.
Previous reports had demonstrated that embryonic transfection with shRNA targeted against the rat homolog of the candidate dyslexia susceptibly gene Kiaa0319 resulted in significant arrest of neuronal migration when assessed 4 days following transfection, with the majority of transfected cells remaining in the VZ (Paracchini et al. 2006). The current experiment indicates that this disruption of neuron migration causes specific types of neuronal migration anomalies in the postnatal brain—specifically PVHs and abnormal laminar locations, and embryonically transfecting neurons with both the Kiaa0319 shRNA and KIAA0319 expression construct rescues this phenotype. In contrast, overexpressing the KIAA0319 protein does not cause gross neuronal migration disorders.
In humans, PVHs and other anomalies of neuronal migration are relatively easy to visualize with computed tomography and magnetic resonance imaging and are confirmed post mortem using standard histological techniques. It has been hypothesized, however, that these malformations may simply act as flags for other, more widespread, disturbances in neocortical organization. For example, disturbances in white matter connectivity associated with neuronal migration disorders have been revealed using diffusion tensor imaging in humans (Lee et al. 2005; Huppi and Dubois 2006), and severe disruptions in connectivity have been demonstrated in various animal models of neuronal migration disorders (Giannetti et al. 1999, 2000; Jenner et al. 2000; Rosen et al. 2000). Moreover, the abundance of radial glia-like morphology in Nestin-positive fibers within the PVH, supports the existence of a more widespread disturbance in neocortical organization. Interestingly, this maintenance of radial glia–like morphology is remarkably similar to that seen following induction a malformation resembling microgyria via a P1 freezing lesion, where Nestin-positive fibers were seen in the microgyria well into adulthood (Rosen et al. 1994).
Inducing neuronal migration anomalies by embryonic transfection with shRNA has enabled us to identify disruptions of laminar positioning that would otherwise not be visualized. In the current experiment, we have demonstrated that following embryonic transfection with Kiaa0319 shRNA, Cux1+ and E18/19 BrdU+ neurons that are normally destined for layer 2/3 collect in PVHs. Although some of these neurons were transfected with Kiaa0319 shRNA, there were large numbers that were not. This replicates and extends previous reports, where we found that embryonic transfection with shRNA targeted against the rat homologue of the candidate dyslexia susceptibility gene Dcdc2 resulted in nontransfected Cux1+ neurons in the PVH (Burbridge et al. 2008).
It could be argued, however, that the Cux1+ and BrdU+ neurons that are eGFP negative, were in fact transfected with Kiaa0319 shRNA but that the cell simply stopped expressing eGFP in the postnatal period. To test this hypothesis, we examined animals that received E15/16 transfections of Kiaa0319 shRNA, an E18/19 injection of BrdU, and were then sacrificed 4 hours later. In these animals, the eGFP+ and BrdU+ cells made up separate and unique populations—there were few cells that were double labeled (Fig. 6A–F). When we examined the brains of identically treated animals in the postnatal period, then, any BrdU+ neuron was not transfected with Kiaa0319 shRNA + eGFP. Because we found large numbers of BrdU+ neurons in the PVH, this strongly supports the hypothesis that these cells failed to migrate due to non–cell autonomous mechanisms. This argument is further bolstered by the presence of GABAergic interneurons in the PVH. Because these neurons are not generated in the VZ (Anderson et al. 2001), it is unlikely that they were transfected with Kiaa0319 shRNA + eGFP. The presence, therefore, of Parvalbumin- and Calretinin-positive interneurons, which are not colabeled with eGFP in the PVH, strongly supports the notion that there are non–cell autonomous effects of embryonic transfection with Kiaa0319 shRNA.
