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A comparative study of collagen synthesis during avian and mammalian secondary palate development : effects.. Benkhaial, Gheith S. 1992-12-31

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A Comparative Study of Collagen Synthesis During Avian and MammalianSecondary Palate Development: Effects of 5-Fluorouracil.ByGheith S. BenkhaialB.D.S., University of Garyounis, 1985A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Oral Biology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1991©Gheith S. Benkhaial, 1991In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.ADepartment ofOral BiologyThe University of British ColumbiaVancouver, CanadaDate Feb 6, 1992DE-6 (2/88)iiABSTRACTA study was undertaken to examine whether collagen synthesis is critical forshelf reorientation. In the initial experiments in quail, a dose of 100µg 5-fluorouracil (5-FU) administered on day 4 of incubation was determined to be thebest dose-time regimen to induce cleft palate. Pregnant hamsters were given81mg/kg 5-FU intramuscularly or 1m1 saline on day 11 of gestation. Control andtreated embryonic palates dissected from hamsters between days 11 and 13 ofgestation, and from quail between days 5 and 10 of incubation, were incubated in agrowth medium supplemented with 14C-proline. The samples were used foreither: 1. Collagen digestion assay to determine the rate of collagen synthesis; 2.Total protein determination; 3. High performance liquid chromatography (HPLC)to determine hydroxyproline (HYP) levels; or 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine different collagenisotypes. In addition, embryos from both hamster and quail were processed forlight microscopy (LM).The LM results showed that, in hamster 5-FU induces cleft palate by delayingthe reorientation of palatal shelves, while in quail the drug widened the gapbetween the palatal shelves. The data on collagen synthesis showed that in controlhamster a spurt in the collagen synthesis was seen in palate between days 12:00 (12day: 0 hour) and 12:04 of gestation, which is the period of shelf reorientation. In 5-FU exposed hamster palates, the rate of collagen synthesis was lower than controlsuntil day 12:04 of gestation followed by a spurt on day 12:12 of gestation. In 5-FU-treated embryos palatal shelf reorientation took place between days 12:16 and 13:00of gestation. In the developing secondary palate of both the control and 5-FU-iiitreated quail, the rate of collagen synthesis peaked on day 8 of incubation. Thecollagen synthesis, however, was lower in 5-FU-treated than in the control palates.HYP levels in both control and 5-FU-treated hamster palate indicated that althoughan equal amount of new collagen was synthesized in both groups, the shelfreorientation was delayed in the drug-treated embryos. The HYP data from controland 5-FU-treated quail indicated that, in addition to new collagen a considerableamount of non-collagenous protein may also have been synthesized during quailpalate morphogenesis. SDS-PAGE showed that only type I collagen wassynthesized during palate development in both the control and 5-FU-treatedhamster and quail.It was suggested that since (1) in birds, a spurt in collagen synthesis occurs inthe absence of shelf reorientation, (2) an equal amount of new collagen wassynthesized in both the control and 5-FU-treated hamster embryos during theperiod of normal reorientation, and (3) in 5-FU-treated hamster embryos, arecovery in collagen synthesis occurs prior to, and a reduction at the time ofinitiation of delayed shelf reorientation, collagen synthesis may not cause shelfreorientation in mammals.ivTABLE OF CONTENTSPAGEABSTRACT^  iiTABLE OF CONTENTS^  i vLIST OF TABLES v iLIST OF FIGURES  viiACKNOWLEDGEMENTS  ixINTRODUCTION^  1Normal Development of the Secondary Palate ^ 1Abnormal Development of the Secondary Palate 135-Fluorouracil^  16Collagen  22Collagen that form fibers with uniform periodic cross striation 24Collagen that do not form uniformally banded fiber system  25Minor cartilage collagen with unknown subunit compositions 27Collagen involvement in organogenesis  27Collagen involvement in the secondary palate development.... 