skeletal development and bone-related gene expression · pdf fileskeletal development and...
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P Gavaia and ML Cancela
Skeletal development and bone-relatedgene expression in fish larvae
Centro de Ciencias do Mar
Functions
Movement and muscle attachment
Body shape
Protection (e.g. scales, spines)
Reproduction
Feeding habits
Calcium and phosphate reservoir (Salmonids)
Composed of: cartilage, bone, chondroid bone, notochord,connective tissues, scales, enamel and dentin
The fish skeleton
Cartilage: composed by an ECM rich in water, collagens (type
II, X), proteoglycans and chondrocytes that differentiatefrom mesenchymal cells
Bone: composed by a mineralized ECM rich in minerals, collagen
type I, and by two types of cells: osteoblasts, thatdifferentiate from mesenchymal cells, and osteoclasts,originating from the macrophage monocyte lineage
Cellular - Osteocytes included in the calcified ECM
Acellular (anosteocytic)- Does not contain osteocytesand has a vascularized woven structure
Fish bone contains collagen type II and chondroitinsulphate
The fish skeleton
Cartilage replacement: Cartilage gradually replaced by bone
Endochondral ossification when calcification begins withinthe cartilage
Perichondral ossification when bone begins to formaround the periphery of the cartilage
(Cranial unpaired bones, pterigophores, mandibula, hypuralia, ….)
The fish skeleton
S. senegalensis27 DAH
S. senegalensis27 DAH
P.J.
Gav
aia
(200
4)
Dermal or intramembranous ossification: Bone develops
directly from the connective tissue
Mesenchymal cells condense, the tissue vascularizes, andcells differentiate directly into osteoblasts
(Cleithrum, cranial paired bones, dermatotrichia e lepidotrichia,supra mandibular, opercula, ….)
Sparus aurata5 DAH
Sparus aurata45 DAH
The fish skeleton
P.J.
Gav
aia
(200
3)
Skeletal malformations
Uncommon in natureMadriaga & Cendrero (1973) Allue (1984)
Appear early in development Frequent in reared larva Reflect culture conditions Leads to:
-Decreased growth rate-Increased mortality-Increased prodution costs-Decreased market price
P.J. Gavaia (2004)
Skeletal malformation in mediterranean species
P.J. Gavaia (2003)
Types of skeletal malformations
• Vertebrae :
• vertebral fusion
• vertebral malformation
• Vertebral column :
• Lordosis
• Kifosis
• Mandibula
• Opercula
• Fins :
• supra/subnumerary rays
• fused rays/pterigophores)
P.J.
Gav
aia
(Unp
ublis
hed)
Senegal sole skeletal development
ClMC
Fp
A
DF
AF EHy
Ha
U
Pt
D
CS
MC BBa
F
Hy
Na
NaHa Hy
CCr
2 DPF 7 DPF 15 DPF
17 DPF
25 DPF
75 DPFP.J. Gavaia et al. (2002). Aquaculture .
CR
Hy1 -2
Phy
A
B
Pp
NS
PpPp
NS
C
Ha
V
HS
NS
D
Senegal sole skeletal deformities
P.J. Gavaia et al. (2002). Aquaculture .
Senegal sole skeletal deformities
44% deformed fish
28% affect caudalvertebrae
P.J. Gavaia et al. (2002). Aquaculture .
Senegal sole skeletal deformities
P.J. Gavaia et al. (2002). Aquaculture .
Skeletal development in Danio rerio
P.J. Gavaia et al. (2006). Gene Expression Patterns .
ALP and TRAP in skeletal development
}Senegal sole
Zebrafish
P.J. Gavaia et al. (2006). Gene Expression Patterns .
SM
V
V
Pq
HS
N
N
B C DHy
R
Skeletal development in Pagrus auriga
N
CoSca
HS
Cl
T
PqCM
Bb
Cb
SM
A
Pt
P.J. Gavaia et al. (2005).
UrAN
CM
SM
AH
H
Skeletal development in Pagrus auriga
ANUrOt
SM
PUCl
Pt R
E F G
P.J. Gavaia et al. (2005).
A B
Skeletal deformities in Pagrus auriga
P.J. Gavaia et al. (2005).