The results reported here are similar to previous reports that examined the pre- and postnatal phenotypes associated with embryonic transfection of candidate dyslexia susceptibility genes. Thus, embryonic knockdown of Dcdc2 (Meng et al. 2005; Paracchini et al. 2006; Burbridge et al. 2008) and Dyx1c1 (Wang et al. 2006; Rosen et al. 2007) disrupted neuronal migration. As with Kiaa0319 embryonic knockdown, the majority of brains transfected with Dcdc2 or Dyx1c1 shRNA develop PVHs, although Dyx1c1 and Kiaa0319 knockdown groups have more extensive malformations. We did find hippocampal malformations following embryonic transfections with shRNA targeted against Kiaa0319, but the incidence was smaller than that associated with knockdown of either Dyx1c1 or Dcdc2. As with the case of Dcdc2 transfected brains, we did not find any evidence of molecular layer ectopias in the current study, whereas they were present in about 25% of the brains transfected with Dyx1c1 shRNA.
Although embryonic knockdown of the 3 candidate dyslexia susceptibility genes always resulted in a population of unmigrated neurons, the disposition of those neurons that did migrate into the cerebral cortex differed. In the case of the previous reports, the peak locations of transfected neurons in the cerebral cortex were superficial to their expected lamina (Rosen et al. 2007; Burbridge et al. 2008). This was not the case in the present report, as neurons embryonically transfected with Kiaa0319 shRNA were distributed in the cerebral cortex identically to control animals transfected with the scrambled version of the Kiaa0319 shRNA. In contrast, we did find disruptions of laminar position following overexpression of KIAA0319, with transfected neurons migrating to positions below their expected location. This was true both for the overexpression and “rescue” group, which were simultaneously transfected with the overexpression construct and Kiaa0319 shRNA. This “undermigration” of neurons transfected in both conditions could either represent the effects of overexpressing the protein, or could be due to the effect of the human protein on the migrating neurons. Because we lack an effective antibody against the protein, we cannot directly distinguish between these 2 possibilities.
It is possible that changes in the methods of quantification may explain the disparity between the previous reports and the current experiment. In the original 2 reports, migration distance of transfected neurons was assessed by determining the number of neurons in successive deciles in a 250-μm-wide sampling grid. In the current experiment, the actual distance that each neuron migrated (as a percent of the total depth of the cortex) was recorded (Supplemental Fig. 2). It could be, then, that the results from the previous reports resulted from undersampling. We have reanalyzed the data from one of these experiments using the present methodology (Burbridge et al. 2008), and found that the results were identical to the previous report (unpublished observations). It is therefore likely that the results reported here reflect a real difference in the postnatal phenotypes between Kiaa0319 and the other candidate dyslexia susceptibility genes.
There is little currently known concerning the functions of these genes, but what is known suggests that they may well disrupt neuronal migration by distinct mechanisms. DCDC2 is one of a group of proteins distinguished by the presence of tandem or single dcx domains, which are critical for binding and stabilizing microtubules (Allen et al. 1998; des Portes et al. 1998; Gleeson et al. 1999; Graham et al. 2004; LoTurco 2004; Reiner et al. 2004; Schaar et al. 2004). The function of Dyx1c1 is not well known, but previous reports suggest that Dyx1c1 is localized in the cytoplasm along microtubules as well (Wang et al. 2006). KIAA0319, on the other hand, is a gene that codes for an integral membrane protein with a large glycosylated (Velayos-Baeza et al. 2008) extracellular region containing multiple PKD domains, a single transmembrane domain, and a small cytoplasmic C-terminus. Recently 3 KIAA0319 splice variants have been identified and one of these codes for a secreted protein (Velayos-Baeza et al. 2008). We do not yet know which splice variant may be responsible for function in migration or dendritic differentiation, however future experiments using different splice variants to rescue the RNAi phenotypes described here can now be used to distinguish among their functions. The full-length KIAA0319 may be involved in cellular adhesion, as PKD domains in polycistin–1 are necessary for adhesion of renal epithelial cells (Wilson 2001). Consistent with a function in cell adhesion in developing cortex, Parrachini et al. (2006) showed a change in the relationship between radial glia and migrating neurons following interference of Kiaa0319 in embryonic neocortex. Similarly, the significant non–cell autonomous component of migration disruption we show here is consistent with a role for the secreted splice variant of Kiaa0319 (Velayos-Baeza et al. 2008).
