Kif7 deletion alters cortical anatomy at E14.5 but preserves the dorso-ventral patterning of the telencephalon.

(A) Kif7 -/- embryos are microphtalmic (black arrow) and exhibit skin laxity (white arrow). (B) External examination of the brain reveals the thinning of the dorsal telencephalon (black arrow, left panel) and the lack of olfactory bulbs (white arrows, right panel). (C) DAPI staining of rostro-caudal series of coronal sections (C1) illustrates the anatomical defects of Kif7 -/- embryonic brains that are quantified in C2-C5. The ventricles of Kif7 -/- embryos are strongly enlarged (C2), their cortical thickness strongly decreased (C3), resulting in minimal brain width (C4) and height (C5) changes. Statistical significance was tested by Two-way ANOVA or mixed model (GraphPad 8.1.0). In C2 (WT, n=4-6, Kif7 -/-; n=5-6 depending on the rostro-caudal level), C3 (WT, n=3; Kif7 -/-, n= 4), mix model reveals a genotype effect (p<0.0001). No genotype effect was observed in C4 (WT, n=5-6; n=5-6 for Kif7 -/- depending on the rostro-caudal level), C5 (WT, n=5-6; Kif7 -/-, n=4-6 depending on the rostro-caudal level), but a significant effect of the rostro-caudal level on brain width (C4, P=0.0441) and height (C5, P=0.092). D. The pallium-subpallium boundary (PSB) identified by the limit of expression of ventral (GSH2, left panels) and dorsal (TBR2, right panels) telencephalic markers remains well-defined in Kif7 -/- embryos. Graphs in C2-C5 represent the means and S.E.M. PSB, pallium-subpallium boundary; CX, cortex; LGE, lateral ganglionic eminence; MGE, median ganglionic eminence; CGE, lateral ganglionic eminence; Th, thalamus. Scale bars, 250 µm.

Clinical diagnosis of patients carrying mutation in the KIF7 gene on both alleles from the literature.

The ablated or mutated domains were identified. Clinical features associated with cortical dysfunction are listed. Among cerebral defects, those observed in the cortex are enlighted. DD, developmental delay; ID, intellectual deficit; CC, corpus callosum; MTS, molar tooth sign.

Histological alterations in the developing cortex of E14.5 Kif7 -/- embryos.

Coronal sections representative of E14.5 wild type (WT) and E14.5 Kif7 -/- embryos imaged on confocal (A) or epifluorescence (B1) microscopes, and on a macroscope (B2). (A) The TBR1(+) staining (red) of the cortical plate is more clustered in Kif7 -/- than in WT embryos, and the MAP2(+) staining (green) of the subplate is absent in the dorsal cortex of Kif7 -/- embryos (white arrow, right column). (B) The TBR2(+) layer (green) of secondary progenitors appears disorganized in the lateral cortex of the Kif7 -/- embryos (white arrowhead in B1). In the dorsal cortex of Kif7 -/- embryos (white arows in B1, B2), the TBR2(+) cells (green) form a disorganized layer that reaches the brain surface (B1) where it intermingles with TBR1(+) cells (B2, red cells). V, ventricle; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; MZ, marginal zone; CP, cortical plate. Scale bars: 100 µm (A), 200 µm (B1, B2).

Kif7 deletion disrupts of the connectivity between the cortex and the thalamus at E14.5.

