@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Dentistry, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Matovinovic, Elizabeth"@en ; dcterms:issued "2009-03-25T23:11:10Z"@en, "1997"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """In the developing chick embryo, FGFR-2 expression patterns correlate with outgrowth of facial prominences: frontonasal mass prominences which form the prenasal cartilage and upper beak express high levels of FGFR-2 receptor while maxillary prominences which form the flattened corners of the beak and palatal shelves express low FGFR-2 transcript levels. Facial epithelium is an abundant source of FGFs and is required to support outgrowth of mesenchymal tissue including cartilage rod formation. Since FGFR-2 is highly expressed in regions of facial outgrowth and epithelium is required for outgrowth of facial prominences, epithelium could be required to maintain FGFR-2 transcripts in facial mesenchyme. To test this hypothesis, we removed epithelium to inhibit outgrowth of regions of the embryonic face, grafted frontonasal mass and maxillary prominences into a host limb bud and then examined changes in FGFR-2 expression using in situ hybridization. We also hybridized adjacent sections with collagen II probe to identify regions undergoing chondrogenesis. Our results indicate that removal of epithelium from frontonasal mass lead to a decrease in FGFR-2 and collagen II expression 24 hours after grafting to host and that neither FGFR-2 nor collagen II expression increased to expected levels at 48 hours. These results suggest that there are signals in the epithelium required for increasing FGFR-2 and collagen II gene transcription and the expression of these genes are linked to outgrowth of facial prominences. We localized FGF8 transcripts in the developing chick face to determine whether FGF8 is a putative epithelial signal required for FGFR-2 expression. Our results indicate that FGF8 ectodermal expression does not overlap FGFR-2 expression in mesenchymal tissue and therefore is not a signal required for the expression of this receptor."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/6546?expand=metadata"@en ; dcterms:extent "5421005 bytes"@en ; dc:format "application/pdf"@en ; skos:note "E P I T H E L I U M IS R E Q U I R E D F O R M A I N T A I N I N G F G F R - 2 E X P R E S S I O N L E V E L S IN F A C I A L M E S E N C H Y M E O F T H E D E V E L O P I N G C H I C K E M B R Y O . by E L I Z A B E T H M A T O V I N O V I C B . A . , The University of Manitoba, 1994 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Oral Health Sciences) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A September 1997 © Elizabeth Mato vino vie, 1997 In presenting this thesis in partial fulfilment of the requirements for an, advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of jJ f The University of British Columbia Vancouver, Canada Da«. Oct ; i i C A o £ 3 B 24HMes 24H Epi + Mes 48H Mes 48HEpi+Mes Expression levels m o • + • ++ • +++ 24HMes 24H Epi + Mes 48HMes 48HEpi+Mes Type of graft Epithelium and Mesenchyme 24h Mesenchyme 24h specimen F G F R - 2 Collagen specimen FGFR-2 Collage no. II no. II 1 + + 1 + + + 2 0 0 2 0 + 3 + + + + 3 + 0 4 + + + + 4 + + + + + 5 0 0 5 t + + 6 + + + + 6 + + + 7 + + + + + + 7 + 0 8 + + 9 + + + + + + Epithelium and Mesenchyme 48h Mesenchyme 48h specimen F G F R - 2 Collagen specimen FGFR-2 Collag no. II no. II 1 + + + + 1 0 0 2 + + + + 2 + 0 3 + + + + + + 3 + + 0 4 + + + + + + 4 + + + + + 5 + + + + + + 5 + + 0 6 + + + + + + 6 + 0 7 + + + + + + 8 + + + + + Table 1: Expression scores of FGFR-2 and Collagen II in individual frontonasal mass specimens. KEY: (0) background expression, (+) low expression, (+ +) moderate expression, ( + + +) high expression, (h) hours, (no.) number Figure 8. Dark field views of stage 20 (A,B), 24 (C,D) and stage 28 (E,F) heads hybridized to antisense F G F 8 riboprobe. A , C , E are frontal sections and B, C , E are parasagittal sections. Scale bar = 250pm Key: (20)stage 20, (24) stage 24, (28) stage 28, (MD) mandible, (MX) maxilla, (fnm) frontonasal mass, (NP) nasal pit, (T) telencephalon, (2nd) second branchial arch, (E) eye A ) Stage 20 frontal section. High F G F 8 expression in epithelium adjacent to the nasal pit, the lateral edges of the frontonasal mass, and at the second branchial