Fig 2: Photographic reconstructions of the PSB in coronal sections at 21 GW. Vimentin (A,C) shows the distribution and orientation of radial glia cells and fibers in the frontal (A) and temporal (C) periventricular zones and the RGF route into the Insula (white arrows). Tbr2 (B,D) marks the PSB (arrow in B), which at this age is less well defined than at earlier fetal stages, and in the temporal lobe does not extend medially beyond the striato-cortical sulcus (asterisks in all panels; in D, the PSB is indicated by a dotted line). Tbr2+ and vimentin+ progenitor cells cross the periventricular fiber layers (Inner fibrous layer, IFL) and extend far into the oSVZ and even the intermediate zone (IZ). Note the complexity of fiber tracts in the temporal lobe, due to the presence of the anterior commissure (AC). The large black dots in the inner (i) SVZ in D are stained blood vessels. Bars: in A: 240 µm, in B: 140 µm, in C: 250 µm, in D: 190 µm.

Fig 3: The RGF on its route to the insula. (A,D,E) Development of the RGF, which is co-extensive with the external capsule (EC). (A) At 11GW, the RGF arises from the PSB, crosses the IC and courses lateral to the putamen (P) (red arrows). PCNA (black) is expressed in the ventricular zone (VZ) and SVZ, but not in the RGF (brown). (B) At 11 GW, the Tbr2+ SVZ at the PSB (green arrow) extends into the IC. The dashed line indicates the outer SVZ (oSVZ) at the PSB. (C) Tbr1+ neurons (black) course along the RGF into the insula. Calretinin+ neurons (yellow) do not form part of this migration. (D) At 12 GW, the sylvian fossa indicates the position of the insula. Two-color staining (PCNA in black, vimentin in brown) shows that the RGF is not a proliferating zone. (E) At 15 GW, the origin of the RGF can be traced back to the intersection of IC and EC (red dotted lines), while the oSVZ has increased in width. (F) At 15 GW, an inner (i) and outer (o) SVZ is particularly wide at the PSB (green arrow). G: 14 GW, Tbr1. At this age, of incipient opercularization, Tbr1 is still expressed by cells in all cortical compartments, and clearly visualizes the separation of pallial and subpallial regions. FC, frontal cortex, TC, temporal cortex. Bars: In A: 270 µm; in B: 160 µm; in C: 50 µm; in D: 700 µm; in E: 350 µm; in F: 150 µm; in G: 450 µm.

Fig 4: Kif26a Regulates Neuronal Radial Migration, Terminal Localization, Morphology and Corpus Callosum Development.(A) Representative images of in utero electroporation (IUE) of control scrambled shRNA (left) and Kif26a shRNA (right) in mouse cortex at E13.5 and analyzed at E17.5 (E13.5-17.5, 4 days post electroporation). Bottom magnified view show electroporated cells are SATB2+ neurons. Scale bars = 100µm.(B) Quantification of the percentage of mCherry-labeled cells expressing neuronal marker SATB2 or IPC marker TBR2. Values represent Mean ± S.D. (n = 5 brains for scrambled, n = 4 for shRNA1, n=6 for shRNA2, n = 5 for shRNA3. Student’s t-test: N.S., not significant).(C) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-17.5. The cortex was evenly divided into 10 bins from basal (bin 1) to apical (bin 10) surfaces and the cell distribution was normalized by the total number of electroporated cells in the analyzed area. Only mCherry+, SATB2+ neurons are quantified. Values represent Mean ± S.D. (n = 7 brains for scrambled, n = 6 for shRNA1 and shRNA2, n = 5 for shRNA3).(D) Quantification of the percentage of labeled neurons displaying bipolar or multipolar morphology, showing loss of bipolar morphology with Kif26a deficient neurons. Values represent Mean ± S.D. (Same samples as (C). Student’s t-test: **, p < 0.005; ***, p < 0.0005).(E) Representative images of IUE in mouse cortex at E13.5 and analyzed at E15.5 (E13.5-15.5, 2 days post electroporation). Scale bar = 100µm.(F) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-15.5. Similar to (C). Only mCherry+, SATB2+ neurons are quantified. Values represent Mean ± S.D. (n = 7 brains for scrambled, n = 4 for shRNA KD. Student’s t-test: ***, p < 0.0005).(G) Representative images of IUE in mouse cortex at E13.5 and analyzed at P5 (E13.5-P5, 10 days post electroporation). Scale bar = 100µm.(H) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-P5. Similar to (C). Only mCherry+, SATB2+ neurons are quantified. Values represent Mean ± S.D. (n = 10 brains).(I) Representative magnified images showing morphology of electroporated neurons at P5. Scale bar = 100µm.(J) Quantification of the percentage of electroporated neurons exhibiting pyramidal morphology at P5. Pyramidal morphology is defined as neurons having 1 basal dendrite and at least 2 apical dendrites from the soma. Values represent Mean ± S.D. (n = 10 brains. Student’s t-test: ***, p < 0.0005).(K) Quantification of the percentage of P5 brains with electroporated axons crossing the corpus callosum (CC). All (10 out of 10) scrambled shRNA brains showed CC crossing, while none (0 out of 10) of Kif26a KD brains showed CC crossing.(L) Representative images showing electroporated neurons send axons across the corpus callosum to the contralateral hemisphere only in scrambled control (top), but not in Kif26a KD (bottom). Insets show magnified view of selected areas. Scale bar = 500µm.See also Figures S3 and S4.

Fig 5: NOVA1 expression is reduced in response to rubella virus (RV) exposure.(A) Differentially expressed genes in different cell types in response to RV treatment without (top panel) and with microglia (bottom panel). IPCs – intermediate progenitor cells. In the presence of microglia, fewer differentially expressed genes in response to RV treatment were identified across all major cell types. In organoids with microglia, NOVA1 trended toward a decrease in IPCs and neurons (labeled in blue in the panel). Kolmogorov-Smirnov test was used on DEGs with p-value <0.05. ***<0.001, NS – not significant, *<0.05. (B) Violin plot for NOVA1 that is differentially expressed in response to RV and presence of microglia. IPCs – intermediate progenitor cells, RG – radial glia, Div. – dividing cells, EN – excitatory neurons. (C) Representative images of RV-exposed organoids with microglia at 2 weeks post-exposure, stained with DAPI for cell nuclei (blue), NOVA1 (magenta), NeuN for neurons (green), and EOMES for intermediate progenitor cells (cyan). (D) Cell number quantification for NeuN+ neurons that were also positive for NOVA1 in control (heat-inactivated RV) or RV condition. Averages of 3-5 sections (technical replicates) across 3 organoids (biological replicates, individual data points) where used for quantification in D-G. Unpaired parametric Student’s t-test was used to compare the two groups in D–G. p-value = 0.088. (E) Cell number quantification for EOMES+ intermediate progenitors that were also positive for NOVA1 in control (heat-inactivated RV) or RV condition. p-value = 0.0042. (F) Cell number quantification for NeuN+ neurons per organoid area displayed in 1000 cells × mm2 or organoid surface area in control (heat-inactivated RV) or RV condition. p-value = 0.0004. (G) Cell number quantification for EOMES+ intermediate progenitor cells per organoid area displayed in 1000 cells × mm2 or organoid surface area in control (heat-inactivated RV) or RV condition. p-value = 0.86.