(A) Panels illustrate the corticofugal projection labeled by DiI crystal (red dots) positioned in the dorsal (A1) and lateral (A2,A3) cortex of wild type (WT, left) and Kif7 -/- (right) embryos on vibratome sections performed 30 days after DiI placement. In Kif7 -/- embryos, less axons project from the dorsal (A1) and lateral (A2) cortex to the subpallium than in WT. Compare enlarged views of the projections below A1 and A2. Cortical injections in Kif7-/- embryos do not label thalamic axons (compare left and right panels in A3) but label a ventral projection (A3, white arrow). (B) Rostro-caudal series of coronal sections immunostained with anti-Netrin G1a (NG1a) antibodies compare the trajectory of thalamo-cortical axons (TCA) in a WT (left) and in a Kif7 -/- (right) embryo. Thalamo-cortical axons reach the pallium-subpallium boundary (PSB) of the WT embryo whereas they are lost in the ventral forebrain of the mutant (right panel, white arrow). C. Representative three-dimensional reconstruction of WT and Kif7 -/- brains immunostained as a whole with NG1a (red) and TBR1 (green) antibodies before transparization and imaging with a light sheet microscope. Compared to TCA in the WT that form a bundle extending in the internal capsule (IC), TCA in the Kif7 -/- brain organize two short bundles, one directed to the IC that stops soon after entering the IC, and the other one oriented caudally and ventrally. PSB, pallium-subpallium boundary; CX, cortex; LGE, lateral ganglionic eminence; MGE, median ganglionic eminence; Th, thalamus; Hy, hypothalamus. IC, internal capsule. Scale bars: 250 µm.

Altered Cxcl12 transcript expression and abnormal cortical distribution of cIN at E14.5 in Kif7 -/- brains.

(A) Panels compare the distribution of Cxcl12 mRNA in wild type (WT, left panel) and Kif7 -/- (right panel) forebrain coronal sections at E14.5. The WT section shows Cxcl12 transcript enrichment in a deep cortical layer already identified as the SVZ. In the Kif7 -/- cortical section, the expression of Cxcl12 transcripts is reduced to the lateral part of the SVZ. (B) The cortical distribution of cIN is visualized in WT and Kif7 -/- mouse embryos using crosses with the Nkx2.1-Cre/R26R-Tomato strain that expresses the fluorescent marker Tomato in MGE-derived cIN. Panels in B1 compare the distribution of Tomato (+) cIN in WT and Kif7 -/- cortical sections prepared at the same rostro-caudal level and in which SVZ is immunostained with TBR2 antibodies (green). Pictures show that the deep migratory stream of cIN terminates in Kif7 -/- brains in the cortical region where the TBR2(+) layer reaches the cortical surface. Quantitative analysis of the mean length of the deep and superficial migratory streams measured from the entry in the pallium to the last detected cIN in the cortex is illustrated in graph B2. Statistical significance is assessed using Two way ANOVA [layers (WT, n=4; Kif7 -/-, n=3; ***, P=0.001) and genotype (*, P=0.0157)]. (C) Representative pictures (C1) of the deep and superfical tangential migratory streams of cIN in the lateral cortex of WT and Kif7 -/- embryos. Pictures illustrate the decreased thickness of the superficial stream, and the reduced distance between the deep-superficial streams in Kif7 -/- embryos. Graph in C2 (WT, n=4; Kif7 -/-, n=4) compares the distribution of the fluorescence intensity along a ventricle/MZ axis (see grey rectangles in C1) using the plot profile function of FIJI. Curves show no change in the distance between the ventricular wall and the deep cIN and a reduction of the distance between the two streams in the Kif7 -/- cortical sections as quantified on the graph [WT, n=4; Kif7 -/-, n=4; Two way ANOVA reveals a significant interaction between genotype and layer (P=0.0233) and multiple comparisons, a statistical difference between genotype only for the distance between de cIN streams (**, P=0.0051)]. Scale bars: 200 µm.

Dynamic behavior of migrating cIN in organotypic cortical slices: comparison between Kif7 -/- slices and control slices with an acute treatment to either activate or block the SHH pathway.

