The fish skeleton; skeletal malformations; skeletal malformations in mediterranean species; Types of skeletal malformations; Senegal sole skeletal development; Senegal sole skeletal deformities; Skeletal development in Danio rerio; ALP and TRAP in skeletal development; Skeletal development in Pagrus auriga; Time course of skeletal development in Pagrus auriga; Spatio-temporal pattern of bgp expression; bgp / mgp expression during development; In situ localization of zebrafish bgp mRNA;In situ localization of sole bgp mRNA; Immuno-detection of Bgp accumulation in zebrafish; Immuno-detection of Bgp accumulation in sole
P.; In situ localization of zebrafish and sole mgp mRNA; Immuno-detection of MGP accumulation in zebrafish; Summary of zebrafish skeletogenesis; Summary of Senegal sole skeletogenesis; In situ localization of zebrafish CollX1 mRNA

Cancela, Leonor

Gavaia, Paulo J.

Sapientia

P Gavaia and ML CancelaSkeletal development and bone-relatedgene expression in fish larvaeCentro de Ciencias do MarFunctionsMovement and muscle attachmentBody shapeProtection (e.g. scales, spines)ReproductionFeeding habitsCalcium and phosphate reservoir (Salmonids)Composed of: cartilage, bone, chondroid bone, notochord,connective tissues, scales, enamel and dentinThe fish skeletonCartilage: composed by an ECM rich in water, collagens (typeII, X), proteoglycans and chondrocytes that differentiatefrom mesenchymal cellsBone: composed by a mineralized ECM rich in minerals, collagentype I, and by two types of cells: osteoblasts, thatdifferentiate from mesenchymal cells, and osteoclasts,originating from the macrophage monocyte lineageCellular - Osteocytes included in the calcified ECMAcellular (anosteocytic)- Does not contain osteocytesand has a vascularized woven structureFish bone contains collagen type II and chondroitinsulphateThe fish skeletonCartilage replacement: Cartilage gradually replaced by boneEndochondral ossification when calcification begins withinthe cartilagePerichondral ossification when bone begins to formaround the periphery of the cartilage(Cranial unpaired bones, pterigophores, mandibula, hypuralia, ….)The fish skeletonS. senegalensis27 DAH S. senegalensis27 DAHP.J. Gavaia (2004)Dermal or intramembranous ossification: Bone developsdirectly from the connective tissueMesenchymal cells condense, the tissue vascularizes, andcells differentiate directly into osteoblasts(Cleithrum, cranial paired bones, dermatotrichia e lepidotrichia,supra mandibular, opercula, ….)Sparus aurata5 DAHSparus aurata45 DAHThe fish skeletonP.J. Gavaia (2003)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 priceP.J. Gavaia (2004)Skeletal malformation in mediterranean speciesP.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. Gavaia(Unpublished)Senegal sole skeletal developmentClMCFpADFAF EHyHaUPtDCSMC BBaFHyNaNaHa HyCCr2 DPF 7 DPF 15 DPF17 DPF25 DPF75 DPFP.J. Gavaia et al. (2002). Aquaculture .CRHy1 -2PhyABPpNSPpSCHaVHSNSDSenegal sole skeletal deformitiesP.J. Gavaia et al. (2002). Aquaculture .Senegal sole skeletal deformities44% deformed fish28% affect caudalvertebraeP.J. Gavaia et al. (2002). Aquaculture .Senegal sole skeletal