Science - USA (2022-04-15)

Gabanyiet al.,Science 376 , eabj3986 (2022) 15 April 2022 7 of 12

• 
  

GH

DMH

ARC

ROSAYFP

I

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**

**

K CRE virusNod2flox

CRE virus Control

036912

100

110

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170

Weeks post-injection

W

eigh

t (

%)

Wei

gh

t (%)

AAV9-hSyn-Cre

in ARC and DMH

of control and Nod2flox mice

De

ltatemp(

oC)

J

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F

ood c

on

sum

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g)

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Unrolled cotton (%)

Control

Heatmaps P-values map

DMH

ARC

Vgat Nod2

ARC

PHY

DMH

FOS expression changes in brain nuclei

-log10(

pValue)

log2(fold change)

-4 -2 0 2 4

Control X

2

1.5

1

0.5

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2.5

DMH

Nod2GFP Nod2GFP

ARC

CD

Vgatcre

DMH

ARC

Nod2GFP

DMH

% of tom

+Nod2

+

(o

ver to

ta

l tom

+cells)

ARC

EF

AB

L

F

ood

consumed (%)

(relative to week 9)

13 24

Weeks post-injection

0

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04812162024

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Weeks post-injection

ABX NO ABX

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* CRE virusNod2

flox

CRE virus Control

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Vgat Nod2

Up

Down

n.s.

CRE virusNod2flox

CRE virus Control

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CRE virus Control

lower orhigher inVgat Nod2

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Fig. 5. Hypothalamic Nod2+neurons are required for weight and temperature
control.(AandB) Fos expression in femaleVgatDNod2mice and controls
at steady state (7 to 8 months;n= 4 per group). (A) Volcano plots of brain
Fos+cell distribution in the brain. (B) Heatmaps indicating the number
of Fos+cells andP-value map of Fos expression in the hypothalamus,
highlighting DMH and ARC. Shown are regions with higher (magenta) or lower
(green) numbers of Fos+neurons in theVgatDNod2group as compared
to the control group. Scale bar, 200mm. (CandD) Representative
immunofluorescence images of ARC (C) and DMH (D) ofNod2GFPmouse,
showing expression of Nod2 (GFP, green); cell nucleus is stained with DAPI

(blue). (E) Representative immunofluorescence image ofVgatcreNod2GFP

mouse brain injected with AAV9.CAG.FLEX.Tdtomato virus in DMH and ARC.

Colocalization of Nod2 (green) with Tdtomato (magenta) is highlighted;

scale bars, 100mm. (F) Frequency of Tdtomato+cells expressing GFP

among total Tdtomato+cells (n= 4 mice). (G) Viral injection scheme.

(H) Representative immunofluorescence image ofRosaYFPmouse brain

injected with AAV9.hSyn.Cre virus in DMH and ARC, showing expression of

YFP (green); cell nucleus is stained with DAPI (blue). (I) Body weight weekly

measures after viral injection (females, 2 to 3 months at week 0;n=8to

11). (J) Food eaten in 40 hours (n=5or6).(K) Body temperature variation in

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