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A l i f k l t l d f itLARVI’09, Ghent, Sept. 9th, ’09
Analysis of skeletal deformity in fish using vitamin A-in fish using vitamin A
induced bone deformity modelyYutaka Haga (TUMSAT), Shao Jun Du g ( ),
(COMB), Shuichi Satoh (TUMSAT), Tomonari Kotani (Fukuyama Univ )Tomonari Kotani (Fukuyama Univ.), Hiroshi Fushimi (Fukuyama Univ.), Toshio Takeuchi (TUMSAT)
Contents of presentationContents of presentation• Molecular mechanism of bone• Molecular mechanism of bone deformity induced by retinoiddeformity induced by retinoid receptor agonist
• Vertebral deformity in fish l i d d b i i idlarvae induced by retinoic acid
Metabolic pathway of vitamin A (VA)O
Metabolic pathway of vitamin A (VA)
O C15H31
O
VA l i β‐carotene
COOH
VA palmitate
COOHOH
R ti l Retinoic acid R ti l Retinal
(RA)Retinol
Mode of action of nuclear receptorsreceptors
Recep
Transcriptiontor
ReceptX
Response E Target gene
por RXR
g g
Two classes of retinoid receptors
RAR:Retinoic Acid Receptor
RAR(RAR α, β, and γ)
RXR:Retinoid X Receptor
RXR(RXRα, β, and γ)
Mode of action of RARs/RXRsQ1 Whi h f RAR/RXRQ1. Which of RAR/RXR is important?
TranscriptionRAR RXRResponse E Target gene
RARg g
Q2 Which kind of geneQ2. Which kind of gene is important?
QUESTION 1QUESTION 1
Which of RAR/RXRWhich of RAR/RXR i ibl fis responsible for bone deformity?
Binding affinity of retinoids used inBinding affinity of retinoids used in the experiment
All-trans RA (atRA) RAR( )
9-cis-RA (9cRA) RAR/RXR
Am80 RAR
M th id (MA) RXRMethoprene acid (MA) RXR
Lower jaw deformity in flounder at 9 and 58 dphat 9 and 58 dph
RXR ligand RAR ligand
9 dph (A, C, E) and 58 dph(B, D, F) Haga et al. 2003
Jaw deformity in flounder by RAR/RXR ligandsRAR/RXR ligands
RAR
80100 RXR
6080
2040
020
DMSO atRA Am80 9cRA MAModified from Haga et al. 2002. & Haga et al. 2003.
Two classes of retinoid receptors
RAR:Retinoic Acid Receptor
RAR(RAR α, β, and γ)
RXR:Retinoid X Receptor
RXR(RXRα, β, and γ)
QUESTION 2QUESTION 2
Which kind of theWhich kind of the genes are
downstream of RAR/RXR?RAR/RXR?
Expression of RAR/RXR and patchedin the lower jawin the lower jaw
Control Am80 MARXR ligandRAR ligand
RAR
RXR
Patched
EF-1α
Expression of pitx-2 in the jaw RAR/RXR li dRAR li d
Control 9cRAatRARAR/RXR ligandRAR ligand
Deformity and altered gene expression induced by VA and phospholipid (DHA) in European sea bass (Villeneuve et al. 2006)
PLVA
Mode of action of RARs/RXRsVA (RA)→RARs/RXRsPL (DHA)→RXRs
Q1. Which of RAR/RXR is important?( )
TranscriptionRAR RXR
is important?
R E T t
TranscriptionRAR RXRResponse E Target gene
Hox, shh, patched, BMP4, IGF, VDR, etc
Q2. Which kind of gene is important?
Stages and skeletal development of Japanese flounder (Fushimi )
11
2
5-1
3-1
3-2
5-2
3-2
6
4
6
Loss of the intervertebral discs in fused vertebrae in red sea breamfused vertebrae in red sea bream
How do intervertebral discs develop?p
Production of zebrafish carryingProduction of zebrafish carrying GFP in intervertebral disc
GFP expression in twhh-GFP transgenic zebrafishzebrafish
Notochord
20dpf 60dpftwhh-GFP twhh-GFP
Intervertebral discs Intervertebral discs
Haga et al. 2009. Transgenic Res. in press
RA induced disruption of GFP i tt i t hh GFPexpression pattern in twhh-GFP
zebrafish
Haga et al. 2009. Transgenic Res. in press
RA induced disruption of GFP expression pattern in twhh GFP
30d f 90d f
expression pattern in twhh-GFP zebrafish
l C t l30dpf 90dpfControl Control
Discs DiscsRA RA
Discs
Haga et al. 2009. Transgenic Res. in press
Summary·Altered gene expression was d d i l h i hi hdemonstrated in larvae showing high percentage of deformitypercentage of deformity
·Twhh GFP transgenic zebrafish is·Twhh-GFP transgenic zebrafish is useful resource to study intervertebral disc development and vertebral fusion in fishvertebral fusion in fish
PerspectiveProcess of production of cultured fishProcess of production of cultured fish
EggsEggsAltered gene
i
J il
expression
Early diagnosis ofJuvenilesEarly diagnosis of skeletal deformityskeletal deformity
Market-size fish