28PURPOSE OF THE STUDY^  30MATERIALS AND METHODS  32Animal Maintenance and Treatment ^  32Hamster^  32Quail 32Embryonic Palatal Tissue Procurement 33Hamster 33Quail^  34Collagen Digestion Assay^  35Measurement of Total Protein 36High Performance Liquid Chromatography^ 37Equipment 37Sample Preparation ^ 37Chromatography 38Collagen Extraction for SDS-PAGE  47SDS-Polyacrylamide Gel Electrophoresis^  47Light Microscopy^ 48RESULTS^  505-FU Dose-Time Response in Quail ^  50Light Microscopic Observation of the Developing Secondary Palate ^ 50Hamster^ 50Control 505-FU-treated^  51VQuail^  53Control^  535-FU-treated  53Measurement of Incorporation of 14C-proline into collagen ^ 56Hamster^  56Control  565-FU-treated^  56Quail  61Control  615-FU-treated  61High Performance Liquid Chromatography^  61Hamster^  66Control  665-FU-treated^  66Quail  67Control  675-FU-treated  67Measurement of Total Protein ^  68Hamster^  68Control  685-FU-treated  68Quail  68Control^  685-FU-treated  69SDS-Polyacrylamide Gel Electrophoresis ^  69Hamster^  69Quail  70DISCUSSION^  75SUMMARY AND CONCLUSION^  89REFERENCES^  92viLIST OF TABLESPAGETable 1^Summary of the morphological development of teratogeninduced cleft palate^  15Table 2^Genetically distinct collagens ^  26Table 3^The flow program for the solvent gradient of buffer A and Bfor chromatography  40Table 4^Effects of 5-fluorouracil on mortility, fetal weight and the palategap in quail ^  52viiLIST OF FIGURESPAGEFigure 1 Metabolic pathways of 5-fluorouracil ^ 21Figure 2 Chromatogram showing hydroxyproline peak in hamstersecondary palate tissue^ 42Figure 3 Chromatogram showing hydroxyproline peak in quailsecondary palate tissue44Figure 4 Chromatogram showing hydroxyproline peak in standardamino acids^ 46Figure 5 Frontal section through the secondary palate ina control hamster embryo on day 11:18 of gestation ^54Figure 6 Frontal section through the secondary palate ina control hamster embryo on day 12:00 of gestation^ 54Figure 7 Frontal section through the secondary palate ina control hamster embryo on day 12:02 of gestation ^ 54Figure 8 Frontal section through the secondary palate ina control hamster embryo on day 12:04 of gestation ^ 54Figure 9 Frontal section through the secondary palate in a5-FU-treated hamster embryo on day 12:00 of gestation ^ 54Figure 10 Frontal section through the secondary palate in a5-FU-treated hamster embryo on day 12:16 of gestation ^ 54Figure 11 Frontal section through the secondary palate in a5-FU-treated hamster embryo on day 12:20 of gestation ^ 54Figure 12 Frontal section through the secondary palate in aa control quail embryo on day 5 of incubation ^ 57Figure 13 Frontal section through the secondary palate ina control quail embryo on day 7 of incubation ^ 57Figure 14 Frontal section through the secondary palate ina control quail embryo on day 8 of incubation ^ 57Figure 15 Frontal section through the secondary palate ina control quail embryo on day 9 of incubation ^ 57Figure 16 Frontal section through the secondary palate in aviii5-FU-treated quail embryo on day 5 of incubation ^ 57Figure 17 Frontal section through the secondary palate in a5-FU-treated quail embryo on day 7 of incubation ^ 57Figure 18 Frontal section through the secondary palate in a5-FU-treated quail embryo on day 8 of incubation ^ 57Figure 19 Frontal section through the secondary palate in a5-FU-treated quail embryo on day 9 of incubation ^ 57Figure 20 Rate of collagen synthesis in developing secondarypalate of hamster embryo ^ 59Figure 21 Rate of collagen synthesis in developing secondarypalate of quail embryo 59Figure 22 Hydroxyproline content in the developing secondarypalate of hamster embryo ^ 62Figure 23 Hydroxyproline content in the developing secondarypalate of quail embryo 62Figure 24 Total protein content in the developing secondarypalate of hamster embryo ^ 64Figure 25 Total protein content in the developing secondarypalate of quail embryo 64Figure 26 SDS-PAGE gel electrophoresis showing type Icollagen in the developing secondary palate ofhamster embryo ^71Figure 27 SDS-PAGE gel electrophoresis showing type Icollagen in the developing secondary palate ofquail embryo ^73ixACKNOWLEDGEMENTSI wish to thank my supervisor, Dr. R. M. Shah for his advise, criticism andsupport throughout the course of this thesis. I would like to express myappreciation to Mrs. E. Feeley and Mr. J. Firth for their various technical help.I am also grateful to Dr. K. Cheng, Director of the University of BritishColumbia's Quail Genetic Stock Center for providing