deformitiesP.J. Gavaia et al. (2002). Aquaculture .Skeletal development in Danio rerioP.J. Gavaia et al. (2006). Gene Expression Patterns .ALP and TRAP in skeletal development}Senegal soleZebrafishP.J. Gavaia et al. (2006). Gene Expression Patterns .SMVVPqHSNNB C DHyRSkeletal development in Pagrus aurigaNCoScaHSClTPqCMBbCbSMAPtP.J. Gavaia et al. (2005).UrANCMSMAHHSkeletal development in Pagrus aurigaANUrOtSMPUClPt RE F GP.J. Gavaia et al. (2005).A BSkeletal deformities in Pagrus aurigaP.J. Gavaia et al. (2005).CleithrumMeckel’sCartílageSupra-mandibularBranchialarchesParietalVertebralcentraVertebralarchesHipuralsEpuralsDorsa finlAna finlPelvic finPectoralfinAge (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 40CartilageBoneTime course of skeletal development in Pagrus aurigaP.J. Gavaia et al. (2005).TissuedistributionBone, teeth Cartilage, kidney, lung,aorta, toothCellularexpressionOsteoblasts,odontoblastsImmature and hypertrophicchondrocytes, vascularsmooth muscle cells(VSMC), endothelial cells,pneumocytes, kidney cells,fibroblasts, cementoblastsSites ofaccumulationCalcified extracellularmatrix (ECM)Calcified ECM of cartilage,bone, dentin andpathological calcificationsTime ofappearanceAfter onset ofmineralizationEarly developmentMarker gene Osteoblastic functionand differentiation,bone formationChondrogenic differentiationBGP MGPBGP vs. MGPSpatio-temporal pattern of bgp expressionPinto et al. (2001). Gene.bgp / mgp expression during development0,010,1110100100010000AgeRelative expressionDr bgpDr mgp0,010,1110100100010000100000AgeRelative expressionSs bgpSs mgp7    8     11   15   18    27   Col18S Et.brbgp18S Et.br3     5    6    7     8   10  12  13 15  20   40   juvmgpZebrafishSenegal sole120 HPFP.J. Gavaia et al. (2006). Gene Expression Patterns .In situ localization of zebrafish bgp mRNA11 DPF9 DPF 11 DPF22 DPF18 DPF 22 DPFMandibula Neural archBasioccipital articulatory process PterigophoresP.J. Gavaia et al. (2006). Gene Expression Patterns .In situ localization of sole bgp mRNA15 DPF9 DPF17 DPF17 DPF 56 DPFBOPHsBABOPNABasioccipital articulatory process Neural archMandibulaHead skeletonP.J. Gavaia et al. (2006). Gene Expression Patterns .Immuno-detection of Bgp accumulation in zebrafishP.J. Gavaia et al. (2006). Gene Expression Patterns .Immuno-detection of Bgp accumulation in soleP.J. Gavaia et al. (2006). Gene Expression Patterns .In situ localization of zebrafish and sole mgp mRNAP.J. Gavaia et al. (2006). Gene Expression Patterns .Senegal soleZebrafish382 718 183878CImmuno-detection of MGP accumulation in zebrafishP.J. Gavaia et al. (2006). Gene Expression Patterns .In situ hybridization (  ) and immunolocalization (  )Summary of zebrafish skeletogenesisP.J. Gavaia et al. (2006). Gene Expression Patterns .Summary of Senegal sole skeletogenesisIn situ hybridization (   ) and immunolocalization (   )P.J. Gavaia et al. (2006). Gene Expression Patterns .In situ localization of zebrafish CollX1 mRNA48 hpf 3 dpf5 dpf4 dpfSimões 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 finsConclusionsFISH BONE HAS DIFFERENT CHARACTERISTICSFROM OTHER VERTEBRATESConclusions• 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