arch. B) Stage 20 parasagittal section. High FGF8 expression is found at the lateral edge of the frontonasal mass and in between the maxillary and mandibular prominence. Strong signal is also present at the isthmus of the mesencephalon and the rombencephalon. C) Stage 24 frontal section. Abundant transcripts are concentrated in nasal pit ectoderm and in epithelium encasing the maxillae. D) Stage 24 parasagittal section. Expression pattern is similar to C . E) Stage 28 frontal section. FGF8 is highly expressed in ectoderm covering the globular processes and the developing maxillae. F G F 8 transcripts are high in lateral frontonasal mass epithelium but are not expressed in the centre of the prominence (arrowheads). F) Stage 28 parasagittal section. Expression is restricted to the medial edge of the maxillae and in the cranial surface of the mandible. 32 33 DISCUSSION Frontonasal mass and maxillary prominences behaved autonomously when grafted to the host limb bud Previous morphological studies indicate that frontonasal mass grafts with epithelium grown for 7 days on a host embryo formed cartilage rods that were 80% of the average prenasal cartilage rod length in vivo (Richman and Tickle, 1989). These data show that the process of outgrowth and differentiation on the host limb bud is remarkably similar to in vivo growth. Our results indicate that after grafting facial prominences with epithelium, FGFR-2 and collagen II transcript levels are similar to levels found in the respective prominences in vivo. For example at 48 hours, maxillary prominences with epithelium express low FGFR-2 and collagen II levels as in vivo and control for possible effects that host limb tissue may be contributing to graft gene expression. In addition, FGFR-2 and collagen II position-specific expression patterns in frontonasal mass and maxillary grafts were preserved in the ectopic location. The FGFR-2 and collagen II expression in the centre of frontonasal mass grafts at 24 hours growth occurs in pre-cartilage cell aggregates. Previous grafting studies have not detected cartilage formation until 48 hours after grafting in alcian green-stained wholemounts (Richman and Tickle, 1989). Position-specific pattern of expression is also preserved in maxillary grafts; here both FGFR-2 and collagen II transcripts maintained peripheral expression patterns concentrated to one edge of the graft. These data are similar to in vivo expression patterns where F G F R - 2 and collagen II transcripts are found at the lateral and medial edges respectively. Why is collagen II expressed in the ventro-medial regions of maxillary prominences? There are no known cartilaginous tissues derived from the ventro-medial part of maxillary prominences (D. Noden, personal communication). Moreover, no cartilage forms when maxillary mesenchyme is grown in micromass culture (Wedden et al, 1987; Langille et al,1989; Richman and Crosby, 1990). The expression of collagen II in the maxillae may be acting as a signalling molecule which controls when and where cyto-differentiative events take place (Thorogood et al. , 1986) or the expression is occurring in non-chondrogenic tissues as it does in the basement membrane of epithelia, dorsal and lateral surface ectoderm, lateral and ventral gut endoderm and other tissues (Kosher and Solursh, 1989; Wood et al.,1991; Cheah et al. , 1991) as a transient expression not indicative of cartilage formation (Cheah et al., 1991). F G F R - 2 expression in dorso-lateral regions of the maxilla is also not correlated with any chondrogenic activity but may contribute to patterning bones derived from the maxillary prominence. Epithelium is required to maintain mesenchymal FGFR-2 levels in the developing frontonasal mass Following 24 hours of growth on a host limb bud, grafts of frontonasal mass mesenchyme have not been re-epithelialized and the mesenchyme exhibits reduced levels of FGFR-2 expression when compared to respective grafts with epithelium. This down-regulation of FGFR-2 expression suggests that frontonasal mass mesenchyme requires signals provided by facial epithelium in order to maintain expression. Rapid down-regulation of gene expression following ectodermal removal has been demonstrated in the limb bud. Within hours of removing the FGF-rich apical ectodermal ridge down-regulation of AP-2, Cek-8 and Msx-1 is observed (Shen et al., 1997; Patel et al., 1996; Ros et al., 1992). While we have not done a detailed time course for FGFR-2 down-regulation in grafts of frontonasal mass mesenchyme, it is likely that decrease in signal would be seen at earlier time points. After 48 hours of growth, frontonasal mass mesenchyme is covered with limb epithelium and gene expression has increased to moderate levels in 50% of the grafts. In contrast, intact frontonasal mass grafts with epithelium had high levels of F G F R - 2 expression, similar to the high levels found in vivo. There are two possible explanations for the modest up-regulation observed in frontonasal mesenchyme grafts at 48 hours. The first possibility is that the limb epithelium inhibited increased expression of FGFR-2. The second possibility is that the initial lack of facial epithelium over the grafts resulted in loss of signals necessary for the temporal up-regulation of FGFR-2 transcription. Our data do not distinguish between these two possibilities. Both immediate and delayed contact of frontonasal mass mesenchyme with limb ectoderm have been shown to inhibit outgrowth of the mesenchyme (Richman and Tickle, 1989; Richman and Tickle 1992). It is clear from these data that dorsal limb ectoderm cannot completely replace facial ectoderm and one of the reasons why outgrowth is inhibited might be due to an inhibitory effect of the limb ectoderm on FGFR-2 expression in the mesenchyme. The alternative possibility, that re-growth of facial epithelium would allow normal gene expression, is not supported by recent data from Imai et al. (1997). Grafts of first arch mesenchyme into other regions of the face do not form cartilage unless they are simultaneously grafted with facial ectoderm. Thus the delay in re-epithelialization in grafts of facial mesenchyme placed within the face may be enough to prevent the cascade of molecular events that normally precede chondrogenesis (including F G F R -2 expression). F G F R - 2 and collagen II expression are differentially affected by the removal of epithelium Both FGFR-2 transcript levels and expression domains in frontonasal mass grafts with epithelium closely overlap collagen II expression levels and domains suggesting that there may be a relationship between FGFR-2 expression and chondrogenesis in this prominence. However, in frontonasal mass grafts without epithelium there is a slight increase of FGFR-2 expression while collagen II transcripts are down-regulated to background levels. These results indicate that the expression patterns of FGFR-2 and collagen II are no longer as strongly correlated in isolated mesenchyme as with the presence of facial epithelia. The differential regulatory effects on FGFR-2 and collagen II that are caused by removing epithelium may reflect the hierarchy of signaling involved in cyto-differentiation. FGFR-2 activity may be further upstream from the collagen II signaling which occurs just prior to cartilage formation. FGFR-2 is specifically expressed across the mid-line of the frontonasal mass in stage 24 embryos and is one of the earliest markers for the future chondrogenic region. In the limb bud, FGFR-2 is also the earliest of the three FGFRs examined to be localized to cartilage condensations (Szebenyi et al., 1995) and precedes the expression of type II collagen (Devlin et al., 1988). We know that cartilage is formed in nearly all grafts of isolated frontonasal mass mesenchyme if they continue developing in the host limb bud (Richman and Tickle, 1989; 1992) which means type II collagen m R N A will be ultimately be up-regulated. This delay in collagen II expression may be linked to the formation of truncated cartilage rods. Endogenous FGFs may be acting on facial mesenchyme FGF2, 4 and 8 are ectodermal signals which may activate mesenchymally expressed FGFR-2 in the developing face. FGF2 is homogeneously expressed throughout facial prominences (Richman et al., 1997) and has mitogenic activity when bound to the mesenchymally expressed isoform of FGFR-2 (Ornitz et al.,1996). F G F R - 2 is present in the frontonasal mass and mandibular prominences and ectopic F G F 2 increases outgrowth of both frontonasal mass and mandibular mesenchyme (Richman et al., 1997) which makes FGF2 a good candidate for activating FGFR-2 in vivo. Dorsal wing ectoderm also contains abundant levels of F G