Role of FGFs in the Development of

Cranial Base Synchondrosis

Mi-Jeong Kwon

The Graduate School

Yonsei University

Department of Dental Science

Role of FGFs in the Development of

Cranial Base Synchondrosis

A Dissertation Thesis

Submitted to the Department of Dental Science

and the Graduate School of Yonsei University

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy of Dental Science

Mi-Jeong Kwon

June 2006

This certifies that the dissertationThis certifies that the dissertationThis certifies that the dissertationThis certifies that the dissertation thesis thesis thesis thesis

of of of of MiMiMiMi----Jeong KwonJeong KwonJeong KwonJeong Kwon is approved. is approved. is approved. is approved.

____________________________________________________________________________________________________________

Thesis Supervisor: Thesis Supervisor: Thesis Supervisor: Thesis Supervisor: HyHyHyHyooooungungungung----Seon BaikSeon BaikSeon BaikSeon Baik

____________________________________________________________________________________________________________

YoungYoungYoungYoung----ChelChelChelChel Park Park Park Park

____________________________________________________________________________________________________________

KeeKeeKeeKee----Joon LeeJoon LeeJoon LeeJoon Lee

____________________________________________________________________________________________________________

SeongSeongSeongSeong----Ho Ho Ho Ho ChoiChoiChoiChoi

____________________________________________________________________________________________________________

KwangKwangKwangKwang----Kyun ParkKyun ParkKyun ParkKyun Park

The Graduate SchoolThe Graduate SchoolThe Graduate SchoolThe Graduate School

Yonsei UniversityYonsei UniversityYonsei UniversityYonsei University

JuneJuneJuneJune 200 200 200 2006666

감사의감사의감사의감사의 글글글글

본 논문이 완성되기까지 지극한 관심으로 보살펴주시고 지금의 제가 있을 수

있도록 많은 가르침 주신 백형선 교수님께 깊은 존경과 감사를 드립니다. 또한

교정학이라는 소중한 배움의 기회를 주신 박영철 교수님과 저로 하여금 현재의

위치에 설 수 있도록 많은 가르침 주시고 이끌어 주신 손병화 교수님, 황충주

교수님, 유형석 교수님, 김경호 교수님, 최광철 교수님께도 진심으로 감사드립니다.

그리고 동물 실험의 기초적인 지침에서부터 실험의 세부적인 사항 하나 하나까지

손수 챙겨 주신 이기준 교수님께 진심으로 감사드립니다. 또한 보다 완성도 있는

논문을 위해 자상하게 조언해 주시고 부족한 논문이지만 용기를 주시고 격려해

주신 최성호 교수님과 박광균 교수님께 진심으로 감사드립니다.

아울러 바쁜 와중에서도 실험을 위해 많은 도움을 준 윤태민 선생과 교정학

교실 의국원 여러분께도 감사의 말을 전합니다.

마지막으로 항상 저를 믿고 후원해 주시며 사랑으로 보살펴주시는 아버님과

친정 아버지, 어머니께 깊은 감사의 말씀을 드립니다. 그리고 늘 곁에서 든든한

버팀이 되어주고 저를 위해 많은 격려와 배려를 아끼지 않은 사랑하는 남편과

바쁜 엄마 밑에서도 건강하고 예쁘게 잘 자라준 지수, 혜수에게 이 작은 결실을

드립니다.

2006년 6월

저자 씀

i

Table of Table of Table of Table of CCCContentsontentsontentsontents

AAAABSTRACTBSTRACTBSTRACTBSTRACT(English)(English)(English)(English)····································· iv

I.I.I.I.INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION ······································· 1

II. MATERIALS AND METHODSII. MATERIALS AND METHODSII. MATERIALS AND METHODSII. MATERIALS AND METHODS

A. Dissection of ICR mouse··························· 5

B. Whole mount staining of mouse head ············ 5

C. Preparation of head specimen for histologic

observation ·········································· 6

D. Immunohistochemistry: PCNA, FGFR 1, 2, 3,

type X collagen ······································ 6

E. Cranial base organ culture & FGF2, 9

treatment············································· 7

F. Statistics ············································ 8

IIIIIIIIIIII. . . . RESULTSRESULTSRESULTSRESULTS

A. Normal growth of cranial base in vivo ············· 9

B. Expression patterns of FGFR1, 2, 3 in the

cranial base cartilage during postnatal growth ··· 10

C. FGF treatment effect in cranial base organ

culture ··············································· 11

IV. DISCUSSIONIV. DISCUSSIONIV. DISCUSSIONIV. DISCUSSION·········································· 13

V. CONCLUSIOSNV. CONCLUSIOSNV. CONCLUSIOSNV. CONCLUSIOSN ········································ 22

REFERNCESREFERNCESREFERNCESREFERNCES ·············································· 24

LEGENDSLEGENDSLEGENDSLEGENDS ················································· 32

ABSTRAABSTRAABSTRAABSTRACT(Korean)CT(Korean)CT(Korean)CT(Korean) ···································· 54

ii

List of List of List of List of Table and Table and Table and Table and FiguresFiguresFiguresFigures

Table 1. Table 1. Table 1. Table 1. Measurements data for morphometric

analysis. ·········································· 44

Figure 1.Figure 1.Figure 1.Figure 1. Dissection of ICR mouse. ······················· 32

Figure 2.Figure 2.Figure 2.Figure 2. Measurements for morphometric analysis. ··· 33

Figure 3Figure 3Figure 3Figure 3. In vivo development of the mouse cranial

base. ············································· 34

Figure 4.Figure 4.Figure 4.Figure 4. Histology of intersphenoidal synchondrosis

during post natal development. ················ 35

Figure 5.Figure 5.Figure 5.Figure 5. Histology of spheno-occipital synchondrosis

during post natal development. ················ 36

Figure 6.Figure 6.Figure 6.Figure 6. Localization of FGFR 1, 2, 3 in the

intersphenoidal synchondrosis and Spheno-

occipital synchondrosis. ························ 37

FigureFigureFigureFigure 7.7.7.7. Organ culture followed by FGF treatment,

depending on developmental stage. ··········· 39

Figure 8. Figure 8. Figure 8. Figure 8. Dose depedent change of FGF2/ FGF9

tratment cultures. ······························· 42

iii

Figure 9.Figure 9.Figure 9.Figure 9. Morphometric analysis chart of dose

dependent change in FGF2 and FGF9

treatment culture of cranial base. ············· 45

Figure 10.Figure 10.Figure 10.Figure 10. Histologic change of intersphenoidal

synchondrosis with FGF 2, 9 treatment,

H-E staining. ·································· 48

Figure 11. Figure 11. Figure 11. Figure 11. Histologic change of spheno-occipital

synchondrosis with FGF 2, 9 treatment,

H-E staining. ·································· 49

Figure 12.Figure 12.Figure 12.Figure 12. Immunohistochemistry of intersphenoidal

synchondrosis with FGF 2, 9 treatment,

PCNA. ··········································· 50

Figure 13.Figure 13.Figure 13.Figure 13. Immunohistochemistry of spheno-occipital

synchondrosis with FGF 2, 9 treatment,

PCNA. ··········································· 51

Figure 14.Figure 14.Figure 14.Figure 14. Immunohistochemistry of intersphenoidal

synchondrosis with FGF 2, 9 treatment,

type X collagen. ································ 52

Figure 15. Figure 15. Figure 15. Figure 15. Immunohistochemistry of spheno-

occipital synchondrosis with FGF 2, 9

treatment, type X collagen. ··················· 53

iv

ABSTRACT

Role of FGFs in the Development Role of FGFs in the Development Role of FGFs in the Development Role of FGFs in the Development ofofofof

Cranial BCranial BCranial BCranial Base Synchondrosisase Synchondrosisase Synchondrosisase Synchondrosis

Mutations of the fibroblast growth factor receptor-2(FGFR2) have been

reported to cause syndromic craniosynostosis such as Apert, Crouzon

syndrome, characterized by unregulated growth of intramembranous bone at

the suture leading to premature fusion of cranial sutures. But human autopsy

studies of craniosynostosis patients and related animal studies have

suggested the possible alteration of the endochondral bone formation in the

cranial base by the mutations in the FGFR2. FGF/FGFRs mediated signaling

pathway has been largely investigated in intramembranous bone, including

calvarial sutures, but relatively little is known about cranial base

development.

