Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex

Abstract

Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia, we show that migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron's migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may in part underlie the neurological defects in Joubert syndrome patients.

Figure 1. Projection neuron placement and axonal outgrowth in Nex-Cre; Arl13b^Lox/Lox cortex

(A-D) P0 somatosensory cortex of Arl13b^Lox/+; Nex-Cre (A,C) or Arl13b^Lox/Lox; Nex-Cre (B,D) mice were immunolabeled for Cux1 (layers II-IV), Ctip2 (layer V), Brn1 (layers II-V) and Tbr1 (layer VI). Neuronal positioning was not altered by Arl13b deletion in projection neurons. (E, F) Serial coronal sections spanning the rostro-caudal extent of E16 Arl13b^Lox/+; Nex-Cre; Tau-mGFP (E) or Arl13b^Lox/Lox; Nex-Cre; Tau-mGFP (F) cortex were immunolabeled for mGFP. No differences in the extent of migration of GFP⁺ projection neurons were seen. Major cortical axonal tracts also appear normal in mutants. (G, H) High magnification image of sections from the lateral cortex of E16 Arl13b^Lox/+; Nex-Cre; Tau-mGFP (G) or Arl13b^Lox/Lox; Nex-Cre; Tau-mGFP (H) cortex immunolabeled for mGFP showing normal migration and initial axonal growth of projection neurons. (I-R) Sagittal sections from E16 (I, J), P0 (K-N) and P7 (O-R) Arl13b^Lox/+; Nex-Cre; Tau-mGFP (I, K, M, O, Q) or Arl13b^Lox/Lox;Nex-Cre; Tau-mGFP (J, L, N, P, R) cortex indicates generally normal development of major cortical axonal tracts. (M-N, Q-R) However, high magnification of internal capsule regions outlined in K-L and O-P, respectively, illustrates disrupted fasciculation in mutants. Sections in G and H were counterstained with DRAQ5 (nuclei). VZ-ventricular zone, IZ-intermediate zone, CP-cortical plate, CC-corpus callosum, IC-internal capsule, FI-fimbria, ST-stria terminalis, AC-anterior commissure. Scale bar=A-D, 225 µm; E-F, M-N, Q-R, 300µm; GL, O-P, 750µm.

Figure 2. Interneuronal placement is disrupted after Arl13b deletion in interneurons

(A-F) P10 control Arl13b^Lox/+;Dlx5/6-CIE (A, C, E) and Arl13b^Lox/Lox;Dlx5/6-CIE (B, D, F) cortices were co-labeled with GFP (Cre⁺) and GABA (A, B) calretinin (C, D) and somatostatin (E, F). Insets show higher magnification images of double-positive cells (arrowheads). (G-I) Quantification of changes in the distribution of double-positive cells across cortical layers. Sections were from somatosensory cortex and counterstained with DRAQ5 [blue]. Data shown are mean ± SEM; * indicates significant when compared with controls at p<0.05 (Student’s t test). p<0.05 (Student’s t test). Scale bar= 100µm.

Figure 3. Arl13 deletion leads to interneuronal migration and branching defects

(A-B) GFP-labeled coronal hemisections show interneuron migration defects in Arl13b^Lox/Lox;Dlx5/6-CIE mutants, with clusters of cells stuck at the pallial-subpallial boundary (arrow, 5B). (C-D) Loss of characteristic interneuronal migratory streams in Arl13b^Lox/Lox;Dlx5/6-CIE cortex. Arrowheads in C indicate streams of migrating interneurons in the MZ, IZ and SVZ of control cortex. Arrows in D indicate disruptions in these streams in mutant cortex. (E-F) Higher magnification images of the control (E) and mutant (F) dorsal cortex illustrating disrupted extent and patterns of migration in mutants. Left asterisks mark pallial-subpallial boundary, right asterisks mark the migration front. (G) Disrupted migration results in altered distribution of interneurons (INs) across the cortical wall in Arl13b^Lox/Lox;Dlx5/6-CIE cortex, with a larger percentage of cells moving through the intermediate zone (IZ) and a corresponding loss of cells in other locations. The overall number of interneurons was reduced in mutants, but mutants show a higher percentage of interneurons in the IZ than controls. (H, I) Camera lucida drawings of sample control (H) and mutant (I) interneurons in the dorsal cortex. (J-L) Quantification of branching defects in Arl13b-deficient interneurons. (J) Number of processes extending from cell soma. (K) Total process length (n=506 cells each from control and mutants). (L) Branching index (average number of branching events in a migrating interneuron per hour). Data shown are mean ± SEM; * indicates significant when compared with controls at p<0.05; ** indicates significant when compared with controls at p<0.01 (Student’s t test). VZ-ventricular zone, SVZ-subventricular zone, IZ-intermediate zone, CP-cortical plate, GE-ganglionic eminence, D.CX-dorsal cortex. Scale bar= A-D, 675µm; E-F, 225µm; G-H, 100µm; J-K, 25µm.

