Supporting Information Kanatani et al. 10.1073/pnas.1420701112 horizontal horizontal COUP-TFII Nkx2.1 COUP-TFII Nkx2.1 CGE LGE MGE MGE POa MGE AEP COUP-TFII/Lhx6 Coronal A B C MGE AEP E13.5 Horizontal section (ventral level) MGE-AEP border zoom E13.5 Horizontal section (dorsal level) E13.5 Coronal section (including POa) MGE Zoom POa Zoom MGE-LGE border zoom Fig. S1. MGE and CGE markers are coexpressed in the POa, but not in the GE. (A and B) Immunohistochemical staining for COUP-TFII (green) and Nkx2.1 (magenta) in an E13.5 horizontal section at the ventral (A) and dorsal level (B). Higher magnification views of the caudal region of the MGE in the boxed areas show that COUP-TFII and Nkx2.1 expression is mutually segregated in the GE at both the ventral and dorsal level. (C) Immunohistochemical staining for COUP- TFII (green) and Lhx6 (magenta) in an E13.5 coronal section, including the POa. Whole section image and higher magnification views of the boxed areas are shown. Lhx6-expressing cells in the MGE do not express COUP-TFII (arrows in MGE zoom). Arrowhead shows the border of the MGE region and Lhx6-expressing cells at the more ventral level expressing COUP-TFII. Multiple images were tiled and combined to show the whole brain slice. [Scale bars, (A–C) 500 μm.] Kanatani et al. www.pnas.org/cgi/content/short/1420701112 1 of 7
Transcript
Supporting InformationKanatani et al. 10.1073/pnas.1420701112
horizontal
horizontal
COUP-TFII Nkx2.1
COUP-TFII Nkx2.1
CGE
LGE
MGE
MGE
POa
MGE
AEP
COUP-TFII/Lhx6Coronal
A
B
C
MGE
AEP
E13.5 Horizontal section (ventral level)
MGE-AEP border zoom
E13.5 Horizontal section (dorsal level)
E13.5 Coronal section (including POa)
MGE Zoom
POa Zoom
MGE-LGE border zoom
Fig. S1. MGE and CGE markers are coexpressed in the POa, but not in the GE. (A and B) Immunohistochemical staining for COUP-TFII (green) and Nkx2.1(magenta) in an E13.5 horizontal section at the ventral (A) and dorsal level (B). Higher magnification views of the caudal region of the MGE in the boxed areasshow that COUP-TFII and Nkx2.1 expression is mutually segregated in the GE at both the ventral and dorsal level. (C) Immunohistochemical staining for COUP-TFII (green) and Lhx6 (magenta) in an E13.5 coronal section, including the POa. Whole section image and higher magnification views of the boxed areas areshown. Lhx6-expressing cells in the MGE do not express COUP-TFII (arrows in MGE zoom). Arrowhead shows the border of the MGE region and Lhx6-expressingcells at the more ventral level expressing COUP-TFII. Multiple images were tiled and combined to show the whole brain slice. [Scale bars, (A–C) 500 μm.]
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Fig. S2. Whole mount identification of the expression patterns of regional markers in the POa. Whole mount in situ hybridization of the regional markersShh, Dbx1, and Er81 at E13.5 with higher magnification views in the POa (Bottom). LGE, lateral ganglionic eminence; POH, the preoptic–hypothalamic borderregion; Sep, septum; SPV, supraoptic paraventricular region; TH, thalamus. The image of each coronal section was prepared by tiling and combining multipleimages to show the whole brain slice. (Scale bars, 500 μm.)
POa1
POa2
POa1
POa2
Sep
SPV
Who
lehe
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pher
eZo
om
THMGE
LGEMGE
CGE
POa POa1
MGECGE
AEP
AEP
87.7±3.3%
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POa1
COUP-TFII & Lhx6 / tdTomato
COUP-TFII / tdTomato
Lhx6 / tdTomatotdTomato single
1.6±1.2%3.0±3.0%
7.7±1.6%1
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ventral
dorsalPOa1
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0 h 48 h
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Focal electroporation into the POa1 at E13.5
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Fig. S3. The migratory profiles of POa1-derived cells differ from those of POa2-derived cells. (A) Focal electroporation was performed into the POa1 of anE13.5 brain hemisphere and cultured for 48 h. Micrographs of the telencephalic hemisphere and of the site of electroporation, indicated by FITC-oligo DNA, areshown. POa1-derived cells tend to migrate caudally into the GE, but their migratory profile is more scattered than that of POa2-derived cells. (B) The distri-bution of POa1 cells. POa1 cells from five brains and 179 cells were dotted in the four sectors. The percentages shown in each sector are mean ± SEM.(C) Schematic diagram of the migratory profile of cells from the POa1. (D) Total proportions of cells expressing COUP-TFII and/or Lhx6. The percentage of cellsexpressing COUP-TFII is lower in the POa1 than in the POa2. [Scale bars, (A) 250 μm.]
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Fig. S4. COUP-TFII overexpression does not affect the neuronal fate of the POa-derived cells. Shown is the immunohistochemical staining for DCX, GFAP, andPDGFRα in the E15.5 caudal cortex to label immature neurons, astroglia, and oligodendrocyte precursors, respectively. The POa-derived cells were labeled withtdTomato at E11.5. The expression levels of these markers are similar in control samples and in samples overexpressing COUP-TFII. (Scale bars, 50 μm.)