F 2 protein (Savage et al.,1993) yet the presence of dorsal wing ectoderm cannot support outgrowth of facial mesenchyme (Richman and Tickle, 1989). Hence, other factors in addition to FGF2 are required for outgrowth of facial mesenchyme. FGF4 can also promote outgrowth of frontonasal mass and mandibular mesenchyme (Richman et al., 1997) and has high mitogenic activity when bound to the mesenchymal isoform of FGFR-2 (Ornitz et al., 1996), however, the in vivo distribution of FGF4 transcripts in the developing chick face has only been detected at the medial-rostral surface of mandibular ectoderm (Barlow and Francis-West, 1997; Francis-West personal communication) where it may have paracrine regulatory effects on neighbouring mesenchymal FGFR-2 expression. More work is needed to determine whether FGF4 is expressed in the frontonasal mass. In the stage 24 face, the expression pattern of FGF-8 is concentrated around the nasal pits (Helms et al., 1997; Richman et al., 1997) adjacent but not overlapping F G F R - 2 expression in the frontonasal mass. Moreover FGF-8 has no mitogenic effects when bound to FGFR-2 (IIIc isoform, Ornitz et al., 1996). Other putative FGFR-2 activators include FGF-6 (de Lapeyriere et al., 1993; Coulier et al., 1994; Han and Martin, 1993), FGF-9 (Hecht et al.,1995; Tagashira et al., 1995), FGF-10 (Ohuchi et a l , 1997) and FHF1-4 (Smallwood et al.,1996) all of which have not been described in the face. The removal of epithelium from facial mesenchyme may have eliminated a ligand required to activate FGFR-2 signalling and dismantled the positive feedback required for FGFR-2 up-regulation. A feedback loop was demonstrated in developing chick skin (Song et al., 1996). The application of exogenous FGF2 lead to up-regulation of FGFR-1 expression. Experiments are underway to test for the presence of a similar feedback loop involving F G F R - 2 in facial mesenchyme. Relevance to Crouzon's syndrome Mutations in fibroblast growth factor receptors (FGFR) have been identified in humans with craniofacial disorders including Crouzon, Jackson Weiss, Pfeiffer and Aperts syndromes (Reardon et a l , 1994; Jabs et al., 1994; Rutland et al., 1995; Wilkie et al., 1995; Oldridge et al., 1995; Galvin et al., 1996). The phenotype of Crouzon's syndrome includes shallow orbits, parrot nose, mandibular prognathism and maxillary hypoplasia (Cohen, 1986). In the developing chick, abundant F G F R -2 transcripts are found in regions of the head affected in Crouzon's syndrome (Wilke et al., 1997). We found that removing epithelia from facial mesenchyme results in diminished F G F R - 2 expression which may cause the truncated outgrowth of cartilage rods that occurs later on in development (Richman and Tickle, 1989). Hence, our results in the chicken embryo may parallel the defects found in Crouzon's syndrome. Other proteins involved in face development In addition to the FGFs , several other proteins have recently been shown to play very important roles in patterning various parts of the embryo. Proteins such as sonic hedgehog (Shh) and bone morphogenetic proteins (Bmp) are present at sites where epithelial-mesenchymal interactions occur and may mediate in vivo F G F functions. A vertebrate homolog of the Drosophila Hedgehog gene (Ingham, 1995), Shh, is expressed in many epithelial tissues, frequently at sites where inductive interactions between epithelial and mesenchymal cells occur (Roelink, 1996). Throughout embryogenesis, Shh is found in organizing centers like Hensen's node, the notochord, the floor plate of the neural tube, and the posterior part of the limb bud. In developing limb buds, Shh mediates polarizing activity (Riddle et al, 1993), and its expression can be induced and maintained by F G F s (Crossley et al, 1996). In addition, Shh can induce F G F and Bmp-2 expression in the limb (Laufer etal, 1994). Since Shh is involved in patterning, interacts with F G F s and Bmps -molecules with distinctly localized transcripts in the developing face, it is necessary to investigate possible Shh functions in facial morphogenesis. Throughout craniofacial development, Shh is exclusively expressed in epithelium. During earlier stages (st. 16), Shh m R N A is expressed in the presumptive oral cavity and in the posterior margin of the 2nd branchial arch which goes on to form facial muscles bones and