Science - USA (2022-01-28)

Eichmülleret al.,Science 375 , eabf5546 (2022) 28 January 2022 8 of 10

pS6, EDNRB pS6 EDNRB

Pat.1

TSC2

+/-

230d

L

D EF

G H

I

KLM

J

A BC

EDNRB

fetal tuber (35GW)

GAD1/2

fetal tuber (35GW)

pS6, GAD1 pS6 GAD1

Pat.1

TSC2

+/-

230d

L

GFAP SCGN Parvalbumin

fetal tuber (25GW)

0

25

50

75

100

P1 P2

% of GCs

GAD1

Batch

A

B

0

25

50

75

100

P1 P2

% of GCs

Batch

A

B

EDNRB

SCGN

Pat.2

TSC2

+/-

230d

L MAP2, SATB2

Pat.1

TSC2

+/-

230d

L

Parvalbumin SCGN

Cortex WM Cortex WM

Ctrl Cortex

TSC unaff. Cx

periles. Cx

Ctrl WM

TSC unaff. WM

TSC WMLCtrl Cortexunaff. Cxperiles. Cx

Ctrl WM

unaff. WMTSC WML

0

500

1500

2500

Cells per mm

(^2) *
CGE
130 230
Excitatory
130 230
MGE
130 230
P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P1 P2
0
25
50
75
100
% of DN
Batch
A
B
C
Tuber WML
PV
SCGN
PV
SCGN
0
500
1000
Cells per mm
2
Tuber WML
PV
SCGN
PV
SCGN
0
50
100
150
DNs per mm
2
ns
Vimentin SCGN Parvalbumin
fetal tuber (35GW)
1
1 2
1
1
2
2
1
1
(^22)
2
Fig. 5. CLIP cells initiate cortical tuber pathogenesis.(AandB) GCs in (A)
organoids and (B) fetal tubers expressed the interneuron lineage marker
GAD1. (C) Quantification of coexpression of GAD1 and pS6 in GCs in organoids
(patient 1,N= 2 independent batchesn= 6 organoids mean = 100%; patient 2:
N= 2 independent batchesn= 8 organoids mean = 100%). (DandE) EDNRB
was expressed in GCs in organoids and in fetal tubers. (F) Quantification of
coexpression of EDNRB and pS6 in GCs in organoids (patient 1,N= 2 independent
batchesn= 4 organoids mean = 95.5%; patient 2:N= 2 independent batches
n= 6 organoids mean = 97.2%). (G) Dysmorphic neurons at 230 days expressed the
CGE marker SCGN. Individual dysmorphic neurons expressed the excitatory marker
SATB2 (stainings at different time points are avilable in fig. S20, C to G).
(H) Quantification of expression of CGE (SCGN and COUP-TFII), excitatory (SATB2),
and MGE markers (PV) in dysmorphic neurons (DNs) at early and late stages in
organoids of two patients. CGE DNs were enriched at 130 days and decreased over
time. Excitatory DNs increased over time. Statistical analysis is provided in fig.
S20, I and J. (I) GFAP identified early dysplastic regions at 25GW located in the white
matter below the cortex. There was a focal enrichment of SCGN-expressing CGE
interneurons, whereas PV expressing MGE interneurons were rare (fig. S21, A and B).
(J) Quantification of MGE marker PV and CGE marker SCGN in ~25GW fetal brain.
Unaffected cortical areas, unaffected white matter, as well as white matter lesions
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