The purpose of this study was to investigate the developmental

progression of the cranial base and the possible involvement of the

FGF/FGFR in regulating the growth and development of cranial base.

By analyzing the time course of normal growth and development of the

endochondral cranial base of mice, the perinatal period is a time of rapid

v

cranial base growth and endochondral ossification and active transition from

cartilage to bone occured in these early postnatal life.

Study of FGFR expression pattern in cranial bone development showed

that FGFR1 and FGFR 2 were predominantly expressed both in the cartilage

phase and in the bone phase, while FGFR3 expression was limited in the

cartilage. These findings implies that FGFR1 and FGFR2 may be involved in

endochondral ossification and osteogenesis as well as chondrogenesis, with

FGFR3 in chondrogenesis.

Experimental culture showed that both FGF2 and FGF9 affects the

maturation in the cranial base. We also found that FGF2 inhibits cartilage

growth mainly by suppressing chondrocyte differntiation and FGF9, unlike

the FGF2, inhibits cartilage growth mainly by early maturation and

differentiation in the synchondrosis.

From these results, FGF9 is considered to be more potent inducer of early

cartilageous maturation compared with FGF2.

Key words; FGF/FGFR, cranial base development, Key words; FGF/FGFR, cranial base development, Key words; FGF/FGFR, cranial base development, Key words; FGF/FGFR, cranial base development, synchondrosis, synchondrosis, synchondrosis, synchondrosis, mousmousmousmouse, e, e, e,

FGF2, FGF9FGF2, FGF9FGF2, FGF9FGF2, FGF9, early maturation, early maturation, early maturation, early maturation

１

Role of FGFRole of FGFRole of FGFRole of FGFs in the Development s in the Development s in the Development s in the Development ofofofof

Cranial Base SynchondrosisCranial Base SynchondrosisCranial Base SynchondrosisCranial Base Synchondrosis

MiMiMiMi----Jeong Kwon, D.D.S., M.S.D.Jeong Kwon, D.D.S., M.S.D.Jeong Kwon, D.D.S., M.S.D.Jeong Kwon, D.D.S., M.S.D.

Department of Dental ScienceDepartment of Dental ScienceDepartment of Dental ScienceDepartment of Dental Science

Graduate School of Yonsei UniversityGraduate School of Yonsei UniversityGraduate School of Yonsei UniversityGraduate School of Yonsei University

(((( Directed by Prof. HyoungDirected by Prof. HyoungDirected by Prof. HyoungDirected by Prof. Hyoung----Seon BaiSeon BaiSeon BaiSeon Baik, D.D.S., M.S.D., Pk, D.D.S., M.S.D., Pk, D.D.S., M.S.D., Pk, D.D.S., M.S.D., Ph.h.h.h. D.D.D.D. ))))

I. INTRODUCTIONI. INTRODUCTIONI. INTRODUCTIONI. INTRODUCTION

Congenital skeletal malformations occur in 2% of all live births and the

majority of these involve the craniofacial complex. With rapid advances in

biotechnology and molecular biology in the past decades, a number of genes

associated with congenital craniofacial malformations have been identified.

Craniosynostosis is the most common form of the congenital craniofacial

deformity and pathogenic mechanisms of various craniosynostosis

syndromes have been largely investigated due to their clinical significance

２

and high prevalence.

Mutations of the fibroblast growth factor receptor-2(FGFR2) have been

reported to cause syndromic craniosynostosis such as Apert, Crouzon

syndrome, characterized by unregulated growth of intramembranous bone at

the suture leading to premature fusion of cranial sutures (Wilkie 1997, Wilkie

et al. 1995, Jabs et al. 1994, Reardon et al. 1994, Rutland et al. 1995,

Lajeunie et al. 1995). Studies in primary calvarial cells derived from patients

with Apert syndrome showed that Apert FGFR2 mutations lead to an

increase in the number of precursor cells that enter the osteogenic pathway,

leading ultimately to increased subperiosteal bone matrix formation and

premature calvaria ossification (Lomri et al. 1998). And Apert FGFR-2

mutations also induce premature apoptosis in human osteoblasts and

osteocytes in vivo and in vitro (Lemonnier et al. 2001a; Kaabeche et al.

2005).

FGFRs are transmembrane tyrosine receptor kinase and activated by a

number of ligands, FGFs, showing complex ligand-binding specificity (Ornitz

et al. 1996). FGFs belong to a gene family comprising of more than 23

members in mammals (Ornitz 2000). They are secreted peptides with

molecular size of approximately 20~35 kDa and expressed in many different

types of tissues during various stages of development. FGFs were first

３

identified as a mitogen for cultured fibroblasts and in other cell types

(Gospodarowicz et al. 1974, Gospodarowicz et al. 1986, Canalis et al. 1988).

In addition to their mitogenic effects, FGFs are involved in diverse biological

processes, including cell motility (Ding et al. 2000), differentiation (Sahni et

al. 1999), migration (Corti et al. 2001), cell survival (Hill et al. 1997),

mesodermal induction (Arman et al. 1998), and pattern formation (Martin et

al. 1998).

The bones in the craniofacial region develop and grow by two processes,

endochondral and intramembranous ossification. Facial bones and cranial

vault are mostly formed through intramembranous ossification, whereas the

basicranium is formed through endochondral ossification. During

intramembranous ossification, there is no cartilaginous template; osteoblasts

in mesenchymal condensations directly secrete bone extracellular matrix

which then matures. During endochondral ossification, mesenchymal cells

first differentiate into chondrocytes to form a cartilage template. This is

subsequently vascularized, followed by recruitment of osteoclasts and

osteoblasts gradually replacing the cartilage scaffold with trabecular bone.

Both types of bone formation are characterized by a series of cellular events,

including commitment of undifferentiated mesenchymal cells to an

chondrogenia/osteogenic lineage, cell proliferation, hypertrophy, and matrix

４

mineralization. These events are tightly controlled and coordinated by a

number of regulatory molecules, such as growth factors, hormones,

transcription factors, and vitamins, and ultimately mature bone.