a continuous supply of quaileggs.I would like to express my gratitude to my brother Ashour Ben-khaial forhis moral and continuous financial support. In addition. I wish to thank all thestaff and students of the Department of Oral Biology and Faculty of Dentistry forproviding a friendly environment.The work conducted for this thesis was supported by a grant from theNSERC of Canada to Dr. R. M. Shah.1INTRODUCTIONNormal Development of the Secondary PalateThe vertebrate secondary palate develops intraorally as bilateral symmetricaloutgrowths from the maxillary processes of the first pharyngeal arch. A review ofliterature indicates that much of the work on the embryology of the secondarypalate was conducted on mammalian embryos (Dursy, 1869; His, 1901; Polzl, 1904;Schorr, 1907; Inouye, 1912; Pons-tortella, 1937; Lazzaro, 1940; Walker and Fraser,1956; Asling et al., 1960; Coleman, 1965; 1967; Andersen and Matthiessen, 1967;Walker, 1969, 1971; Dostal and jelinek, 1970; Shah and Chaudhry, 1974a; Holmstedtand Bagwell, 1977; Gulamhusein and England, 1982; Kiso et al., 1984; Young et al.,1991a). These studies indicate that in order to form the mammalian secondarypalate, three sequential events during embryogenesis are essential. These eventsare :1. A bilateral vertical outgrowth of palatal buds (shelves) from the maxillaryprocesses. Eventually, as the tongue and lower jaw grow forward(anteriorly), the palatal shelves hang vertically along the sides of the tongue;2. A change in the direction of development of the palatal shelves from avertical to a horizontal plane (reorientation), above the dorsal surface of thetongue; and3.^Union of the opposing horizontal shelves to separate the oral and nasalcavities.The literature further indicates that the prenatal development of thesecondary palate has also been analyzed in a few other vertebrates. In fish, the2palatal shelves form vertically along side the tongue but never reorient or fuse(Shah et al, 1990). Consequently, through the ontogeny of fish, the palate remainsopen, i.e., physiologically clefted. Similar morphogenesis was also noted inamphibians, eg., frogs, by LeCluyse et al (1985). In reptiles, eg., alligator, the palatalshelves originate as a horizontal projections ad initium over the dorsal surface ofthe tongue and grow toward each other to eventually unite and separate the oraland nasal cavities (Ferguson, 1981; Shah and Ferguson, 1988). In other reptiles,however, the palate remains open through their ontogeny (Shah, 1984; Shah et al.,1990). In birds, as in the alligator, the palatal shelves also develop horizontally adinitium over the dorsal surface of the tongue. The shelves then grow toward oneanother over the dorsal surface of the tongue, and approximate. Subsequently,however, unlike alligator and mammals, they never fuse. Instead, a physiologicalcleft persists in the roof of the avian mouth through their ontogeny (Shah andCrawford, 1980; Koch and Smiley, 1981; Shah et al., 1985a, 1987, 1988; Shah andCheng, 1988)It is clear from the foregoing analysis that the morphogenesis of thesecondary palate amongst vertebrates is different. In addition, the reorientation ofpalatal shelves from a vertical to a horizontal plane is a unique developmentalfeature of mammals.The manner by which mammalian palatal shelves reorient from a verticalto a horizontal position has been an area of controversy. The historical literatureon this subject was reviewed by Lazzaro (1940), Stark and Ehrmann (1958), Walkerand Fraser (1957) and Shah (1979a, b). From their observations of fixed humantissues, His (1901) and Inouye (1912) suggested that the palatal shelves, due to their3inherent elasticity, "rotate" from the side of the tongue to a position above thetongue. This view, however, was disputed by Polzl (1904) who indicated that atransformation in the "form" of the vertical shelves brings them to a horizontalposition. He further suggested that the transformation in the "form" was broughtabout by a differential growth of the tongue and mandible.Pons-tortella (1937) forwarded another explanation for the reorientation ofpalatal shelves. He suggested that the vertical shelf "regresses" and then a newhorizontal shelf grows from its medial surface at the approximate level of thedorsum of the tongue.In 1940, Lazzaro reviewed the earlier literature on the development ofsecondary palate and