(A) Tomato(+) cIN migrating in living cortical slices were tracked manually using the MTrackJ plugin and their trajectories color-coded as shown in legend to characterize their preferred direction (tangential, oblique, radial, immobile) and cortical layer localization (VZ-SVZ, IZ, CP). The picture in A1 illustrates the z-projection of trajectories reconstructed in a control slice, superimposed to the last picture of the movie. Cortical interneurons migrating tangentially in the superficial migratory stream (MZ) could not be tracked because of high density. A2. Graphs compare the percentage of each kind of trajectory recorded in the lateral cortex of control slices (control, see also Suppl. Movie S3), Kif7 -/- slices (Kif7 -/-, see also Suppl. Movie S4), control slices treated acutely with either murine SHH (SHH, see also Suppl. Movie S5) or cyclopamine (Cyclo, see also Suppl. Movie S6). Significance of the differences between the four distributions were assessed by a Chi-square test, X2 (24, n=1224), P=0.0004998, ***). All experimental conditions differed from the control (Fisher test, p =0.0004998, ***). Slices from three WT animals in control condition or treated with drugs and from three Kif7 -/- animals were analyzed; the number of analyzed cells is indicated above bars. A3. Schemes summarize the main results observed in each experimental condition. Trajectories are represented with the same color code as in A1, and line thickness is proportional to the percentage of cells exhibiting each type of trajectory. Immobile cells are figured by a coil. (B) Box and whisker plots indicate the mean speed (B1) and the frequency (B2) and duration (B3) of resting phases for cIN migrating tangentially in the deep stream (red box and whisker plots, left) or to the cortical plate (green box and whisker plots, right). Statistical significance assessed by Krustkal-Wallis tests in each cluster. ****, P<0.0001; ***, B2 left P=0.0005; **, B1 right P=0.0088, B2 right, P=0.0037, B3 left P=0.0034; *, B1 left P=0.0233, B2 right P=0.0470, B3 left P=0.0134, B3 right P=0.0394. Number of analyzed cells is indicated on plots. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. Scale bar: 300 µm.

Expression of Shh transcripts (A-C) and distribution of SHH protein (D) in the developing forebrain.

(A,B) Distribution in median (A) and caudal (B) coronal sections of Shh mRNA detected by in situ hybridization with an antisens Shh probe at E13.5 (A1,B1) and by RNAscope at E14.5 (A2-3,B2). Shh transcripts are strongly expressed in the medial ventral forebrain (SVZ and mantle zone of the MGE and septum, A1, A2), in the mantle zone of the CGE (B1,B2), in the zona limitans intrathalamica (ZLI in B1) and in the ventral midline of the 3rd ventricle (V3 in B1). RNAscope further confirmed the strong expression of Shh mRNA (A2, green) in MGE and septum regions that strongly express Lhx-6 mRNA (A2, red). Confocal observations in the SVZ and mantle zone of the MGE showed that Shh mRNA (green) is co-expressed with the Lhx6 mRNA (red) in a significant number of cIN (yellow cells in A3). (C) Confocal analyses at higher magnification of the double detection by RNAscope of Shh and Lhx-6 mRNA. Cells were identified on stacked images (Δz=1 µm) using Nomarski optic. In the lateral cortex close to the PSB (C1, z projection of 10 confocal planes) and in the dorsal cortex (C2, z projection of 10 confocal planes), a very small proportion of cells expressing Lhx-6 mRNA also express Shh mRNA (white arrows in C1,C2). Counting in the deep stream (SVZ-IZ) and in the MZ is shown in graph C3 (9-17 fields in three sections). A few progenitors in the cortical VZ express Shh mRNA at very low level (arrowhead, C1). (D) E14.5 forebrain coronal sections immunostained with antibodies directed against the N-ter domain of the activated SHH protein are imaged by epifluorescence (D1) and confocal (D2) microscopy. At low magnification (D1), SHH-Nter is slightly enriched in the ventral midline (black arrow) and at the ventricular angle (black arrowhead) near the PSB. Confocal analysis of SHH-Nter immunostaining (D2) shows immuno-detection in blood vessels and the presence of numerous bright dots all over the cortical neuropile. In the cortical neuropile, small bright dots align radially in the VZ (enlarged in D3), tangentially in the SVZ-IZ, and radially in the CP. On the ventricular side of the PSB and of the lateral-most part of the LGE (D3), larger SHH-Nter(+) brigth elements are aligned radially. Confocal images are a merged stacks of 10 images distant of 0.2 µm. CX, cortex; LGE, MGE and CGE, lateral, medial and caudal ganglionic eminence; V3, third ventricle; ZLI, zona intra-thalamica; Th, thalamus; Hyp, hypothalamus; PSB, pallium-subpallium boundary; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. Scale bars: 500 µm (A, B, D1), 20 µm (C), 250 µm (D1), 50 µm (D2).