FGF/FGFRs mediated signaling pathway have been largely investigated

in intramembranous bone, including calvarial sutures, but relatively little is

known about cranial base development. However earlier human autopsy

studies of craniosynostosis patients and related animal studies suggested

the possible alteration of the endochondral bone formation in the cranial

base by the mutations in the FGFR2 (Kreiborg et al. 1976, Avantaggiato et

al. 1996). Moreover, the synchondrosis fusion and the developmental

deficiency in the basicranium of FGFR2c null mice were also reported. The

purpose of this study was to investigate the developmental progression of

the cranial base and the possible involvement of the FGF/FGFR in

regulating the growth and development of cranial base.

５

II. MII. MII. MII. MATATATATEEEERIALS AND METHODSRIALS AND METHODSRIALS AND METHODSRIALS AND METHODS

A. Dissection of ICR mouse A. Dissection of ICR mouse A. Dissection of ICR mouse A. Dissection of ICR mouse

Timed pregnant ICR mice were purchased, housed and handled according

to approved Animal Study Protocols. From the head portion of ICR mouse,

the calvaria was removed and brain and associated tissues were removed to

expose the cranial base. Portions of the cranial base including the

basisphenoid, basioccipital, and anterior portions of the exoccipital

ossification centers with their intervening synchondroses were isolated and

submerged in multi-well plates. Cranial bases from embryo of 17th day

(E17) to mouse of postnatal 56 day (P56, 8week) were used (Fig.1).

BBBB. Whole mount staining of mouse head . Whole mount staining of mouse head . Whole mount staining of mouse head . Whole mount staining of mouse head

Cranial bases were cleaned by removing soft tissues and fixed in 95%

ethanol for 2-3 days and stained in a 0.015% alcian blue solution for 1-2

days. The skulls were dehydrated in 100% ethanol for 2 days and cleaned in

1 % KOH solution for 2 days. Mineralized bone was stained with 0.001 %

alizarin red S in 1 % KOH solution for 2 days and further cleared in a series

of glycerin solutions (25, 50, 80 %) for 24 hours per solution. The stained

skulls were stored in 100% glycerin and observed under a dissection

microscope (Leica, Wetzler, Germany). Alcian blue stained cartilage matrix

６

and alizarin red stained calcified bone and cartilage.

C.C.C.C. Preparation of head specimen for histologic observation Preparation of head specimen for histologic observation Preparation of head specimen for histologic observation Preparation of head specimen for histologic observation

Parasaggital sections (6μm) were prepared from intersphenoidal and

sphenooccipital synchondroses, stage of development of P1, P4, and P10

that had been fixed in 4% paraformaldehyde and embedded in paraffin.

Sections were stained with Hematoxylin and Eosin (HE) for histology.

D. Immunohistochemistry: PCD. Immunohistochemistry: PCD. Immunohistochemistry: PCD. Immunohistochemistry: PCNA, FGFR 1, 2, 3, TNA, FGFR 1, 2, 3, TNA, FGFR 1, 2, 3, TNA, FGFR 1, 2, 3, Type X ype X ype X ype X

collagencollagencollagencollagen

To assess cell proliferation, proliferating cell nuclear assay (PCNA) was

performed. The slides were incubated with anti-rabbit primary antibody, and

visualized with biotinylated anti-rabbit IgG (Santa Cruz, California, USA)

followed by the peroxidase reaction (brown color for Ki67-positive nuclei).

To address the roles of FGFRs in cranial base development, we examined

their protein expression patterns in parasagittal sections. The distribution of

FGFRs was then compared in markers of hypertrophic chondrocytes and

bone, type X collagen (Col X).

Polyclonal anti-rabbit antibodies specific for FGFR1, FGFR2, FGFR3

(Santa Cruz, California, USA) were introduced to the sections at a range of

７

concentrations from 1:50 to 1:200.

The primary antibody was incubated overnight at 4°C in humidified

conditions, washed in a phosphate-buffered saline solution, and incubated

with a species-specific biotinylated secondary antibody for 10 minutes at

room temperature. Antibody amplification detection kits (Zymed, San

Francisco, USA) specific for mouse specimens were used according to the

manufacturer’s instructions. Negative controls were prepared by omitting the

primary antibody in favor of preimmune serum/phosphate-buffered saline.

The DAB reaction was stopped by rinsing in distilled water.

Rabbit polyclonal antibodies directed against type X collagen (Col X) was

used at 1:500 dilution. Images of stained specimens were captured using a

Leica DC 300F digital camera (Leica, Wetzler, Germany).

E. Cranial base organ culture & FGF treatmentE. Cranial base organ culture & FGF treatmentE. Cranial base organ culture & FGF treatmentE. Cranial base organ culture & FGF treatment

Cranial bases obtained from new born mice were cultured and treated with

either FGF2 or FGF9, to investigate the possible role of the FGF2, 9 and

FGFR1, 2, 3 in the growth of the cranial base in the early posnatal life.

Cultures were maintained in 1ml BGJb medium (Gibco Invitrogen, California,

USA), supplemented with 100 g/ml ascorbate, 1 mM beta-glycerophosphate,

antibiotics and antimycotics. To investigate the treatment effect at each

８

developmental stage, cranial bases from stage of E18.5, P1 and P4 were

treated with either FGF2 or FGF9. For further investigation of dose-

dependent effects of FGFs, cultures were treated with either FGF2 or FGF9

at the concentration of 10, 20, 50, 100 and 200ng/ml, respectively, for 1

week. Medium was renewed every two days. At the end of the treatments,

cultured tissues were stained with Alcian blue and alizarin red according to

protocols previously described. Sections of intersphenoidal and

sphenooccipital synchondrosis were prepared and H-E staining,

immunohistochemical anaysis for PCNA and type X collagen were done for

histologic study. For morphometric analysis, length, width, and area of

related structure are measured (Fig.2). Images of specimens were captured

using a Leica DC 300F digital camera (Leica, Wetzler, Germany), and

measurements were obtained using Image-Pro Plus ver 4.5.0 software

(Image & Graphics, Georgia, USA). Each measurement was done in triplicate

and the entire experiment was repeated three times.

F. StatisticsF. StatisticsF. StatisticsF. Statistics

Values are expressed as means ± SD. The data were representative of at

least three separate experiments. Statistically significant differences were

determined by Student’s t test.

９

III. RESULTSIII. RESULTSIII. RESULTSIII. RESULTS

A. Normal growth of cranial bA. Normal growth of cranial bA. Normal growth of cranial bA. Normal growth of cranial base in vivoase in vivoase in vivoase in vivo

Whole mount staining of cranial base developmentWhole mount staining of cranial base developmentWhole mount staining of cranial base developmentWhole mount staining of cranial base development

The cranial base develops from a cartilaginous template that is replaced by

bone through the process of endochondral ossification. During the first week

of postnatal development, there was accelerated growth and ossification in

the cranial base, compared to the latent periods of life. The intraoccipital

synchondroses were nearly ossified by 4 weeks of postnatal development

whereas the intrasphenoidal and the spheno-occipital synchondrosis

retained a thin layer of cartilage into adulthood (8 weeks). Active transition

from cartilage to bone occured during early postnatal life up to 1 week. Until

1 week, the cranial base grew in all dimensions. However, after 1 week,

growth was predominant in the anterior-posterior dimension with little

increase in width (Fig.3).

