Supplementary Figure S1: Pial and periventricular vascular networks and the dual GABA neuron streams of the early embryonic mouse telencephalon

Transcription

1 Supplementary Figure S1: Pial and periventricular vascular networks and the dual GABA neuron streams of the early embryonic mouse telencephalon a) The profile of GABA neurons arriving at the pallial-subpallial boundary in an E12 telencephalon of GAD65-GFP mouse (25 µm). b) At E13, the two GABA neuron streams extend further into the developing cortex. c) By E13.5-E14, the dual GABA neuron streams can be clearly visualized in the dorsal telencephalon all the way up to the medial edge. a-c) White arrows show the formation of the deep stream of GABA neurons that come to occupy the SVZ region and pink arrows point out the formation of the superficial stream of GABA neurons in the MZ. The arrows also highlight the leading front of the dual migratory streams. d) Low magnification image of vascular profile from an E13 telencephalon (50 µm) shows the arrangement of periventricular vascular network shaped to facilitate GABA neuron migration. Note the curvature of vessels as they near the pallial-subpallial boundary (white arrows) and the periventricular vascular network organization in the dorsal telencephalon. The area depicted by white arrows is highlighted in e and white dotted region is highlighted in f. e) A higher magnification image of the profile of periventricular vessels at the pallialsubpallial boundary that herd the bulk of GABA neurons (white arrows) as a uniform stream into the dorsal telencephalon. f) Periventricular vessel lattice pattern in the dorsal telencephalon (white arrows). The SVZ GABA interneuron stream (white arrows) is "herded" within the periventricular vessel lattice in the dorsal telencephalon. Scale bars: a, 100 µm (applies also to a-f).

2 Supplementary Figure S2: Intact periventricular vessel network formation in the dorsal telencephalon is a pre-requisite for GABA neuron migration from ventral to dorsal telencephalon To test if GABA neurons use the periventricular vessel network as a substrate for migration, we resorted to our transplantation expertise. An E12 dorsal telencephalic explant from a CD1 mouse was transplanted with an E13 ventral telencephalic explant from a GAD65-GFP mouse in transplantation experiment 1 (a). An E10 dorsal explant from a CD1 mouse was kept in culture for 48 hours. We have shown previously (reference 5) that an E10 dorsal explant cultured on its own, without the ventral explant lacks periventricular vessels. This pre-cultured dorsal explant was transplanted with an E13 ventral explant from a GAD65-GFP mouse for the next 24 hours in transplantation experiment 2 (b). The idea was to see if GFP+ve cells from the ventral explant could enter into a dorsal explant that lacked the periventricular vascular network. a) A co-label of isolectin B4 and GAD65/67 antibodies show that in the presence of periventricular vessels in the dorsal explant (white arrows), many GABA neurons have migrated into the dorsal explant after 24 hrs, some of them are in close contact with vessels (blue arrows). b) When the E10 dorsal explant pre-cultured for 48 hrs was transplanted with a ventral explant, few periventricular vessels entered from ventral to dorsal explant (purple arrows) but intact periventricular vessel palisade could not form. And indeed, very few GABA neurons (pink arrows) migrated into the dorsal explant due to lack of presence of a well-formed periventricular vessel network. c) The mean density of GAD65/67 +ve cells in the dorsal explant was significantly decreased in the absence of a pre-formed periventricular vessel network (n=15 transplants, mean ± SD, P , Student s t-test). Cell viability was commendable in the transplants at the end of the experiment. Due to their fragile nature, transplants were always transferred for paraffin wax histology. Therefore GAD65-GFP cells had to be detected with a GFP antibody (Abcam) as the GFP fluorescence from this transgenic mouse was lost during the rigorous paraffin steps.

3 Supplementary Figure S3: No cell death or differences in migration of GABA neurons was observed by TNP-470 treatment. (a-b) None of the cultured GABA neurons were positive for propidium iodide uptake or showed abnormal cell morphology in control cultures (a) or when treated with TNP-470 (b). (c-e) The distribution of migrating GABA cells in areas surrounding GE explants (white arrows) was comparable in both control (c) and TNP-470 (d) treated conditions. Migrated cells in sectors I and II were expressed as a ratio (e). Data represents mean ± SD. Scale bars: a, 100 µm (applies also to b-d)

4 Supplementary Figure S4: CDK5R1 and neuronal development The map is based on the enrichment distribution for CDK5R1 (enriched in periventricular endothelial cells) and its role in neuronal development sorted by 'Statistically significant Maps' set created by the software Metacore ( Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

5 Supplementary Figure S5: SSTR2 regulation of cell proliferation The map is based on the enrichment distribution for somatostatin receptor 2 (enriched in periventricular endothelial cells) and its regulation of cell proliferation sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

6 Supplementary Figure S6: Role of PDGFs in cell migration The map is based on the enrichment distribution for PDGF (enriched in periventricular endothelial cells) and its role in cell migration sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

7 Supplementary Figure S7: CXCR4 signaling in immune response The map is based on the enrichment distribution for CXCR4 (enriched in periventricular endothelial cells) signaling sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

8 Supplementary Figure S8: Endothelial cell contacts by non-junctional mechanisms The map is based on the enrichment distribution for cell adhesion/endothelial cell contacts by non-junctional mechanisms sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

9 Supplementary Figure S9: Chemokines and cell adhesion The map is based on the enrichment distribution for chemokines and cell adhesion molecules sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

10 Supplementary Figure S10: Extracellular matrix (ECM) remodeling The map is based on the enrichment distribution for cell adhesion /extracellular matrix remodeling sorted by 'Statistically significant Maps' set created by the software Metacore. Experimental data from all files is linked to and visualized on the maps as thermometer-like figures. Thermometerbar: 1, Pial endothelial cells and 2, Periventricular endothelial cells. The bar height indicates strength of PLIER signal. Summary of the symbols/keys are provided in Supplementary Figure S11.

11 Supplementary Figure S11: Summary of symbols/keys depicted in Supplementary Figures S4-S10.

12 Transcription factors Channels/Transport Cell Surface Others Dlx1, Dlx2, Nkx2.1, Foxg1, Npas1, Ssbp2, Csdc2, Emx2, Etv1, Nr2e1 Gabrg2, Gabrb3,Gabra1, Gabra5,Gria1, Grip1, Grik1, Kcnq2, Cacnb4, Scn8a, Scn3b Nxph1, Neto1, Cntnap4, Cxcr4,Chl1, Ptprd, Astn1, Cdk5r1 Slco5a1,Cntn2, Fezf2, Klhl1, slc32a1, Dact1, Sstr2, Sez6l2c Supplementary Table S1: A list of some top genes commonly known to be expressed and/or traditionally confined to GABA neurons/ interneurons and their precursors that were upregulated in periventricular endothelial cells.

13 Transcription factors Channels/Transport Cell Surface Others Foxd1, Lhx6, Mafb, Etv4, Nfil3, Dlx6as Gabra2, Gria4 Gprc5b, Tas1r1,V2r1b Slc6a11, Nr2f2, Sema3e Supplementary Table S2: A list of some top genes commonly known to be expressed and/or traditionally confined to GABA neurons/ interneurons and their precursors that were upregulated in pial endothelial cells.