The btbd9 Gene Knockdown Alters Iron-Dopamine Homeostasis in Neural Substrates of
Restless Legs Syndrome at Early Developmental Stages of Zebrafish
Myeong-Kyu Kim, M.D. & Ph.D.
Department of Neurology, Chonnam National University Medical School, Gwangju, South Korea
Introduction
• Restless legs syndrome, also known as Willis-Ekbom disease (RLS/WED), is a common
neurological disorder that is characterized by an urge to move the legs, usually
associated with unpleasant sensations within the legs.
• RLS/WED is commonly associated with periodic limb movements of sleep (PLMS),
autonomic dysfunction or abnormal eating behaviors.
• Although accumulating evidence indicates that RLS/WED is a dopamine-based
disorder related to iron metabolism, the exact pathophysiology of RLS/WED remains
largely unknown, especially for the autonomic dysfunction or abnormal eating behaviors.
• GWASs suggested that variants of BTBD9 are associated with RLS/WED, PLMS, or
iron stores.
• The aims of the current study were to determine whether btbd9 plays a role in
dopamine-iron homeostasis during the development of zebrafish, and, if so, by what
mechanism, and to see if zebrafish is a good animal model for RLS/WED.
Methods
• One-cell stage zebrafish embryos were injected with control morpholino oligonucleotide
(MO) or btbd9 MO, and then subjected to whole mount in situ hybridization with fth1a,
drd2a, and drd2l riboprobes at the indicated stages.
- fth1a: Ferritin Heavy Polypeptide 1a Gene
- drd2a & drd2l: the zebrafish-specific dopamine D2 receptor genes
• The expression levels of fth1a, drd2a, and drd2l (also known as drd2b) were compared
between wild-type (WT) and btbd9 morphant zebrafish embryos at 1 or 2 day-postfertilization (dpf).
Results
 Expression of fth1a in embryos X
1. At 1 dpf
• The expression pattern of fth1a was comparable between WT and btbd9
morphants across the whole brain and spinal cord.
• However, the expression of fth1a was much stronger over the
intermediate cell mass (ICM) in btbd9 morphants than in WT embryos
(P<0.001; Fig. 1A, B).
2. At 2 dpf
• fth1a expression levels were comparable across the whole body between
WT and btbd9 morphant embryos (P>0.05; Fig. 1C, D).
Figure 1.
Figure 5.
Results (cont’d)
 Expression of Dopamine D2 Receptors
1. At 1 dpf
• The expression pattern of drd2a & drd2l was comparable between WT and
btbd9 morphant embryos (P>0.05 in each; Fig. 2A, B & Fig. 3A, B)
2. At 2 dpf
• The expression of drd2a in the transitional area between the
myelencephalon and the rostral spinal cord (RSC) was more marked in
btbd9 morphants than in WT embryos at 2 dpf (P<0.05, Fig. 2C–H).
• The expression levels of drd2l in diencephalic structures (DES) were
much stronger in WT than in btbd9 morphant embryos (P<0.05; Fig. 3C, D,
E, G).
• However, the expression level of drd2l in the myelencephalon was much
stronger in btbd9 morphants than in WT embryos (n=5 and 6, respectively;
P<0.001; Fig. 3C–H).
C
D
E
F
Figure 6.
 Effect of btbd9 knockdown on the development of DMV
• The intensity and extent of the GFP fluorescence in the DMV were comparable
between WT (Fig. 5A-C) and btbd9 morphant Tg(isl1-GFP) embryos (Fig. 5D-F)
at 2 dpf, suggesting that btbd9 knockdown does not interfere with the
development of the cranial motor neurons, including the DMV in zebrafish at 2
dpf.
 Rescue experiment
• Co-injection of btbd9 MO and RNA into one-cell stage embryos restored the
normal levels of drd2l expression in the in btbd9 morphant embryos at 2
dpf DMV (P<0.05; Fig. 6A–D).
Figure 3.
B
C
B
 Co-expression of drd2l and isl1* in myelencephalic structures
• At 2 dpf, the expression of isl1 co-localized with the expression of drd2l in
the btbd9 morphant embryos, especially in the myelencephalon (Fig. 4A, B),
indicating that the myelencephalic structure with strong drd2l expression is
the dorsolateral motor nuclei of the vagus (DMV).
* An early marker of motor neurons in vertebrates
Figure 2.
A
A
A
B
 btbd9 knockdown in zebrafish
 firstly delayed the expression of fth1a in the primitive erythropoietic system,
ICM, which may cause iron deficiency in the brain of zebrafish morphant
embryos
 then altered the expression of D2 receptors in
1. the diencephalospinal dopaminergic pathway,1 a potential neural substrate of
RLS/WED, and
2. the dorsolateral motor nucleus of the vagus, a potential neural substrate of
autonomic dysfunction or abnormal eating behavior of RLS/WED.2,3
 Our data suggest
 that btbd9 is involved in maintaining iron-dopamine homeostasis in zebrafish at
early developmental stages, and
 that a disruption in the dopaminergic link between the DES, RSC, or DMV at
early developmental stages may underlie the pathophysiology of RLS/WED.
D
C
D
Conculsion
References
Figure 4.
E
F
G
H
E
F
G
H
1. Clemens S, Rye D, Hochman S (2006) Restless legs syndrome: revisiting the dopamine hypothesis
from the spinal cord perspective. Neurology 67:125–130.
2. Walters AS, Rye DB (2009) Review of the Relationship of Restless Legs Syndrome and Periodic Limb
Movements in Sleep to Hypertension, Heart Disease, and Stroke. Sleep 32:589-597.
3. Provini F, Antelmi E, Vignatelli L, Zaniboni A, Naldi G, Calandra-Buonaura G, Vetrugno R, Plazzi G,
Montagna P (2009) Association of restless legs syndrome with nocturnal eating: a case-control study.
Mov Disord 24:871-877.