proposed three possible mechanisms by which thereorientation of shelves might be achieved. These mechanisms were: (1) "externalforce", i.e., pressure exerted by the tongue, (2) growth changes involving regressionof the ventral portion of the shelves and their outgrowth in a horizontal plane, assuggested by Pons-tortella (1937), and (3) a "rotation" of shelves due to some"intrinsic force". Lazzaro himself favoured the last mechanism and attributed themovement of shelves to an increase in the extracellular matrix (ECM) within thedeveloping palatal shelf.On the basis of their observations on different strains of mice, Walker andFraser (1956) argued that the transition of shelves from a vertical to a horizontalposition is too rapid to be due solely to mandibular and tongue growth as suggestedby Polzl (1904). They also discarded the theory that the vertical shelf regresses andlater a new shelf grow in the horizontal plane, as proposed by Pons-tortella (1937).In their opinion, the reorientation of shelf from a vertical to a horizontal plane4was achieved by a process of remodelling. The remodelling of shelves involvedformation of a "bulge"on the medial wall of the vertical shelf, over the tongue,with a simultaneous "retraction" of the ventral part of the shelf. They did not findany evidence suggesting the dropping of the tongue or mandible (Lazzaro, 1940), orthe pressure by the tongue (Peter, 1924), to allow the vertical shelves to becomehorizontal. Instead, Walker and Fraser (1956) supported Lazzaro's (1940)proposition that the palatal shelves change their position because of an "intrinsicforce".Since the publication of Walker and Fraser's (1956) work, continued effortshave been made to investigate both the extrinsic and intrinsic factors that may beinvolved in reorienting the palatal shelves.A proposal that external forces such as muscular pressure by the tongue onthe palatal shelves might cause shelves to reorient themselves (Lazzaro, 1940) hasreceived attention of numerous workers. Lazzaro suggested that the withdrawal ofthe tongue before the shelf movements and subsequent pressure on the undersurface of the palatal shelves by the tongue might push the vertical shelves into ahorizontal position. He proposed five mechanisms for downward tonguewithdrawal from its position between the vertical shelves: (1) a lowering of themandible and the tongue; (2) a forward displacement of the tongue; (3) a lifting ofthe roof of the oral cavity; (4) changes in form of the tongue due to musculardevelopment, and (5) muscular movement of the tongue.Subsequent studies have supported many of the above proposals to someextent. For example, from their studies on rats, Asling et al (1960) and Coleman(1965) suggested that a lowering of the mandible and the tongue occur primarily5due to "differential growth" of both the tongue and mandible, a possibilityindicated earlier by Polzl (1904). A marked growth spurt of the mandible, relativeto the maxilla, at the time of shelf elevation would allow the tongue to descendtowards the floor of the mouth and clear the way for the vertical shelves to becomehorizontal (Asling et al., 1960; Coleman, 1965; Diewert, 1976).This proposition, however, was disputed by Humphrey (1971). She observedthat in human embryos an increased mandibular growth follows rather thanprecedes the downward tongue withdrawal from its position between the verticalshelves. Rather the tongue withdrawal occurs as a part of fetal mouth openingreflex. She indicated that the tongue provides an "active force" by its movement tobring shelves in the horizontal plane, a view earlier proposed by Walker (1969,1971) in his studies on the palate development in mice and rabbit embryos. Theseauthors supported the idea that the mandible could be lowered due to lifting ofhead from against the chest. The tongue is simultaneously withdrawn frombetween the shelves, thus creating a space and making it possible for reorientationof the vertical shelves to a horizontal position. Taylor and Harris (1973) andDiewert (1976) suggested that differential growth of various areas of the craniofacialregion would allow realignment of the relationship between the tongue, mandibleand palatal shelves for the latter to reorient from a vertical to a horizontal plane.While the studies continued toward defining the role of extrinsic factorsduring the reorientation of