Figure 4. Arl13b is required for interneuron migration through a microgradient of endogenous guidance cues from dorsal cortex

(A) Left: Schematic of microfluidic chamber migration assay. Dissociated wild-type dorsal cortical cells (D. Cx, blue) are plated in one side of a chamber and dissociated interneurons from the GE of control (Arl13b^Lox/+; Dlx5/6-CIE or Arl13b^+/−) or mutant (Arl13b^Lox/Lox; Dlx5/6-CIE or Alr13b^−/−) cortex are plated in the other side. Right: Brightfield/fluorescent image of the assay soon after plating of neurons. Interneurons were exposed to a microfluidic gradient of cues released by dorsal cortical neurons. Interneurons migrate toward the dorsal cortical cells via microchannels (vertical lanes) separating the chambers. (B-G) Compared to control Arl13b^Lox/+ (B), GFP⁺ interneurons from Arl13b^Lox/Lox; Dlx5/6-CIE brains (C) migrated significantly shorter distances (D). Similar defects in migration occurred in interneurons from Arl13b^−/− brains (E-G). Interneurons from Arl13b^−/+(E) or Arl13b^−/−(F) brains were immunolabeled (red) with anti-GAD67 antibodies. Data shown are mean ± SEM; * indicates p<0.01 (Student’s t test). Scale bar= B-C, 15µm; E-F, 30µm.

Figure 5. Ciliary function of Arl13b is critical for the regulation of interneuron migration

(A) Schematic of a non-ciliary form Arl13b (Arl13b^V358A). (B) In ciliated IMCD3 cells transfected with control Arl13b-GFP and Arl13b^V358A-GFP constructs, Arl13b-GFP localizes to cilia (arrow), but Arl13b^V358A-GFP does not. Nuclei were counterstained with DAPI. (C) Expression of wild type Arl13b rescued the migratory defect in Arl13b deficient (Arl13b^Lox/Lox; Dlx5/6-CIE) interneurons. In contrast, expression of Arl13b^V358A did not rescue the defect. Data shown are mean ± SEM (n=42 [Control: Arl13b^Lox/+; Dlx5/6-CIE], 79 [Arl13b deficient: Arl13b^Lox/Lox; Dlx5/6-CIE], 104 [Arl13b^Lox/Lox; Dlx5/6-CIE +Arl13b], 49 [Arl13b^Lox/Lox; Dlx5/6-CIE+Arl13b^V358A]); * indicates significant when compared with controls at p<0.001; ** indicates significant when compared with Arl13b mutant at p<0.001 (Student’s t test). (D-H) Dominant negative N-terminal domain (Arl13b-ND) of Arl13b disrupts selective targeting of Arl13b to primary cilia and inhibits interneuronal migration. (D) Cartoon of experimental protocol. Arl13b-ND/GFP or GFP DNA was focally electroporated into the MGE of E14.5 coronal slices. Images of GFP⁺ interneuronal migration into dorsal cortex were acquired at 24 and 48 hours. (E-H) Interneurons expressing GFP alone leave the MGE at 24 h (E, left panel) and migrate into the dorsal cortex by 48 h (E, right panel, and higher magnification image of dorsal cortex in H). Interneurons co-expressing Arl13b-ND mostly remain in the GE at 24 h (F, left panel) and show less migration into dorsal cortex at 48 h (F, right panel, and higher magnification image in I). Compared to control (arrows, H), fewer GFP⁺ interneurons are seen in the dorsal cortex in Arl13b-ND slices (I). (G) Quantification of changes in the extent of interneuronal migration. Migration index indicates the number of cells migrating greater than 350 µm past the GE-dorsal cortex boundary. Data shown are mean ± SEM (n=36 slices/ group); * p<0.05 (Student’s t test). GE, ganglionic eminence; D.Cx, dorsal cortex. (J-K) Disrupted interneuron migration in Ift88^−/− brains. (I) Calbindin⁺ interneurons (red, arrow) migrate from the ganglionic eminence into the dorsal cortex. This migration is severely disrupted in Ift88^−/− brain (K). Insets (I, K) show the presence and absence of Arl13b⁺ cilia in wild type and mutant brains, respectively. Sections were counterstained with DAPI or DRAQ5 (blue). Scale bar= B-C, 34 µm; E-F, 400 µm; H-I, 175 µm; J-K, 180 µm.