COUP-TFII
ANeg
ative
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shRNA
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sistan
t COUP-T
FII
+ shR
NA
GAPDH
GATTGTTCTCAACTTCAACttcaagagaGTTGAAGTTGAGAACAATC
GTCCCAGTGTGCTTTGGAAttcaagagaTTCCAAAGCACACTGGGAC
CACTACCGTTGTATAGGTGttcaagagaCACCTATAACAACGGTAGTshRNA control
COUP-TFII knockdown
Nrp2 knockdown
shRNA name Sequence
C
Negati
ve co
ntrol
Nrp2 Nrp2+ s
hRNA
shRNA-re
sistan
t Nrp2
+ shR
NA
Nrp2
GAPDH
B
gatAgtActGaaTttTaac
gtcGcaAtgCgcAttAgaashRNA-resistant COUP-TFII
shRNA-resistant Nrp2
Construct name Sequence
D
Fig. S5. Characterization of shRNA knockdown efficiency in Neuro2a cells. (A and B) Western blot analyses of COUP-TFII (A) and Nrp2 (B). COUP-TFII shRNAand Nrp2 shRNA efficiently deplete COUP-TFII and Nrp2 protein expression, respectively. The shRNA vectors did not knock down shRNA-resistant COUP-TFII andNrp2. (C) Target sequences of shRNA for the control, COUP-TFII, and Nrp2 vectors. (D) Sequences of shRNA-resistant COUP-TFII and Nrp2 vectors. Capital lettersindicate mutations generated in the original COUP-TFII and Nrp2 sequences.
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Fig. S6. Detachment of POa-derived cells from the CMS and their lateral migration following overexpression or knockdown of Nrp2 is similar to that observedfollowing overexpression or knockdown of COUP-TFII. A coronal section series of E13.5 brains following Nrp2 overexpression (Nrp2 OE) or knockdown(Nrp2 KD) via electroporation at E11.5. Higher magnification of the CMS and CGE area shows that POa-derived cells overexpressing Nrp2 localize in the CMS(Nrp2 OE), and POa-derived cells in which Nrp2 is depleted migrate away from the CMS (Nrp2 KD). (Scale bars, 250 μm.)
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Fig. S7. Migration defects caused by knockdown of COUP-TFII or Nrp2 in POa-derived cells are rescued by shRNA-resistant COUP-TFII or Nrp2 expression, re-spectively. (A and B) Representative micrographs of E13.5 brains electroporated with CAG-tdTomato together with shRNA COUP-TFII and CAG-shRNA–resistantCOUP-TFII (COUP-TFII KD + COUP-TFII rescue) or shRNA Nrp2 and shRNA-resistant Nrp2 (Nrp2 KD + Nrp2 rescue). (C) Representative distributions of POa-derivedcells under COUP-TFII or Nrp2 KD conditions rescued by the shRNA-resistant constructs. Percentages are based on the following number of samples: COUP-TFIIKD + COUP-TFII, four brains, 371 cells; Nrp2 KD + Nrp2 rescue, three brains, 313 cells. Asterisks indicate a significant difference from the control [*P < 0.05,**P < 0.01; COUP-TFII KD (Fig. 5) vs. COUP-TFII + COUP-TFII rescue, P = 0.0050 (sector 3); Nrp2 KD (Fig. 7) vs. Nrp2 KD + Nrp2 rescue, P = 0.00042 (sector 3); Tukey–Kramer test]. (D) Schematic diagrams of COUP-TFII or Nrp2 knockdowns and their rescue by shRNA-resistant COUP-TFII or Nrp2 overexpression. [Scale bars, (A and B)500 μm.]
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Fig. S8. Expression levels of COUP-TFII and Nrp2 affect the distribution of POa-derived cells into the medial amygdala. (A) Cell distribution of the POa-derivedcells electroporated at E11.5 was examined in coronal sections of the medial amygdala at E13.5. Immunohistochemistry for Lhx6 was performed to visualize thedorsal part of the medial amygdala. When COUP-TFII or Nrp2 was OE, the POa-derived cells labeled with tdTomato at E11.5 accumulated in the medial part ofthe region with Lhx6-expressing cells. In contrast, the POa-derived cells sparsely distributed when COUP-TFII or Nrp2 was knocked down (KD). (B) Proportions oftdTomato-positive POa-derived cells in medial amygdala among all tdTomato-positive cells on each coronal section were calculated. Asterisks indicate asignificant difference between the control and the OE or KD samples (*P < 0.05, **P < 0.01; COUP-TFII OE, P = 0.0002; Nrp2 OE, P = 0.0003; COUP-TFII KD, P =0.036; Nrp2 KD, P = 0.006, six brains each, Dunnett’s test). CGE, caudal ganglionic eminence; MeA, medial amygdala. (Scale bars, 100 μm.)
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Fig. S10. Migration analysis of Nrp2 knockout mice. Immunohistochemical staining for Nrp2-tauGFP (green), calbindin (red), and COUP-TFII (blue) in an E13.5horizontal section of a heterozygous (+/−) or homozygous (−/−) Nrp2-Δ mouse brain. No significant difference in migration was observed. Ctx, cortex; GE,ganglionic eminence. (Scale bars, 200 μm.)
Fig. S11. Nrp1 overexpression shows almost the identical effect with the Nrp2 overexpression in the POa-derived cells. In utero electroporation of Nrp1overexpression in the POa at E11.5 and observed at E13.5 is shown. Representative micrographs of E13.5 brains electroporated with CAG-tdTomato togetherwith CAG-Nrp1 are shown. Ctx, cortex; GE, ganglionic eminence; MeA, medial amygdala. (Scale bars, 500 μm.)
POa
POa
Ctx Ctx
POa
In utero electroporation, E12.5 to E15.5
BF
Caudal end zoom
Fluo
Whole hemisphere
Ctx
Fig. S9. The CMS from the POa observed in the E15.5 brain electroporated at E12.5. In utero electroporation with CAG-tdTomato was performed into the POaat E12.5, and the brain was observed at E15.5. BF, bright field; Ctx, cortex; Fluo, fluorescence. (Scale bars, 500 μm.)
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