palatal shelves, attention was also directed towardevaluation of factors intrinsic to the shelves. These factors included both theproliferative and migratory behavior of palatal mesenchymal cells and thesynthesis of extracellular matrix.6In 1907, Schorr indicated that an increase in the rate of cellular proliferationwithin the palatal shelf tissue may account for reorientation. This possibility hasreceived some attention in the literature. Mott et al (1969), Jelinek and Dostal(1974), Nanda and Romeo (1975) and Luke (1989) observed increased rates of cellproliferation, in the developing palate of mice and rats, several hours prior topalatal shelf reorientation. On the other hand, Walker and Fraser (1956) andHughes et al (1967) observed only a few mitotic figures in the shelf tissue prior to,or during, reorientation and consequently they did not attach any significance toSchorr's suggestion. Also, neither an increased rate of mitosis (Cleaton-Jones, 1976)nor an increase in the synthesis of DNA, indicative of the cell proliferation rate(Shah et al., 1989a, b) have been observed immediately prior to, or during, thereorientation of shelves.Walker and Fraser (1956), on the basis of suggestion made earlier by His(1901) and Inouye (1912), speculated that the intrinsic shelf force may reside in theelastic fiber of ECM. This speculation was, however, discarded by Frommer andMonroe (1969) who failed to demonstrate the presence of elastic fibers in thedeveloping palatal shelves.Following La77aro's (1940) and Walker and Fraser's (1956) speculation, thatthe intrinsic shelf force may reside in the ECM of the developing palatal shelves, aconsiderable amount of attention was focused on involvement of both theglycosaminoglycans (GAG) and collagen during palatogenesis. From their animalstudies, and using histochemical and/or biochemical techniques, numerousresearcher (Larsson, 1962; Jacobs, 1964; Nanda, 1971; Pratt et al., 1973; Ferguson,1978; Brinkley, 1980; Jacobson and Shah, 1981; Brinkley and Morris-Wiman, 1984;7Turley et al., 1985) observed a spurt in the synthesis of GAG prior to, and duringthe reorientation of shelves. Although, initially, it was thought that thepredominant GAG associated with reorientation of shelves was sulfated i.e.,chondroitin sulfate (Larsson, 1962; Jacobs, 1964), a consensus emerged in thesubsequent literature that the major GAG involved during the palatal shelfreorientation was hyaluronic acid (Nanda, 1971; Pratt et al., 1973; Ferguson, 1978;Brinkley, 1980; Jacobson and Shah, 1981; Turley et al., 1985). Cell culture studieshave also shown that the palate mesenchymal cells can be stimulated to producehyaluronic acid (Greene et al., 1982; Sasaki and Kurisu, 1983; Yoshikawa et al., 1986,Pisano and Greene, 1987). The hydrophilic properties of hyaluronic acid results inalterations in osmotic concentrations with consequent swelling of the ECM andcorresponding decrease in mesenchymal cell density (Brinklely, 1980). It has alsobeen proposed in the literature that GAG may facilitate the movement ofmesenchymal cells (discussed below) during palatal shelf reorientation (Shah,1979a, b; Brinkley, 1980; Venkatasubramanian and Zimmerman, 1983). In spite ofthese observations, the precise nature of the role played by GAG molecules duringthe reorientation of the palatal shelves is unclear.Since an in vivo increase in the synthesis of collagen was observed duringpalatogenesis, collagen was also implicated as a candidate to account for theinternal shelf force responsible for palatal shelf reorientation (Shapira, 1969; Prattand King, 1971; Shapira and Shoshan, 1972; Hassell and Orkin, 1976; Silver et al.,1981). Collagen synthesized in vivo in the reorienting shelf was type I (Hassell andOrkin, 1976). During the vertical growth, however, some type III was also observedin the shelf (Silver et al., 1981). In organ culture of palatal explants (Uitto and8Thesleff, 1979), and in cell culture of palate mesenchyme (Sasaki and Kurisu, 1983)both types I and III collagen were observed. It appears that the observation ofisotypes I and III depends on in vivo or in vitro methods, or on the stage of palataldevelopment. Although the studies to date have noted a continuous increase inthe synthesis of collagen during palatogenesis, the role which the increasingcollagen synthesis may play in the reorientation of palatal shelves