Histologic finding Histologic finding Histologic finding Histologic finding ofofofof the cranial base the cranial base the cranial base the cranial base synchondrosis synchondrosis synchondrosis synchondrosis

Resting chondrocytes in the central zone undergo proliferation, leading to

an appearance more flattened and compact than that of the central region of

the synchondrosis. Chondrocytes then hypertrophy with an increase in cell

１０

size and an increase in extracellular matrix production. Width of cartilageous

layer was gradually reduced. Positive PCNA staining in the zone of active

proliferation was gradually reduced with decrease in the cell population of

proliferating chondrocyte, ultimately proliferation activity was remarkably

reduced in the center of intersphenoidal synchondrosis of cranial base at the

stage of P10 (Fig. 4, 5).

B. ExpressB. ExpressB. ExpressB. Expression patterns of FGFR1ion patterns of FGFR1ion patterns of FGFR1ion patterns of FGFR1, 2, , 2, , 2, , 2, 3 in the cranial base 3 in the cranial base 3 in the cranial base 3 in the cranial base

cartilage during postnatal growthcartilage during postnatal growthcartilage during postnatal growthcartilage during postnatal growth

FGFR1, -2 and -3 were all present during development of the

endochondral basicranium. FGFR1 and 2 were localized in the reserved

proliferation and hypertrophy zones of the cranial base cartilage and also in

the newly formed endosteal bone surfaces. On the other hand, FGFR3

expression was limited in the cartilage, mainly in the reserve and

proliferation zone of synchondrosis. These expression patterns imply that

possible involvement of the FGFRs in both cartilage and bone formation

during basicranial growth( Fig .6 ).

１１

C. C. C. C. FGF treatmentFGF treatmentFGF treatmentFGF treatment effect in cranial base organ culture effect in cranial base organ culture effect in cranial base organ culture effect in cranial base organ culture....

FGF treatment effect FGF treatment effect FGF treatment effect FGF treatment effect and morphometric analysis and morphometric analysis and morphometric analysis and morphometric analysis

In organ culture followed by FGF treatment accelerated maturation of the

cultured cranial base was observed. Gross changes were similar in stage of

E18.5, P1, P3 and FGF treatment resulted in reduction of the cartilage zone.

Both FGF2 and FGF9 seemed to accelerate cranial base maturation in the

cranial base. Especially, FGF9, at the concentration of 50ng/ml, exhibited

more marked reduction of syncondroses layer independent of developmental

stage(Fig. 7).

In dose-depedent changes in cranial base from stage of P1 mice, 10, 20,

50ng/ml of FGFs lead to incremental reduction of cartilage zone and

accelerate cranial base maturation. Concentration of 50ng/ml, FGF9 appeared

to exhibit the greatest effect in the reduction of synchondroses, compared to

the other concentrations (Fig. 8).

In morphometric analysis, the cartilage length was specifically reduced,

regardless of the intervening bone lengths. in FGF9 treatment samples are

remarkably reduced with compared FGF2 treatment samples(Table. 1, Fig.

9).

１２

Histology of Histology of Histology of Histology of synchondrosis in FGF2/FGF9 treated culture synchondrosis in FGF2/FGF9 treated culture synchondrosis in FGF2/FGF9 treated culture synchondrosis in FGF2/FGF9 treated culture

Histology of intersphenoidal synchondrosis and sphenooccipital

synchondrosis shows that both FGF2 and FGF9 can affect cartilage growth,

but histiogic differerences between FGF2 and FGF 9 were observed. FGF9

treated cartilage shows that the thickness of the reserve and proliferation

zone was remarkably reduced indicating accelerated maturation. Whereas

FGF2 treated cartilage shows relatively little change in reserve and

proliferation zone compared to control, with increased hyprertrophic

zone(Fig. 10, Fig. 11). In PCNA of intersphenoidal synchondrosis and

spheno-occipital synchondrosis with FGF 2/9 treatment, proliferating cells

were gradually reduced with regard to the increase in FGF concentration

within reserve and proliferation zone. But characteristic early maturation of

proliferating cell was observed in FGF9 treatment cultures (Fig. 12, Fig. 13).

Immunohistochemical analysis of Col X in intersphenoidal synchondrisis

and sphenooccipital synchondrosis with FGF 2 and 9 treatment shows that

hypertrophic zone were increased in both. Both FGF2 and FGF9 induduced

increase in Col X in the hypertrophic layer of synchondrosis. FGF9 treatment

culture, remarkably increased prehypertrophic zone are also observed as

well as increased hypertropic zone, and these findings are predominant in

50ng/ml FGF treated cultures (Fig. 14, Fig. 15). These findings imply that

１３

FGF 9 can induce early maturation.

IV. DISCUSSIONIV. DISCUSSIONIV. DISCUSSIONIV. DISCUSSION

Cranial base development in vivo and its importanceCranial base development in vivo and its importanceCranial base development in vivo and its importanceCranial base development in vivo and its importance

The synchondroses are the major sites of growth of late embryonic and

postnatal bone growth in the cranial base, like the sutures in the calvaria.

The cranial base synchondroses have a key role in determining the overall

skull shape as well as the relationship each to its neighbouring bone. The

spheno-occipital synchondrosis is especially important as it not only is

responsible for the most growth but remains patent fot the longest periods of

postnatal life, into adolescence(between 13 and 17 years of age in humans).

Normal development of the cranial base requires the coordinated growth

and maturation of multiple skeletal elements. The embryonic cranial base is a

cartilaginous structure formed by the fusion of the parachordal plates around

the notochord. During the fetal period, ossification centers form within the

cartilage, and the segments separating these centers are referred to as

１４

synchondroses. In the postnatal period, endochondral ossification of the

synchondroses contributes to the expansion of the ossification centers and

growth of the cranial base. By analyzing the time course of normal growth

and development of the endochondral cranial base of mice, we have identified

the perinatal period as a function of cranial base growth and endochondral

ossification.

Histologic observation of PCNA, zone of active proliferation was gradually

reduced over time and remarkably reduced by developmental stage of P10,

which supports the hypothesis that the cranial base is not the primary site

of growth center.

Involvement of the FGFRs in endochonral ossification of Involvement of the FGFRs in endochonral ossification of Involvement of the FGFRs in endochonral ossification of Involvement of the FGFRs in endochonral ossification of

craniacraniacraniacranial base synchondrosis l base synchondrosis l base synchondrosis l base synchondrosis

In the endochondral basicranium, FGFR1, -2 and -3 isoforms are all

present during its development. Previous studies showed that Fgfr2c

transcripts were detected in the resting and proliferative zone chondrocytes ,

perichondrium as well as osteoblasts in the erosive zone of the cranial base

synchondroses with high intensity in differentiating osteoblasts at the sutural

osteogenic fronts of the calvarial bones. Fgfr3b and Fgfr3c were shown to be

１５

found chiefly in proliferating chondrocytes in mouse embyo (Britto et al.

2001; Rice et al. 2003).

Our Data are similar with these findings, however, this is the first study

that shows the FGFR expression patterns in the active postnatal growth

period and this implies that FGFR may be involved in the cranial base

cartilage growth like long bone, or condylar cartilage.