nerves (Wall and Hogan, 1995). At stage 25, Shh is expressed in epithelium at the inferior borders between the frontonasal mass, lateral nasal prominences, and maxillary prominences where it may mediate fusion of these tissues (Helms et al., 1997). At stage 29, Shh expression is highly restricted to the caudal edge of frontonasal mass ectoderm, maxillae and in the midline of the stomodeum (Helms et al., 1997). In sum, early Shh expression in the presumptive oral cavity may participate in putative signalling pathways delegating overall facial symmetry while Shh expression which occurs in already formed facial prominences may be involved in outgrowth and patterning. Interestingly, Shh mouse knockouts display a cyclops phenotype (Chiang et al., 1996) which already indicates that this protein is involved in establishing the mid-line of the face. Since patched, the proposed receptor for Shh (Stone et al., 1996; Marigo et al., 1996), is almost exclusively expressed in epithelium, it is unlikely that the ligand-receptor model postulated for F G F signalling where epithelium provides the ligand required for the activation of a mesenchymally expressed receptor applies to Shh mechanism of action. How epithelial expression of Shh interacts with underlying mesenchymal tissue is an intriguing question which has yet to be addressed. Bmps are also expressed at inductive sites of epithelial-mesenchymal interactions. Bmp-2 and Bmp-4 are members of the T G F p super family which are growth factors involved in various aspects of development (Hogan, 1996). In the developing chick face, Bmp-2 and Bmp-4 m R N A expression is complex, shifting from epithelium to mesenchyme in regions involved in outgrowth (Francis-West et al., 1994). Recently, ectopic applications of Bmp-2 and 4 have been shown to participate in a signaling cascade involving F G F 4 that controls outgrowth and patterning of the facial primordia (Barlow and Francis-West, 1997) and it has been postulated that Bmp-2 and FGF4 may interact to regulate development of the mandible (Barlow and Francis-West, 1997) as they do during limb development (Laufer et al., 1994). Signaling cascades found at various sites of epithelial-mesenchymal interactions may also be present in face development. In Drosophila imaginal discs, the Bmp related factor 'decapentaplegic' (Dpp) is an important downstream target of hedgehog (HH), a homologue of Shh, action. Here, Dpp appears to be involved in several organizing effects of H H (Roelink, 1996). In the chick, Shh induces ectopic mesenchymal Bmp expression in the developing hindgut (Roberts et al., 1995), and in the developing wing bud (Laufer et al., 1994). Since Shh can regulate Bmp expression at various sites of epithelial-mesenchymal interactions, perhaps Bmp is a downstream target of Shh function in the developing face. Moreover, since F G F s can induce Shh expression in developing limbs, Shh may be a downstream target for F G F functions in the face. Therefore, the relationship that exists between these proteins in other regions of the developing embryo may also be present in the developing face. S U M M A R Y In sum, my thesis addresses the role of overlying epithelium in the regulation of mesenchymal F G F R - 2 expression during face development. M y results suggest that there are signals in overlying epithelium required for the maintenance of F G F R - 2 expression in the developing chick face. We believe that the epithelium may provide a ligand which activates F G F R - 2 , triggering a positive feedback mechanism that is dismantled when mesenchyme is stripped of epithelium and consequently, F G F R - 2 receptor expression is not up-regulated to levels found i n mesenchyme with endogenous epithelium. We postulate a putative mechanism for how epithelial and mesenchymal interactions occur; epithelium may provide the ligand necessary for activating receptors present in underlying mesenchyme. We localized F G F 8 m R N A in the developing face to determine whether this protein co-localizes with F G F R - 2 and found that it does not. 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Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Epithelium is required for maintaining FGFR-2 expression levels in facial Mesenchyme of the developing chick embryo"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/6546"@en .