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

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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

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PpPp

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Ha

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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

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N

N

B C DHy

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Skeletal development in Pagrus auriga

N

CoSca

HS

Cl

T

PqCM

Bb

Cb

SM

A

Pt

P.J. Gavaia et al. (2005).

UrAN

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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

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Age

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Age

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7 8 11 15 18 27 Col

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bgp

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3 5 6 7 8 10 12 13 15 20 40 juv

mgp

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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

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Pat

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Immuno-detection of Bgp accumulation in zebrafish

P.J.

Gav

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. Gen

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Pat

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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