Supplementary Figure 1 Kif1a RNAi effect on basal progenitor differentiation Related to Figure 2. Representative confocal images of the VZ and SVZ of rat cortices transfected at E16 with scrambled or Kif1a shrna, and immunostained 4 days later for intermediate progenitor marker, Tbr2 (n=5 for scramble and Kif1a shrna). The percentage of Tbr2+/GFP transfected cells was significantly reduced in Kif1a knockdown brains compared to control. SVZ: subventricular zone; VZ: ventricular zone. Scale bars 15µm.
Supplementary Figure 2 Common effects of different Kif1a vectors Related to Figure 4. A and B. Representative coronal sections of E20 rat brains injected at E16 with two different control and shrna constructs. Scramble and Kif1a-shRNA was cloned into the prnat- U6.1/Neo-GFP (A), or into the mu6pro vector and co-injected with pcag-rfp (B). Kif1a shrnas in either vector resulted in a similar accumulation of multipolar neurons in the SVZ / lower IZ. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone. Scale bars 100 m.
Supplementary Figure 3 Non-cell-autonomous effect of RNAi for Dcx but not nuclear envelope dynein recruitment factors
Related to Figure 3. E16 rat embryonic brains were subjected to in utero electroporation with shrnas for BicD2, Nup133, Cenp-F, Dcx, or TrkB. Brain slices were stained at E20 for anti-tbr1. Expression of BicD2, Nup133, Cenp-F, Dcx, and Trkb shrnas resulted in a marked reduction of electroporated cells in the IZ and the CP, with many cell bodies in the SVZ retaining a multipolar morphology. Non-transfected cells expressing Tbr1 were still distributed normally within the upper IZ/lower CP for BicD2, Nup133, Cenp-F, and TrkB shrnas. However, DCX shrna resulted in Tbr1 expression in non-transfected cells within the SVZ/lower IZ, suggesting a non-cell autonomous effect. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone. Scale bar 50 m.
Supplementary Figure 4 Limited overlap of sequentially versus cotransfected cells Related to Figure 5. A and B. Representative coronal sections of E20 rat brains at E16 co-injected (A) or sequentially injected (B) with RFP and GFP plasmids. (A) Co-transfection with plasmids encoding RFP and GFP resulted in the majority of transfected cells to be double-labeled (yellow) while sequential transfection (B) labels largely non-overlapping cell populations. CP: cortical plate; IZ: intermediate zone. Scale bars 100 m.
Supplementary Figure 5 Kif1a RNAi does not affect radial glial cell morphology and organization
Representative coronal sections of E20 rat brains transfected at E16 with scrambled or Kif1a shrna, and immunostained 4 days later for the intermediate filament marker, vimentine (A); or transfected at E16 with BLBP-GFP alone or in combination with Kif1a shrna (B). No disruption in the organization of radial glial fibers was observed in brains subjected to Kif1a RNAi, with both basal and apical processes resembling those observed in control. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone. Scale bars 100 m.
Supplementary Figure 6 BDNF does not rescue INM but rescues non-cell-autonomous neuronal effect Related to Figure 8. E19 coronal rat brain slices electroporated at E16 with Dcx shrna (A), NeuroDcre + Kif1a shrna (B), the human mutation R18W (C), or Kif1a shrna (D), and cultured for 24 hours in the presence of recombinant BDNF (50 ng/ml) in PBS, or control vehicle alone, and then fixed and examined by microscopy. A-C. BDNF treatment rescued the non-cell autonomous effect caused by Dcx shrna (A), conditional knockdown of Kif1a specifically in neurons (NeuroD-cre + Kif1a-shRNA; B), and the human mutation R18W (C), as evidenced by restoration of migration in non-transfected cells and normal Tbr1 distribution. D. BDNF treatment does not rescue the INM defect caused by Kif1a shrna. E. Quantification of the distance of RGP nuclei from the ventricular surface (VS) at E19 ((0-10: Kif1A shrna+pbs 53.4 ± 5.5%, Kif1A shrna+bdnf 50.06 ± 3.7%, p=0.4857; 10-20: Kif1A shrna+pbs 19.66 ± 3.3%, Kif1A shrna+bdnf 24.59 ± 1.04%, p=0.286; 20-30: Kif1A shrna+pbs 14.72 ± 1%, Kif1A shrna+bdnf 15.38 ± 2.7%, p=0.3429; >30: Kif1A shrna+pbs 12.21 ± 3.5%, Kif1A shrna+bdnf 9.97 ± 1.7%, p=0.3429; n=4 for scramble and Kif1a shrna), revealing that RGP nuclei still remain accumulated at the VS after BDNF treatment. Scale bars 15 m (A) 100 m (C, D and E).
Supplementary Figure 7 Bdnf KD does not affect INM at E20 but affects neuronal migration at P7 Related to Figure 8. Coronal rat brain slices electroporated at E16 with Bdnf shrna and analyzed at
E20 (A), or P7 (C). A and B. E20 brain sections stained with RGP cell marker, Pax6 (A) and neuronal marker, TuJ1. The high magnification panels show examples of Pax6 and Tuj1 positive cells (arrows). There is no significant difference between the percentage of RGP cells or neurons electroporated with control or Bdnf shrna, suggesting that Bdnf shrna does not affect the ratio of symmetric vs asymmetric divisons. C. Immunostaining for the neuronal marker, NeuN and the upper cortical layer marker CDP, at P7 reveal a distribution of the transfected cells through the white matter and the 6 layers after Bdnf RNAi, but no non-cell autonomous effect. D. Diagrammatic representation of normal and KIF1A/BDNF-altered neurogenesis and migration. Upper panel: temporal progression of control (white) RGP cell behavior, showing cell cycle-dependent nuclear oscillations (INM), followed by symmetric or asymmetric mitotic division and, in the latter case, migration to the multipolar stage in the SVZ/lower IZ and ultimately, to differentiating bipolar neurons in the CP, where Tbr1 (green) is normally expressed. Lower panel: Kif1a knockdown RGP cells (blue) progress through cell cycle without nuclear oscillations and exhibit far fewer asymmetric mitotic divisions. Postmitotic neurons arrest at the multipolar stage in the SVZ/lower IZ, but prematurely express Tbr1. Surrounding control cells (white) also exhibit migration arrest and ectopic Tbr1 expression, an effect phenocopied by Bdnf knockdown and strikingly rescued by BDNF addition. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone; WM: white matter. Scale bars 100 m (A, C and E), 15 m (inset).