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