BIO20122451 Supplementary Material

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• Supplementary Figure S1 -

  Fig. S1. In utero electroporation to label and analyze CA1 pyramidal neurons in mouse hippocampus and suppression of Vps35 expression in HEK293 cells and in CA1 neurons expressing miR-Vps35. (A) Schematic illustration of in utero electroporation. Plasmids were in utero electroporated into E15.5 progenitor cells located in the ventricular zone. The electroporated progenitor cells then differentiate to neurons and migrate to pyramidal cell layer (PCL) in CA1 region postnataly. GFP labeled CA1 neurons send out axons to the contralateal hippocampus, passing through (HCC) where they form a dense axonal bundle, similar to corpus callosum (CC) axons from medial cortical (MC) neurons. Neurons and their dendrites in the hippocampal CA1 region and their axons in hippocampal commissure (HCC) region were examined in P10, P14, or P25 postnatal stages. (B) A representative GFP (counter stained with Topro3) image showing that electroporated neurons were located in PCL of hippocampal CA1 region and medial cortex (MC) at P10. (C) An enlarged image showing dendritic layering of GFP labeled CA1 neurons according to nucleus staining (topro-3, shown in blue): basal dendrites in the Stratum oriens (OR), cell bodies in the pyramidal cell layer (PCL), proximal dendrites in the Stratum radiatum (RA) and distal dendritic tufts in the stratum lacunosum-moleculare (L-Mol). Arrowheads demarcate pia surface: the boundary of CA1 and dentate gyrus (DG). (D) Western blot analysis showing reduced Vps35 expression in HEK293 cells co-transfected Flag-Vps35 with the indicated miR-Vps35 plasmids. (E) Immunostaining analysis with anti-VPS35 antibody showing a decreased Vps35 expression in CA1 neurons expressing miR-VPS35-1. Note that the VPS35 immunosignal (shown as red punctae) in the control neuron (green) was strong, but it was undetectable in the miR-VPS35-1 expressing neuron (green). Scale bar: 10 µm.

• Supplementary Figure S2 -

  Fig. S2. Suppression of Vps35 expression affects early development of CA1 neurons. (A) Electroporation of miR-Vps35-1 (E15.5) caused shorter apical dendrites of CA1 neurons at P7 as compared to control apical dendrites. Scale bar: 100 µm. (B) Quantification of apical dendrite growth by measuring normalized length of apical dendrites at P7. The dendrites of miR-VPS35-1 expressing neurons were significantly shorter than control dendrites (*P<0.01). (C) Quantification of neuronal migration at P10 showing that miR-Vps35-1 expression caused slightly but significant increase (*P<0.01) in the percentage of mislocalized neurons in hippocampus, however, no significant migration defect was observed in miR-Vps35-3 transfected neurons.

• Supplementary Figure S3 -

  Fig. S3. Suppression of BACE1 expression by its miRNA does not affect miR-Vps35's knock-down efficiency. (A) Western blot analysis of lysates of HEK293 cells co-transfected miR-BACE1 and BACE1-mCherry, which demonstrates the knock-down efficiency of miR-BACE1-1. (B) Immunostaining analysis with anti-VPS35 antibody showing a decreased Vps35 expression in CA1 neurons expressing miR-VPS35-1+Control as well as expressing miR-Vps35-1+miR-BACE1. Scale bar: 10 µm.