The role of FGF/FGFR in cranial bone development has been investigated

by mice harboring activating mutations in FGF signaling pathways. Deletion

of FGFR2c or expression of gain function mutated FGFR2c in mice results in

multiple skeletal and craniofacial abnormalities (Eswarakumar et al, 2002; Yu

et al, 2003). In A dominant mutation that affects alternative splicing in

FGFR2 causes synostosis in mice (Hajihosseini et al. 2001; Yu and Ornitz

2001). Synchondrosis fusion and developmental deficiency in the

basicranium of FGFR2c null mice implies primary anomalies in the

basicranium, simultaneously with those of cranial vault. Loss-of-function

mutations in FGFRs have been less informative. Embryos lacking FGFR1 or

FGFR2 die prior to skeletal development (Deng et al. 1994; Arman et al.

1999). Mice lacking FGFR3 do not have obvious defects in calvarial bones

(Colvin et al. 1996; Deng et al. 1996). FGFR3 was identified as a negative

regulator of chondrogenesis and osteogenesis (Deng et al, 1996; Chen et al,

１６

1999). The role for FGFR3 in cartilage growth plate development was

confirmed by knockout experiments (Deng et al. 1996), in which disruption

of FGFR3 expression led to prolonged endochondral bone growth and

concomitant elongated long bones. Analysis of both gain-of-function and

loss-of-function mutations in FGFR3 at late gestational and postnatal stages

of mouse development show that the net consequence of signaling through

this receptor is to limit chondrocyte proliferation and differentiation (Naski

and Ornitz. 1998; Ornitz 2001). The inhibitory role to chondrocyte

proliferation is mediated through STAT-1 pathway (Sahni et al. 1999).

FGFR3 has also been demonstrated to inhibit expression of Ihh and Bmp4 in

proliferating chondrocytes of the growth plate (Naski et al. 1998). However,

no abnormalities in the intramembranous bones of the craniofacial skeleton

were found, indicating that osteoblast function appeared unaffected by the

presence or absence of FGFR3.

In our study, the FGFR1 and FGFR 2 were predominantly expressed both

in the cartilage phase and in the bone phase, while FGFR3 is limited in the

cartilage. These findings imply that the FGFR1 and FGFR2 may be involved

in endochondral ossification and ostoegenesis in the chondrogenesis phase,

whereas FGFR3 are mainly involved in chondrogenesis .

１７

FGF 2, 9 FGF 2, 9 FGF 2, 9 FGF 2, 9 eeeeffectffectffectffectssss on the synchondroses of cranial base on the synchondroses of cranial base on the synchondroses of cranial base on the synchondroses of cranial base

development development development development

The roles of FGF ligands in skeletal development are unclear. Exogenous

FGF effect has been reported mainly in long bone and calvaria, while little is

known about the specific role of FGF involved in cranial base formation.

Coffin et al. were the first to produce a dwarf mouse by overexpression of

FGF2 through a constitutive phosphoglycerate kinase promoter (Coffin et al.

1995) and showed that the transgenic mice over-expressing FGF-2 exhibit

markedly shortened long bones associated with growth plate defects, and

enlarged calvaria over the occipital bones, which is usually associated with

either cranial base shortening or with suture fusion. Continuous FGF2

treatment stimulates osteoblast replication, decreases the differentiation

markers, alkaline phosphatase and type 1 collagen, and stimulates osteoclast

formation and bone resorption in calvaria (Hurley et al. 2002). Intermittent

FGF2 treatment stimulates bone formation both in vitro (Zhang et al. 2002)

and in vivo(Hurley et al. 2002). Previous studies showed that FGF2 is a

negative regulator of endochondral bone growth with a specific effect on

chondrocyte differentiation at the epiphysial growth plate (Chen et al. 1999;

Li et al. 1999; Iwata et al. 2000; Chen et al. 2001). Cephalic neural crest

１８

cells from quail embryos have been shown to respond to exogenous FGF-2

in a dose dependent manner; lower doses induce proliferation and higher

doses induce cartilage differentiation (Sarkar S et al. 2001). Blocking of Fgf2

with neutralized beads prevents osteogenesis in long bone (Moore et al,

2002), however, deletion of FGF2 produced no effect in bone length (Zhou et

al. 1998).

Taken all together, FGF2 promotes cell proliferation and inhibits

chondrocytic differentiation, especially terminal differrentiation, under most

cell-culture conditions and also in vivo (Coffin et al. 1995, Nagai et al, 1995,

Mancilla et al. 1998).

On the other hand, about FGF9 relatively little is known. FGF9 is

expressed in immature chondrocytes in condensing mesenchyme at the time

of initiation of endochondral differentiation (Colvin et al. 1999). The

skeletons of newborn Fgf9-/- mice are slightly smaller than of wild type

littermates and their proximal skeletal elements are disproportionately short.

In calvaria, FGF9 is expressed and which is upregulated in the endocranial

portions of the in the sutural mesenchyme me and is downregulated during

postnatal development (Ornitz 2001; Kim et al. 1998). Recent studies in

calvarial bone cell culture shows that FGF2 and FGF9 stimulated proliferation

of the cell populations consisting of more mature osteoblasts, but not those

１９

with undifferentiated precursor cells. Continuous treatment with FGF2/9

inhibited expression of several osteoblast marker genes and mineralization.

However, brief pretreatment with FGF2/9 led to marked stimulation of

mineralization, suggesting that FGFs enhance the intrinsic osteogenic

potential (Fakhry et al. 2005).

However, mice lacking these FGFs show no apparent defects in skeletal

development (Colvin et al. 2001). Functional redundancy between these

FGFs may, in part, account for the lack of phenotype. Therefore, roles for

these FGFs in the early stages of chondrogenesis have not yet been defined.

In our study, cartilage of synchondrosis are reduced which implies that

both FGF2 and FGF9 are related to maturation in the cranial base, but

histologic differences were observed between FGF 2 and FGF9. Although

precise mechanism is unclear not yet, these findings imply that FGF2 inhibit

cartilage growth by controlling of chondrocyte differentiation and that FGF9

affect cartilage growth predominantly by controlling chondrocyte

proliferation and early maturation and differentiation.

In addition, ligand concentration seemed to be related cartilage growth, and

50ng/ml was appeared to be most effective concentration. These findigs are

considered to be a result of biphasic effect between ligands and receptors.

Higher concentration is not always effective, since FGF ligand-receptor

２０

interactions are influenced by many factors other than ligand concentration,

such as heparin and heparin sulphate moieties of matrix and membrane-

bound proteoglycans and other cell-surface proteoglycans. Further studies

are needed to elucidate these influencing factors.

FGF9 is more potent FGF9 is more potent FGF9 is more potent FGF9 is more potent inducer of cartilage maturation inducer of cartilage maturation inducer of cartilage maturation inducer of cartilage maturation

compared with FGF2compared with FGF2compared with FGF2compared with FGF2

In morphometric study of dose dependent change, FGF9 is thought to be

more potent inducer of bone formation. However, it is unclear what makes

these differences.

One possibity of these differential effects of FGF9 comes from activation

of differential types of FGFR. FGF2 is known to ligand with affinity to

universal receptor( FGFR1, 2, 3) but FGF9 is ligand with relatively high

affinity with FGFR2, 3 (Ornitz et al. 1996). Therefore our findings may be

result of depending whether FGFR1 is activated or not.