remainsunknown.The most recent hypothesis to explain the intrinsic shelf force was themigration of mesenchymal cells from the tip portion of the vertical palatal shelvesinto the medial bulge to form the horizontal shelves. Lassard and associates (1974)and Krawczyk and Gillon (1976) indicated that the palatal mesenchymal cellssynthesize the contractile proteins, actin and myosin, which may be responsible forcell migration during the reorientation of palatal shelves. Babiarz et al (1975)observed a calcium-dependent adenosine triphosphatase activity in themesenchymal cells around the time of shelf reorientation thus supporting thepossibility of presence of a contractile system. Subsequently, Shah (1979b) observedthat in a reorienting shelf, the shape of the mesenchymal cells alter from aspherical to elongated, and the cells developed cytofilaments in its sub-plasmamembrane region. The cytofilaments were oriented along the long axis ofthe cells. Also, the elongated cells formed junctions and appeared to be flowinginto the medial bulge. An increased synthesis of hyaluronic acid duringreorientation of palatal shelves (noted above) could facilitate migration of palatemesenchymal cells (Shah, 1979b; Brinkley, 1980; Venkatasubramanian andZimmerman, 1983; Pisano and Greene, 1987). Since alterations in both cellular9morphology and contents along with an increased synthesis of ECM were believedto be associated with cellular movement during organogenesis (Trinkaus, 1984),these changes together were suggested to contribute to the intrinsic shelf forceduring reorientation of palatal shelves (Shah, 1979b). A spurt in the synthesis ofvarious ECM molecules in the developing palate, it was proposed, could be one ofthe critical aspects in the cascade of cellular and molecular events, that may allowmarshalling of the internal shelf force for reorientation of the mammalian palatalshelves (Shah, 1979b; Brinkley and Morris-Wiman, 1984).During the past decade, studies were also focused on defining theinvolvement of hormones and growth factors in regulation of behaviour ofmesenchymal cells of the developing palate in mammals. It was generallyrecognized in the literature that epidermal growth factors (EGF), transforminggrowth factors (TGF), cyclic adenosine monophosphate (cAMP), prostaglandines,catecholamines and neurotransmitters may be involved in regulation of growthand differentiation of mammalian palatal cells during development (Greene et al.,1982, 1989; Zimmerman and Wee, 1984; Pratt et al., 1984; Shah et al., 1985b; Pisanoand Greene, 1986, 1987; Pratt, 1987; Greene, 1989; Gehris et al., 1991). For example,levels of cAMP are altered during palatal development (Greene and Pratt, 1979;Olson and Massaro, 1980; Shah et al., 1985b). Adenylate cyclase activity, whichcatalyzes the synthesis of cAMP from adenosine triphosphate (ATP), has beendemonstrated in the developing palate by both cytochemical and biochemicaltechniques (Waterman et al., 1976; Greene and Pratt, 1979; Palmer et al., 1980).Adenylate cyclase activity is maximum prior to and during fusion of the palatalprocesses and temporally corresponds to the increase in levels of cAMP. The10activities of adenylate cyclase, and cAMP, in palatal cells can be modulated byseveral agents. For example, treatment of embryonic palate mesenchymal cells invitro with prostaglandin PGE2 and prostacycline results in dose-dependentaccumulation of intracellular cAMP (Greene et al., 1981). Palmer et al (1980)observed that PGE1, E2 and F2 a stimulated adenylate cyclase activity in the intacthamster palate. Phenylbutazone, an agent which inhibits prostaglandinbiosynthesis, produces a high incidence of cleft palate (Montenegro et al., 1976), bypreventing fusion of palatal processes in vitro (Montenegro et a1.,1982). George andChepenik (1985) have observed the presence of several phospholipases whichhydrolyse membrane phospolipids yielding free fatty acids utilized inprostaglandin synthesis by the embryonic palate mesenchymal cells. Theseobservations on the activity of prostaglandins in the developing palate indicates arole for these compounds in cAMP mediated growth and differentiation of thesecondary palate (Chepenik et al., 1984; Pisano and Greene, 1986).Catecholamines have also been implicated as modulators of cAMP duringpalatogenesis. For example, quantitative alterations in catecholamines, i.e.,dopamine, norepinephrine, and