Cleithrum
Meckel’sCartílage
Supra-mandibular
Branchialarches
Parietal
Vertebralcentra
Vertebralarches
Hipurals
Epurals
Dorsa finl
Ana finl
Pelvic fin
Pectoralfin
Age (days post hatching)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
CartilageBone
Time course of skeletal development in Pagrus auriga
P.J. Gavaia et al. (2005).
Tissuedistribution
Bone, teeth Cartilage, kidney, lung,aorta, tooth
Cellularexpression
Osteoblasts,odontoblasts
Immature and hypertrophicchondrocytes, vascularsmooth muscle cells(VSMC), endothelial cells,pneumocytes, kidney cells,fibroblasts, cementoblasts
Sites ofaccumulation
Calcified extracellularmatrix (ECM)
Calcified ECM of cartilage,bone, dentin andpathological calcifications
Time ofappearance
After onset ofmineralization
Early development
Marker gene Osteoblastic functionand differentiation,bone formation
Chondrogenic differentiation
BGP MGP
BGP vs. MGP
Spatio-temporal pattern of bgp expression
Pinto et al. (2001). Gene.
bgp / mgp expression during development
0,01
0,1
1
10
100
1000
10000
Age
Rel
ativ
e ex
pres
sion
Dr bgpDr mgp
0,01
0,1
1
10
100
1000
10000
100000
Age
Rel
ativ
e ex
pres
sion
Ss bgpSs mgp
7 8 11 15 18 27 Col
18S Et.br
bgp
18S Et.br
3 5 6 7 8 10 12 13 15 20 40 juv
mgp
Zebr
afis
hSe
nega
l sol
e
120 HPF
P.J. Gavaia et al. (2006). Gene Expression Patterns .
In situ localization of zebrafish bgp mRNA
11 DPF9 DPF 11 DPF
22 DPF18 DPF 22 DPF
Mandibula Neural arch
Basioccipital articulatory process Pterigophores
P.J. Gavaia et al. (2006). Gene Expression Patterns .
In situ localization of sole bgp mRNA
15 DPF
9 DPF
17 DPF
17 DPF 56 DPF
BOP
Hs
BA
BOP
NA
Basioccipital articulatory process Neural arch
MandibulaHead skeleton
P.J.
Gav
aia
et a
l. (2
006)
. Gen
e Ex
pres
sion
Pat
tern
s .
Immuno-detection of Bgp accumulation in zebrafish
P.J.
Gav
aia
et a
l. (2
006)
. Gen
e Ex
pres
sion
Pat
tern
s .
Immuno-detection of Bgp accumulation in sole
P.J. Gavaia et al. (2006). Gene Expression Patterns .
In situ localization of zebrafish and sole mgp mRNA
P.J. Gavaia et al. (2006). Gene Expression Patterns .
Senegal sole
Zebrafish
38
2 7
18 18
38
7
8
C
Immuno-detection of MGP accumulation in zebrafish
P.J. Gavaia et al. (2006). Gene Expression Patterns .
In situ hybridization ( ) and immunolocalization ( )
Summary of zebrafish skeletogenesis
P.J. Gavaia et al. (2006). Gene Expression Patterns .
Summary of Senegal sole skeletogenesis
In situ hybridization ( ) and immunolocalization ( )
P.J. Gavaia et al. (2006). Gene Expression Patterns .
In situ localization of zebrafish CollX1 mRNA
48 hpf 3 dpf
5 dpf4 dpf
Simões et al. (2006). Under publication
• Skeletogenesis in fish starts early in embrionicdevelopment
• First calcified structures are normally associated tofeeding and movement
• Formation of bony skeletal structures occurs by bothendochondral and intramembranous ossification
• The formation of the axial skeleton begins later inlarval life and parallels the formation of unpaired fins
Conclusions
FISH BONE HAS DIFFERENT CHARACTERISTICSFROM OTHER VERTEBRATES
Conclusions
• High rates of deformities are observed in cultured fish
• Future work should focus on understanding themechanisms responsible for the incidence ofmalformations on early larval stages, in order to reducetheir appearance
• Marker gene expression can be used to follow normaldevelopment and characterize the developing stuctures
• Abnormal gene expression and/or protein accumulationmay reflect early malformations and therefore be usedas indicators