KIF7 deletion affects the cortex development at E16.5.

(A) Coronal sections at E16.5 of transgenic Nkx2.1-Cre/R26R-Tomato control (left panels) and Kif7 -/- (right panels) brains show that the hippocampus does not form properly in Kif7 -/- animals and the cerebral cortex remains thinner. The migration of Tomato(+) cIN to the dorso-medial cortex had progressed between E14.5 and E16.5, but remains delayed with regard to E16.5 control brains. (B) Coronal view of whole-mount imaging of control and Kif7 -/- brains immunostained with NG1a (red) and TBR1 (green) antibodies shows that thalamo-cortical axons extended to the cortex in both control (upper panel) and Kif7 -/- (bottom panel) brains. An abnormal projection of thalamic axons to the ventral telencephalon is still present in Kif7 -/- animals is (white arrow in B). (C) Immunostaining of thalamo-cortical axons on coronal brain sections reveals hampered colonization of the deep cortical layers above TBR2(+) layer (green) by thalamic axons (NG1a in white) in the Kif7 -/- embryos. Scale bars: 100 µm.

Western blot analysis was performed on the cortex and MGE of wild type (WT) and Kif7 -/- embryos at E14.5. In Kif7 -/- cortex, the clived form of GLI3 (GLI3-R at 83 KDa) was reduced as compared with control cortex whereas on the contrary, the full length GLI3 (GLI-FL at 190 KDa) was increased. The levels of GLI-FL and GLI3-R were strongly lower in the MGE compared to the cortex in WT and Kif7 -/-. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (bottom blots).

Kif7 deletion is associated with cortical heterotopia at E14.5.

(A) On coronal sections of the telencephalon of wild type (WT) animals, TBR2(+) cells form a well-defined layer restricted to the cerebral cortex. Most TBR2(+) cells are densely packed in the SVZ, whereas some TBR2(+) cells are dispersed in the ventricular zone. (B) In about 20% of Kif7 -/- embryos, heterotopia identified by a disorganization of the TBR2(+) layer were observed in various regions of the cerebral cortex, either dorsal (B1) or lateral (B2,B3). Depending on heterotopia, TBR2(+) were either displaced toward the brain surface (B2) or toward the ventricle (B3). Scale bar: 200 µm.

Alterations of the thalamo-cortical projection at E14.5 in Kif7 -/- brains.

(A) Immunostaining of coronal brain sections at a caudal level with NG1a (red) and PAX6 (green) antibodies shows that TCA mistargeting in the ventral telencephalon is not related to abnormal PAX6 expression in the zona incerta between the dorsal and ventral thalamus. (B) Coronal view of whole-mount imaging of transparised brains immunostained with NG1a (red) and TBR1 (green) antibodies that label respectively the thalamo-cortical projection and cortical plate cells. While all labeled thalamo-cortical axons (TCA) extend in the internal capsule (IC) and a significant proportion of them enter the cerebral cortex in the wild type brain (WT), TCA in the Kif7 -/- brain split in two bundles in the basal telencephalon. A bundle stops shortly after entering the IC (white arrow head) whereas the second bundle extends ventrally (white arrow). Scale bar: 200 µm.

SHH-Nter immunostaining of brain coronal sections at E12.5.

Representative pictures of SHH-Nter immunostaining imaged with a macroscope. High signal is observed in the zona limitans intrathalamica (left, arrow) and along the third ventricle (right, arrow). Scale bar: 200 µm.

Kif7 deletion affects the cortex development at P0.

A. Tomato (+) cIN are abnormally distributed in the Kif7 -/- cortex: their density is strongly decreased in the infragranular cortical layers that express CTIP(+) principal neurons (green) and whose thickness is drastically reduced, presumably accounting for cortical thinning. B. Thalamo-cortical axons (TCA) labeled with NG1a antibodies (white) are able to reach the dorsal cortex in the Kif7 -/- brain. They nevertheless appear bundled and unable to enter and colonize the cortical plate, as compared with TCA distribution in the littermate wild type brain (WT). Scale bar: 200 µm.