Figure 6. Changes in the primary cilia localization of interneuronal migration related guidance cue receptors and second messenger activity in Arl13 deficient interneurons

(A) Primary cilia (red) of control Arl13b^Lox/+; Dlx5/6-CIE (top) and mutant Arl13b^Lox/Lox; Dlx5/6-CIE (bottom) interneurons were fluorescently labeled with Smoothened-tdTomato (red) and co-labeled with a panel of eight different antibodies against known interneuron migration cue receptors (white). Arrowheads point to cilia/receptor co-localization. These images depict the range of localization patterns observed for all receptors, none were restricted to a single part of the cilium (B) Fluorescent intensity (FI) line scans through Smo⁺ primary cilia in interneurons indicates the level of co-localization. Overlay of the red (Smo⁺ cilia) and blue (receptor labeling) FI measurement peaks indicates co-localization. The left panel in B illustrates 5-HTR-6 FI peaks in detail; FI peaks for other receptors in controls and mutants are shown at right. (C) Percent of control and mutant interneuronal cilia with receptor co-localization. Data shown are mean ± SEM (n=4). (D) Primary cilia (blue) in wild type and inducibly deleted, Arl13 deficient MEFs were also co-labeled with receptor (white) antibodies. Acetylated tubulin or ACIII antibodies were used to immunolabel cilia in MEFs. Arrowheads point to cilia/receptor co-localization. Arl13b deletion altered the percentage of MEF cilia containing these receptors (E). Data shown are mean ± SEM (n=3). (F) Erk1/2 phosphorylation decreases in Arl13b mutant interneurons. (G) Quantification of the decrease in Erk1/2 phosphorylation. Densitometric measurements of phosho-Erk (pERK) bands were normalized to Erk2 expression. (H) cAMP levels increase in Arl13b^Lox/Lox; Dlx5/6-CIE GE interneurons. Data shown are mean ± SEM; * indicates p<0.01 (Student’s t test). Scale bar = 2.4 µm (A); 1.25 µm (D).

Figure 7. Interneuronal migration guidance cue receptor transport is defective in Arl13b-deficient primary cilia

Wild-type and Arl13b^−/− cells express 5-Htr6-GFP in primary cilia. A subregion of the primary cilium was photobleached (green arrowhead, A-B) and fluorescence recovery over time was monitored. Compared to fluorescence recovery in control cilia (A, arrow), recovery was significantly delayed in mutant cilia (B, arrow). (C) Quantification of the recovery half-time. Data shown are mean ± SEM (n=14 [WT], 21 [−/−]); ** indicates p<0.01 (Student’s t test). Time interval between each panel is 1.125 seconds. Scale bar = 1µm.

Figure 8. Expression of mutated human Arl13b allele disrupts interneuron migration

(A) Pathogenic ARL13B mutations R79Q, W82X, R200C cause Joubert syndrome. (B-E) Panels from timelapse imaging of control (Arl13b^Lox/+; Dlx5/6-CIE; B), Arl13b deficient interneurons (Arl13b^Lox/Lox; Dlx5/6-CIE; C), and Arl13b deficient interneurons expressing either WT (D) or W82X (E) human Arl13b (red). sterisks (B-E) indicate cell soma of migrating interneurons. Time elapsed between panels is in minutes. (F) Reduction in the rate of migration in Arl13b deficient interneurons is rescued by expression of human Arl13b. R79Q, W82X and R200C mutant Arl13b constructs fail to rescue, suggesting that these mutations impair interneuron migration. Data shown are mean ± SEM (n=40 cells for each condition) * indicates p<0.001 compared with control; ** indicates p<0.001 when compared with Arl13b deficient cells (Student’s t test). Scale bar=75µm.