FGFR1 is expressed in prehypertrophic and hypertrophic chondrocytes

whereas FGFR3 is expressed in proliferating chondrocytes, especially

FGFR1 and FGFR3 have very distinct domains of expression with little

overlap (Peters et al. 1992, 1993; Deng et al. 1996). This juxtaposition of

２１

FGFR1 and FGFR3 expression domains are related with unique functions. In

previous studies, expression of Fgfr1 in hypertrophic chondrocytes suggests

a role for FGFR1 in survival of the hypertrophic chondrocyte, in regulating a

feedback signal to control the rate of differentiation, in regulating the

production of the unique extracellular matrix products of these cells, or in

signaling their eventual apoptotic death (Peters et al. 1992; Delezoide et al.

1998). Therefore through FGFR 1 mainly acts in the prehypertrophic and

hypertrophic chondrocyte. In our histologic findins, FGF2 tratment cultures

shows that proliferation reduction was relatively weak, this implies that

mainly hypertrophic zones are affected, which is consistent with previous

findings.

Another possibility comes from that FGFR3 ectodomain can compete with

FGFR1 for ligand binding (Pandit et al. 2002). Therefore FGF 9 is relatively

amplified interaction with FGFR3 whereas FGF2 interacts with FGFR1 and

FGFR3. As mentioned above, the primary effect of signaling through FGFR3

is to negatively regulate chondrocyte proliferation and differentiation (Naski

and Ornitz. 1998; Ornitz 2001). In Our histologic findings, both proliferation

and differentiation are remarkably reduced in FGF9 treatment cultures,

which support the more amplified action of FGFR3.

Since little is known about FGF function and interaction with receptors in

２２

cranial base development, further study is needed and the mechanism of FGF

signaling is only part of complex network of biological response, therefore

further study will be need to elucidate cross- talk of signaling molecule,

and feedback loops, availability of target genes.

V. CONCLUSIONV. CONCLUSIONV. CONCLUSIONV. CONCLUSION

The purpose of this study was to investigate developmental progression of

the cranial base and the possible involvement of the FGF/FGFR in regulating

the growth and development of cranial base in the early post natal life .

By analyzing the time course of normal growth and development of the

endochondral cranial base of mice, the perinatal period is a time of rapid

cranial base growth and endochondral ossification. Active transition from

cartilage to bone occurs in these early postnatal life.

Concerning the role of FGF/FGFR in the cranial base development, this

study demonstrates that FGFR1 and FGFR 2 is predominantly expressed

both in the cartilage phase and in the bone phase, while FGFR3 is limited in

the cartilage. These findings imply that FGFR1 and FGFR2 may be involved

２３

in endochondral ossification and osteogenesis as well as chondrogenesis,

with FGFR3 in chondrogenesis.

Experimental culture with FGF treatment showed that both FGF2 and FGF9

affect the maturation in the cranial base while FGF9 is more potent inducer

of cartilage early maturation compared with FGF2. We also found that FGF2

inhibits cartilage growth mainly by suppressing chondrocyte differntiation

and FGF9, unlike the FGF2, inhibits cartilage growth mainly by early

maturation and differentiation in the synchondrosis.

２４

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３２

LEGENDSLEGENDSLEGENDSLEGENDS

FigureFigureFigureFigure 1.1.1.1. DDDDissection of ICR mouseissection of ICR mouseissection of ICR mouseissection of ICR mouse....

In preparation for culture, from the head portion of ICR mouse(A), the

calvaria was removed(B) and brain and associated tissues were removed to

expose the cranial base (C). The posterior portion of cranial base including

the basisphenoid, basioccipital, and anterior portions of the exoccipital

ossification centers with their intervening synchondroses was isolated(D).

AAAA BBBB

CCCC DDDD

３３

Figure 2.Figure 2.Figure 2.Figure 2. Measurements for morphometric analysisMeasurements for morphometric analysisMeasurements for morphometric analysisMeasurements for morphometric analysis

Each measurements’ explanations are as follows.

a : length of intersphenoidalsynchondrosis

b : length of basisphenoidal bone

c : length of sphenooccipital synchondrosis

d : length of basioccipital bone

e1: width of interoccipital synchondrosis,

left side

e2: width of interoccipital synchondrosis,

right side

f : area of basioccipital bone

３４

P1 P2

E17 P1 P4E17 P1 P4E17 P1 P4E17 P1 P4

P4 P7 P14

1 week 1 week 1 week 1 week 2 week 3 week 2 week 3 week 2 week 3 week 2 week 3 week

P21 P56

4444 week 8week 8week 8week 8 weekweekweekweek

Figure Figure Figure Figure 3333. In vivo development of . In vivo development of . In vivo development of . In vivo development of the the the the mouse mouse mouse mouse cranial basecranial basecranial basecranial base....

Mouse cranial bases were stained with alcian blue for cartilage and alizarin

red for calcified cartilage and bone at E17, P1, P2, P4, 1, 2, 3, 4 and 8 week

stages of development. During the first week of postnatal development, there

was accelerated growth and ossification of the cranial base. The intraoccipital

synchondroses were nearly ossified by 4 weeks of postnatal development

whereas the intrersphenoidal and the spheno-occipital synchondroses retained

a small amount of cartilage into adulthood, 8 weeks. (IS: intersphenoidal, SO:

sphenooccipital, IO: interoccipital synchondrosis)

ISISISIS

SOSOSOSO

IOIOIOIO

３５

Figure Figure Figure Figure 4444.... Histology of intersphenoidal synchondrosis Histology of intersphenoidal synchondrosis Histology of intersphenoidal synchondrosis Histology of intersphenoidal synchondrosis duringduringduringduring post natalpost natalpost natalpost natal

developmentdevelopmentdevelopmentdevelopment....

H-E staining at the developmental stage of P1(A), P4(C), P10(E) and PCNA

of P1(B), P4(D), P10(F) respectively. Resting chondrocytes in the central

zone undergo proliferation. The hypertrophic zone is invaded by vessels\ and

the cartilaginous matrix is eroded away. Osteoblasts differentiate and start to

lay down bone matrix. Zone of active proliferation is gradually reduced and

synchondrosis cartilage layer is narrowed over time. Remarkably reduced

proliferation activity in the central zone of synchondrosis cartilage is

observed at stage of P10(F).

AAAA BBBB

CCCC DDDD

FFFF EEEE

３６

Figure Figure Figure Figure 5555. Histology of . Histology of . Histology of . Histology of ssssphenophenophenopheno----occipital synchondrosisoccipital synchondrosisoccipital synchondrosisoccipital synchondrosis during post natalduring post natalduring post natalduring post natal

developmedevelopmedevelopmedevelopmentntntnt

H-E staining at the developmental stage of P1(A), P4(C) and PCNA of

P1(B), P4(D) respectively. Zone of active proliferation in cartilage is

gradually reduced and synchondrosis width is reduced over time.

AAAA BBBB

CCCC DDDD

３７

A. Localization of FGFRs in the intersphenoidal synchondrosis

Reserve

& proliferative zone

Erosive

& osteogenic zone

Immunohistochemistry

of FGFR1

Immunohistochemistry

of FGFR2

Immunohistochemistry

of FGFR3

Figure 6. Localization of FGFR 1, 2, 3 in the intersphenoidal synchondrosFigure 6. Localization of FGFR 1, 2, 3 in the intersphenoidal synchondrosFigure 6. Localization of FGFR 1, 2, 3 in the intersphenoidal synchondrosFigure 6. Localization of FGFR 1, 2, 3 in the intersphenoidal synchondrosis is is is

and and and and ssssphenophenophenopheno----occipital synchondrosis. occipital synchondrosis. occipital synchondrosis. occipital synchondrosis.