epinephrine within the embryonic palatal tissuehave been observed during development (Zimmerman and Wee, 1984; Pisano andGreene, 1987). Catecholamines can stimulate adenylate cyclase in the developingpalate (Waterman et al., 1976; Palmer et al., 1980). Murine embryonic palatemesenchymal cells in vitro have been shown to respond to various catecholamineswith dose-dependent elevations of intracellular cAMP. In these cells, P-adrenergicreceptors have also been characterized (Greene, 1989). Also, it has been shown that11the neurotransmitters, serotonin and acetylcholine, were capable of stimulatingpalatal shelf reorientation while y-amino-n-butyric acid (GABA) exerted aninhibitory effect (Zimmerman and Wee, 1984). Further, the levels of bothserotonin and GABA in the embryonic palate changes during palatogenesis(Zimmerman and Wee, 1984). It was suggested that serotonin would stimulatepalate mesenchymal cell motility, by altering the intracellular levels of cAMP andcGMP and by increasing cellular protein carboxymethylation to bring about theshelf reorientation (Zimmerman and Wee, 1984).Growth factors are also involved in regulation of palate development.Human and murine embryonic palate mesenchymal cells contain EGF receptorsand are responsive to growth stimulation by EGF (Nemo et al., 1980; Yoneda andPratt, 1981). Hassell (1975) and Hassell and Pratt (1977) observed that theprogrammed cell death in the medial edge epithelium (MEE) can be prevented bythe addition of EGF to organ culture of developing palate. Subsequently, Yonedaand Pratt (1981) indicated that EGF influences mouse palatal MEE differentiationvia action on the underlying mesenchyme. The effect of EGF on the synthesis ofECM in the developing palate in organ culture has also been reported (Silver et al.,1984; Turley et al., 1985). Silver et al (1984) observed that mouse palatal shelvesgrown in the presence of EGF were substantially larger with increases occurring inDNA content as well as in protein synthesis. They also observed that the netcollagen synthesis in palatal organ culture appears to be stimulated by EGF.Recently, transforming growth factor (TGF) a and 13's have been shown tobe involved in palate development (Gehris et al., 1991). TGF-13 has been12immunolocalized in embryonic mesenchyme of neural crest origin destined todevelop into a number of craniofacial structures including the secondary palate(Heine et al., 1987; Fitzpatrick et al., 1990; Williams et al., 1991). Greene et al (1989)observed that TGF-13 and basic fibroblast growth factor (bFGF), stimulatesproliferation of human embryonic palate mesenchymal cells in vitro. In contrast,however, murine embryonic palate mesenchymal cells growth is inhibited by TGF-13. Pelton et al (1990) observed a differential expression of the three TGF-13 genes(TGF-131,132, (33) in both the mesenchymal and epithelial cells of the palatal shelvesand suggested that they may be involved in the morphogenesis of the secondarypalate. Gehris et al (1991) suggested that TGF-13s may have both autocrine andparacrine mode of action during palate development. They further indicated thatTGF-f31, synthesized in epithelial tissue, may exerts influence on the underlyingmesenchyme. Also, a similar mode of action for TGF-132 has been suggested byGehris et al (1991) whereby this gene product, synthesized in the mesenchyme(Pelton et al., 1990; Fitzpatrick et al., 1990), may exert its effects on the overlyingepithelium.While the studies on growth and differentiation of developing palate weremainly conducted in mammals, those in the other vertebrates are only a few. Ithas been observed that the developing palatal tissues of vertebrates differ in theirbiological behaviour. For example, unlike mammals where the DNA synthesis inthe MEE ceases approximately 24 hours prior to reorientation and fusion of thepalatal shelves (Hudson and Shapiro, 1973; Pratt and Martin, 1975; Shah et al.,131985b), DNA synthesis in avian MEE continues at a steady pace through themorphogenesis of the secondary palate (Shah et al., 1985b, 1987; Shah and Cheng,1988). In mammals, CAMP levels change during palatogenesis (Greene and Pratt,1979; Olson and Massaro, 1980; Shah et al., 1985b), but in birds the cAMP activitiesin the developing palate remains unaltered (Shah et al., 1985b, 1987; Shah andCheng, 1988). The programmed cell death