Skeletal development and bone-related gene expression in fish larvae

Universidade do Algarve

P Gavaia and ML Cancela
Skeletal development and bone-related
gene 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 differentiate
from mesenchymal cells
Bone: composed by a mineralized ECM rich in minerals, collagen
type I, and by two types of cells: osteoblasts, that
differentiate from mesenchymal cells, and osteoclasts,
originating from the macrophage monocyte lineage
Cellular - Osteocytes included in the calcified ECM
Acellular (anosteocytic)- Does not contain osteocytes
and has a vascularized woven structure
Fish bone contains collagen type II and chondroitin
sulphate
The fish skeleton
Cartilage replacement: Cartilage gradually replaced by bone
Endochondral ossification when calcification begins within
the cartilage
Perichondral ossification when bone begins to form
around the periphery of the cartilage
(Cranial unpaired bones, pterigophores, mandibula, hypuralia, ….)
The fish skeleton
S. senegalensis27 DAH S. senegalensis27 DAH
P.J
. G
ava
ia (
20
04
)
Dermal or intramembranous ossification: Bone develops
directly from the connective tissue
Mesenchymal cells condense, the tissue vascularizes, and
cells 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
. G
ava
ia (
20
03
)
Skeletal malformations
 Uncommon in nature
Madriaga & 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
. G
ava
ia
(U
npu
bli
she
d)
Senegal sole skeletal development
ClMC
Fp
A
DF
AF E
Hy
Ha
U
Pt
D
CS
MC BBa
F
Hy
Na
Na
Ha Hy
C
Cr
2 DPF 7 DPF 15 DPF
17 DPF
25 DPF
75 DPF
P.J. Gavaia et al. (2002). Aquaculture .
CR
Hy1 -2
Phy
A
B
Pp
NS
Pp
S
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 caudal
vertebrae
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 D
Hy
R
Skeletal development in Pagrus auriga
N
CoSca
HS
Cl
T
PqCM
Bb
Cb
SM
A
Pt
P.J. Gavaia et al. (2005).
Ur
AN
CM
SM
AH
H
Skeletal development in Pagrus auriga
AN
UrOtSM
PU
Cl
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’s
Cartílage
Supra-
mandibular
Branchial
arches
Parietal
Vertebral
centra
Vertebral
arches
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
Cartilage
Bone
Time course of skeletal development in Pagrus auriga
P.J. Gavaia et al. (2005).
Tissue
distribution
Bone, teeth Cartilage, kidney, lung,
aorta, tooth
Cellular
expression
Osteoblasts,
odontoblasts
Immature and hypertrophic
chondrocytes, vascular
smooth muscle cells
(VSMC), endothelial cells,
pneumocytes, kidney cells,
fibroblasts, cementoblasts
Sites of
accumulation
Calcified extracellular
matrix (ECM)
Calcified ECM of cartilage,
bone, dentin and
pathological calcifications
Time of
appearance
After onset of
mineralization
Early development
Marker gene Osteoblastic function
and 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
R
el
at
iv
e 
ex
pr
es
si
on
Dr bgp
Dr mgp
0,01
0,1
1
10
100
1000
10000
100000
Age
R
el
at
iv
e 
ex
pr
es
si
on
Ss bgp
Ss 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
Ze
br
af
is
h
Se
ne
ga
l s
ol
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
. G
ava
ia e
t a
l. (
20
06
). G
ene
 Ex
pre
ssi
on 
Pat
ter
ns 
.
Immuno-detection of Bgp accumulation in zebrafish
P.J
. G
ava
ia e
t a
l. (
20
06
). G
ene
 Ex
pre
ssi
on 
Pat
ter
ns 
.
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 CollX1 mRNA
48 hpf 3 dpf
5 dpf4 dpf
Simões et al. (2006). Under publication
• Skeletogenesis in fish starts early in embrionic
development
• First calcified structures are normally associated to
feeding and movement
• Formation of bony skeletal structures occurs by both
endochondral and intramembranous ossification
• The formation of the axial skeleton begins later in
larval life and parallels the formation of unpaired fins
Conclusions
FISH BONE HAS DIFFERENT CHARACTERISTICS
FROM OTHER VERTEBRATES
Conclusions
• High rates of deformities are observed in cultured fish
• Future work should focus on understanding the
mechanisms responsible for the incidence of
malformations on early larval stages, in order to reduce
their appearance
•Marker gene expression can be used to follow normal
development and characterize the developing stuctures
• Abnormal gene expression and/or protein accumulation
may reflect early malformations and therefore be used
as indicators


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Skeletal development and bone-related gene expression in fish larvae

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Universidade do Algarve