FGFR2 were detected mostly in the reserve and proliferative zone and

osteogenic zone with high intensity. FGFR1 was also detected in cartilage

phase and in the bone phase but relatively weak. FGFR3 was found chiefly in

proliferating chondrocytes of the synchondroses, developmental stage of P4.

３８

B. Localization of FGFRs in the sphenooccipital synchondrosis

Reserve

& proliferative zone

Erosive zone

& osteogenic zone

Immunohistochemistry

of FGFR1

Immunohistochemistry

of FGFR2

Immunohistochemistry

of FGFR3

３９

A. FGF2, 9 treatment at E18.5

Figure 7.Figure 7.Figure 7.Figure 7. Organ culture followed by FGF treatment Organ culture followed by FGF treatment Organ culture followed by FGF treatment Organ culture followed by FGF treatment, depending on, depending on, depending on, depending on

developmental stage (Aizarin red and alcian developmental stage (Aizarin red and alcian developmental stage (Aizarin red and alcian developmental stage (Aizarin red and alcian blue staning).blue staning).blue staning).blue staning).

Cranial bases from stage of E18.5(A), P1(B) and P3(C) were cultured with

FGF2

20ng/ml 50 ng/ml

FGF9

20 ng/ml 50ng/ml

４０

B. FGF2, 9 treatment at P1

FGF2/FGF9 treatment. FGF treatment result in reduction of cartilage zone, and

gross changes were similar independent of developmental stage. Both FGF2

and FGF9 seemed to accelerate cranial base maturation in the cranial base.

Especially, FGF9, at the concentration of 50 ng/ml, induced more marked

reduction of synchondroses.

FGF2

20ng/ml 50 ng/ml

FGF9

20 ng/ml 50ng/ml

４１

C. FGF2, 9 treatment at P3

FGF2

20ng/ml 50 ng/ml

FGF9

20 ng/ml 50ng/ml

４２

FGF2 FGF9

10 ng/ml

20ng/ml

50 ng/ml

４３

Figure Figure Figure Figure 8888.... Dose Dose Dose Dose----depedent change of depedent change of depedent change of depedent change of FGF2FGF2FGF2FGF2/ / / / FGF9 trFGF9 trFGF9 trFGF9 treatedeatedeatedeated cultures cultures cultures cultures....

Both FGF2 and FGF9 result in cartilage zone reduction, and 10, 20 ,50 ng/ml

of FGF appear to incremental reduction of cartilage zone and accelerate

cranial base maturation in a dose-dipendent manner. Used all specimen are

dissected from the developmental stage of P1. The scale bar represents 1mm.

100 ng/ml

200 ng/ml

４４

A. Measured data depending on concentration of FGF 2 treatment

control 10ng/ml 20ng/ml 50ng/ml 100ng/ml 200ng/ml

A (mm) 1.013 0.778 0.840 0.716 0.681 0.743

b (mm) 1.139 1.166 1.299 1.286 1.178 1.125

c (mm) 0.563 0.458 0.452 0.382 0.396 0.361

d (mm) 2.035 2.166 1.958 2.073 1.966 1.929

e1 (mm) 0.721 0.690 0.584 0.606 0.638 0.463

e2 (mm) 0.728 0.732 0.639 0.531 0.565 0.513

f (mm2) 3.846 3.667 3.653 3.758 3.726 3.347

B. Measured data depending on concentration of FGF 9 treatment

control 10ng/ml 20ng/ml 50ng/ml 100ng/ml 200ng/ml

a (mm) 1.013 0.827 0.779 0.646 0.646 0.653

b (mm) 1.139 1.334 1.313 1.299 1.299 1.334

c (mm) 0.563 0.396 0.389 0.326 0.326 0.340

d (mm) 2.035 2.002 2.000 2.021 2.021 1.967

e1 (mm) 0.721 0.444 0.426 0.396 0.396 0.403

e2 (mm) 0.728 0.526 0.434 0.425 0.425 0.467

f (mm2) 3.846 3.861 3.803 3.773 3.773 3.784

TTTTable1. Measurements data able1. Measurements data able1. Measurements data able1. Measurements data forforforfor morphometric analysis morphometric analysis morphometric analysis morphometric analysis....

The data shown are means(n=3) and are representative of three similar

experiments. Measurements were performed using Image-Pro Plus ver 4.5.0.

４５

A. Dose-dependent change of measurement ‘a’ , length of intersphenoidal

synchondrosis

a

0.0

0.2

0.4

0.6

0.8

1.0

1.2

FGF2 FGF9

dose

mm

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

B. Dose-dependent change of measurement ‘b’, length of basisphenoidal

bone

b

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

FGF2 FGF9

dose

mm

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

FigFigFigFigure 9ure 9ure 9ure 9. . . . MorphoMorphoMorphoMorphometric analysis chart of dose dependent change in metric analysis chart of dose dependent change in metric analysis chart of dose dependent change in metric analysis chart of dose dependent change in FGF2 and FGF2 and FGF2 and FGF2 and

FGF9 FGF9 FGF9 FGF9 treatment culture of cranial base.treatment culture of cranial base.treatment culture of cranial base.treatment culture of cranial base.

４６

C. Dose-dependent change of measurement ‘c’ , length of sphenooccipital

synchondrosis

c

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FGF2 FGF9

dose

mm

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

D. Dose-dependent change of measurement ‘d’ , length of basioccipital bone

d

0.0

0.5

1.0

1.5

2.0

2.5

3.0

FGF2 FGF9

dose

mm

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

Mainly, measurements of synchondrosis, ‘a’, ‘c’, and ‘e’ are reduced with

treatment (A,C,E). FGF9 treatment results in more reduction of the

synchondrosis measurements. * p<0.05 when compared with controls.

*

４７

E. Dose-dependent change of measurement ‘e’ , width of interoccipital

synchondrosis

e

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

FGF2 FGF9

dose

mm

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

F. Dose-dependent change of measurement ‘f’, area of basioccipital bone

f

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

FGF2 FGF9

dose

mm2

0 ng/ml

10 ng/ml

20 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

* * * * * *

４８

Figure Figure Figure Figure 10101010. Histologic change of intersphenoidal synchondrosis with FGF 2, 9 . Histologic change of intersphenoidal synchondrosis with FGF 2, 9 . Histologic change of intersphenoidal synchondrosis with FGF 2, 9 . Histologic change of intersphenoidal synchondrosis with FGF 2, 9

treatmenttreatmenttreatmenttreatment, H, H, H, H----E staining. E staining. E staining. E staining.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). FGF9 treatead cartialge (C,E) shows that

reserve and proliferation zone appears marked reduction. Whereas FGF2

treated cartilage shows relatively little change in reserve and proliferation

zone, instead. shows increased hyprertrophic zone (B,D).

BBBB CCCC

EEEE

AAAA

DDDD

４９

Figure Figure Figure Figure 11111111. Histologic change of spheno. Histologic change of spheno. Histologic change of spheno. Histologic change of spheno----occipital synchondrosis with FGF 2, 9 occipital synchondrosis with FGF 2, 9 occipital synchondrosis with FGF 2, 9 occipital synchondrosis with FGF 2, 9

treatment, Htreatment, Htreatment, Htreatment, H----E staining. E staining. E staining. E staining.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). Similar patterns are observed with

intersphenoidal synchondrosis. FGF9 treatead cartialge (C,E) shows that

reserve and proliferation zone appears marked reduction.