Prior to the current experiment, the effects of embryonic knockdown and overexpression of candidate dyslexia susceptibility genes on neuronal morphology had not been addressed. Here we find that transfection with Kiaa0319 shRNA results in hypertrophy of apical, but not basal, dendrites when compared with the other 3 conditions. It could be that our results could be affected by biases in the orientation of the sections, the laminar positions of the neurons being measured, or in the relatively narrow section thickness. There was no difference in the laminar distribution of neurons that were measured between the groups, and the analysis of the subsection of those neurons in the upper laminae were identical to those from the entire population. Our results could be explained by biased sectioning only if the orientations of Kiaa0319 shRNA–transfected neurons were systematically different than those of neurons in the other groups. We have no evidence of differential orientation between the experimental groups. The relatively narrow section thickness does result in a less elaborate dendritic branching when compared with, for example, biocytin injection. Although there is no evidence of systematic bias between the groups, replication of these results in thicker sections of biocytin-injected neurons would be warranted. Further confirmation of the effect of Kiaa0319 expression on dendritic outgrowth awaits experiments in cultured neurons.
The mechanisms underlying the dendritic differences are not known, but could reflect direct effects of Kiaa0319 on both neuronal migration and dendritic outgrowth. Along these lines, the relationship between neuronal migration disorders and dendritic hypertrophy has been previously established. Takashima et al. (1991) reported the coexistence of hemimegalencephaly, a disorder of neuronal migration, and dendritic hypertrophy in 6 post mortem patients. More recently, PTEN deficient mice—a model for macrocephaly and Lhermitte–Dudos disease—exhibit both ectopic neurons and hypertrophic dendrites in the cerebral cortex (Kwon et al. 2006). The p21-activated kinase (Pak1), a cytoskeletal regulator, has significant effects on neuronal migration, elaboration of axons and dendrites, and the formation of dendritic spines (Banerjee et al. 2002; Causeret et al. 2009), and Lis-1 deficient mice have hippocampal migration disorders as well as altered dendritic arborization (Fleck et al. 2000). ROBO1, a candidate dyslexia susceptibility gene, plays important roles in both neuronal migration and process outgrowth (Long et al. 2004; Hannula-Jouppi et al. 2005; Andrews et al. 2008). Alternatively, it could be that the changes in the elaborations of the dendrites are secondary to the neuronal migration disorder that is induced by Kiaa0319 knockdown. For example, long-term potentiation has been shown to increase various measures of dendritic morphology in the cerebral cortex (Monfils et al. 2004), and it is well known that damage to the developing brain can affect dendritic arborization in connected regions (Marin-Padilla 1997). One way to distinguish between these possibilities would be to assess dendritic morphology of both transfected and nontransfected neurons in the same brain. Future experiments involving sequentially transfecting pups will enable this question to be directly addressed. Alternatively, one could conditionally re-express Kiaa0319 in animals embryonically transfected with Kiaa0319 shRNA after the completion of neuronal migration and during the dendritic differentiation (P7–P21).
In summary, we have demonstrated that embryonic knockdown of the candidate dyslexia susceptibility gene homolog Kiaa0319 results in a neuronal migration disorder and hypertrophy of apical dendrites. Both cell autonomous and non–cell autonomous mechanisms appear to play a role in the formation of the neuronal migration disturbances, but their relative contribution to apical dendritic hypertrophy is not known. Overexpression of KIAA0319 does not produce PVH or other gross disturbances in neuronal migration, but does act to limit the distance that neurons migrate into the CP. At present, all of the candidate dyslexia susceptibility genes whose functions have been investigated have been found to play a role in neuronal migration. This is the first demonstration that one of these genes has an effect on subsequent neuronal morphology, which may provide another biological substrate for the functional differences seen in this disorder. Whether other dyslexia susceptibility genes have similar effects on neuronal morphology is not known, but will provide a fruitful avenue for future investigations.
National Institutes of Health (HD20806) to A.M.G., J.J.L., and G.D.R; and Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program and Fannie and John Hertz Foundation/Myhrvold Family fellowship supported Z.W.G.
The authors thank Ankur Thomas for the validation of the plasmids. The authors thank the reviewers for their helpful comments on a previous version of the manuscript. Conflict of Interest: None declared.