AAAA

BBBB CCCC

DDDD EEEE

５０

DDDD

Figure 1Figure 1Figure 1Figure 12222. . . . Immunohistochemistry Immunohistochemistry Immunohistochemistry Immunohistochemistry of intersphenoidal synchondrosis with FGF of intersphenoidal synchondrosis with FGF of intersphenoidal synchondrosis with FGF of intersphenoidal synchondrosis with FGF

2, 9 treatment, PCNA.2, 9 treatment, PCNA.2, 9 treatment, PCNA.2, 9 treatment, PCNA.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). In reserve and proliferation zone, gross

active proliferating cells are gradually reduced with FGF treatment. But

characteristic early maturation of proliferating cell is observed in FGF9

treatment cultures (D, E).

AAAA

BBBB

EEEE DDDD

CCCC

５１

Figure 1Figure 1Figure 1Figure 13333. Immu. Immu. Immu. Immunohistochemistry of sphenonohistochemistry of sphenonohistochemistry of sphenonohistochemistry of spheno----occipital synchondrosis with FGF occipital synchondrosis with FGF occipital synchondrosis with FGF occipital synchondrosis with FGF

2, 9 treatment, PCNA.2, 9 treatment, PCNA.2, 9 treatment, PCNA.2, 9 treatment, PCNA.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). Similar patterns are observed with

intersphenoidal synchondrosis. characteristic early maturation of proliferating

cell is observed in FGF9 treatment cultures (D, E).

AAAA

CCCC

DDDD EEEE

BBBB

５２

Figure 1Figure 1Figure 1Figure 14444. Immunohistochemistry of intersphenoidal synchondrosis with FGF . Immunohistochemistry of intersphenoidal synchondrosis with FGF . Immunohistochemistry of intersphenoidal synchondrosis with FGF . Immunohistochemistry of intersphenoidal synchondrosis with FGF

2, 9 treatment, type X collagen. 2, 9 treatment, type X collagen. 2, 9 treatment, type X collagen. 2, 9 treatment, type X collagen.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). FGF2 treatment cultures, increased

hypertrophic zone, immunostained for type X collagen with brown, are

observed(B). Whereas FGF9 treatment culture, remarkably increased

prehypertrophic zone are also observed(D).

BBBB CCCC

DDDD EEEE

AAAA

５３

Figure 15Figure 15Figure 15Figure 15. Immunohistochemistry of spheno. Immunohistochemistry of spheno. Immunohistochemistry of spheno. Immunohistochemistry of spheno----occipital synchondrosis with FGF occipital synchondrosis with FGF occipital synchondrosis with FGF occipital synchondrosis with FGF

2, 9 treatment, type X collagen.2, 9 treatment, type X collagen.2, 9 treatment, type X collagen.2, 9 treatment, type X collagen.

P1 stage cranial base(A) was cultured for seven days and exposed to 50,100

ng/ml FGF2(B,C) and FGF9(D,E). FGF9 treatment culture, charateristic

increased prehypertrophic zone are observed. And these findings are

predominant in 50ng/ml FGF treated cultures(D).

AAAA

BBBB CCCC

DDDD EEEE

５４

국문요약

두개저두개저두개저두개저 연골결합연골결합연골결합연골결합 발육시발육시발육시발육시

섬유아세포섬유아세포섬유아세포섬유아세포 성장인자성장인자성장인자성장인자(FGF)(FGF)(FGF)(FGF)의의의의 효과효과효과효과

연세대학교연세대학교연세대학교연세대학교 대학원대학원대학원대학원 치의학과치의학과치의학과치의학과

권권권권 미미미미 정정정정

((((지도교수지도교수지도교수지도교수 백형선백형선백형선백형선))))

섬유아세포 성장인자 수용체(fibroblast growth factor receptor, FGFR) 유전

자의 돌연변이는 에이퍼트(Apert) 증후군과 크로우즌(Crouzon) 증후군 등에서

특징적으로 두개골 유합증을 나타내는 것으로 알려져 있으며 이는 두개 봉합 부

위의 골막성 성장이 비정상적으로 과증식되어 나타나는 증상이다. 그러나 에이퍼

트 증후군 증례의 부검 보고 및 관련된 유전자 변형 동물 연구에서는 골막성 성

장 외에 두개저의 연골성 골 성장에도 변화가 있음을 제시하고 있다.

현재까지 섬유아세포 성장인자 및 수용체의 역할과 관련하여 두개 봉합 부위의

골막성 성장에 관해서는 많은 연구가 있었으나 두개저의 연골성 성장에는 어떠한

영향을 미치는지에 관해서는 거의 연구된 바가 없다.

이에 본 연구에서는 인류와 유사한 두개 구조를 가진 ICR mouse를 사용하여

두개저 성장의 정상적인 발육 양상을 살펴보고 섬유아세포 성장인자(fibroblast

growth factor, FGF) 및 섬유아세포 성장인자 수용체(FGFR)가 두개저의 연골

결합(cranial base synchondrosis) 성장에 어떠한 영향을 미치는지 알아보고자

하였다.

정상적인 두개저 성장 관찰 결과, 두개저 성장에 있어서 출생직후에 가장 뚜렷

５５

한 연골의 골화가 나타났으며 이 시기에 가장 많은 양의 성장이 일어남을 알 수

있었다. 섬유아세포 수용체의 발현양상에 있어 섬유아세포 성장인자 수용체

1(FGFR1)과 2(FGFR2)는 연골 및 골 형성 부위에서 발현된 반면 섬유아세포

성장인자3(FGFR3)는 주로 연골부위에서 발현되었으며 이는 섬유아세포 수용체

인자들이 각각 골 형성 및 연골 형성에 특이하게 관여하고 있음을 보여 주는 결

과이다.

또한 섬유아세포 수용체 각각에 대해 특이한 친화성을 갖는 섬유아세포 성장인

자2와 9(FGF2, FGF9)가 두개저 발육에 어떠한 영향을 미치는지 알아보고자 농

도 별로 배양을 시행하였으며 그 결과 두 인자 모두 연골의 성장억제를 나타냈으

며 특히 섬유아세포 성장인자 9에서 특징적인 억제 양상을 보였다. 연골 성장 억

제양상에 있어서는 조직학적으로 두 인자 간에 차이를 보였으며 섬유아세포 성장

인자2(FGF2)는 주로 연골세포의 마지막 분화 단계를 억제하는 것으로 나타났고

섬유아세포 성장인자9(FGF9)는 연골 세포의 분화 억제 외에 특징적으로 증식 억

제 및 조기 성숙을 일으키는 양상을 보였다.

핵심되는핵심되는핵심되는핵심되는 말말말말; ; ; ; 두개저두개저두개저두개저 연골연골연골연골결합결합결합결합,,,, 섬유아세포섬유아세포섬유아세포섬유아세포 성장인자성장인자성장인자성장인자////수용체수용체수용체수용체, , , , 쥐쥐쥐쥐, , , , 섬유아세포섬유아세포섬유아세포섬유아세포

성장인자성장인자성장인자성장인자2, 92, 92, 92, 9, , , , 조기조기조기조기 성숙성숙성숙성숙