observed during mammalianpalatogenesis (Mato et al., 1966; Chaudhry and Shah, 1973, 1979; Shah andChaudhry, 1974a, b) is absent during palate development in birds (Shah andCrawford, 1980; Koch and Smiley, 1981; Shah et al., 1985, 1987, 1988; Shah andCheng, 1988) as well as fish (Shah et al., 1990) and reptiles (Ferguson, 1981). Thus,these observations underscore differing life histories of palatal tissues duringembryonic development.Abnormal Development of the Secondary Palate:In human, clefting of the palate is a major birth defect. It allows acommunication between both the oral and nasal cavities, thus, affecting thefunctions of mastication, deglutition, respiration and phonation.Researchers have been using teratogens, that induce cleft palate in animals,as a tool to understand both the normal and abnormal aspects of palatogenesis.Almost all research work on teratogen induced cleft palate have been carried out inmammals. From these studies, it is suggested that a teratogen may affect any oneof the three events of normal palatogenesis in mammals to induce a cleft palate inthe offspring (Table 1). The mechanism(s) by which these teratogens induce cleftpalate, however, are largely unknown. In addition, it has also been suggested that14a cleft palate may result from rupture of previously fused palate (Veau, 1931;Kitamura, 1966, 1991; Goss, 1977).During the last thirty years much of the efforts were focused onunderstanding the mechanism of glucocorticoid-induced cleft palate.Glucocorticoids, when administered to various mammalian species duringmidgestation induce cleft palate in the offspring (Baxter and Fraser, 1950; Shah andKilistoff, 1976). The mechanism by which glucocorticoids induce cleft palate is,however, unclear. It has been shown that, in the developing palate,glucocorticoids can affect cell proliferation (Jelinek and Dostal, 1974; Nanda andRomeo, 1978), ECM synthesis (Larsson, 1962; Jacobs, 1964; Shapira, 1969; Pratt et al.,1973; Jacobson and Shah, 1981; Sasaki and Kurisu, 1983), lysosomal enzymesynthesis (Shah and Chaudhry, 1974a; Herold and Futran, 1980; Goldman et al.,1981; Ads et al., 1983; Shah et al., 1991a, b), cyclic nucleotide levels (Erickson et al.,1979; Greene et al., 1981) and mobilization of arachidonic acid, the precursor forprostaglandin synthesis (Piddington et al., 1983; George and Chepenik, 1985) whichaffects the growth of the cells. Glucocorticoid action may be mediated via itsbinding to receptors in the palatal tissues (Goldman et al., 1978; Bekhor et al., 1978;Salomon and Pratt, 1979; Shah and Burton, 1980) which may affect aforementionedcellular functions.Several phospholipase inhibitory proteins (PLIP) have recently been shownto mimic the teratogenic effects of glucocorticoids on the developing palate in vivo.PLIP inhibited terminal differentiation of the MEE of palatal shelves in vitro, aneffect which could be reversed by addition of arachidonic acid to the culturemedium (Gupta et al., 1984). On the basis of these results, it was postulated that15effect which could be reversed by addition of arachidonic acid to the culturemedium (Gupta et al., 1984). On the basis of these results, it was postulated that16Table 1.^Summary of the Morphological Aspects of Teratogen-induced CleftPalate Development.Affected Stage of^ Agents^ ReferencePalate Development1. Vertical2. Reorientation3. Fusion6-mercaptopurine, bromo-deoxyuridine, hadacidin.cortisone, triamcinolone,vitamin A, folic aciddeficiency, diazo-oxo-norleucine, 5-fluorouracil,cyclophosphamide,radiation, 6-amino-nicotinamide.Burdett and Shah, 1988;Burdett et al., 1988; Shahet al., 1991a, b.Walker and Fraser, 1957;Walker and Crain, 1960;Asling et al., 1960; Callasand Walker, 1963;Kochhar and Johnson,1965; Dostal and Jelinek,1972; Ferguson, 1977;Shah, 1979c; Diewert, 1979Diewert and Pratt, 1979;Shah and Wong, 1980;Shah et al., 1989.hydrocortisone,^Shah and Travill, 1976a;phenylbutazone Montenegro et al., 1982.17glucocorticoids may inhibit both the arachidonic acid release and prostaglandinbiosynthesis, and consequently affect various cellular functions including ECMsynthesis, during the critical period of palate morphogenesis (Gupta et al., 1984) toinduce cleft palate.5-Fluorouracil:In 1957, Duschinsky and Pleven reported the synthesis of a pyrimidineanalogue, 5-fluorouracil (5-FU). The drug is a white, odourless, crystalline powder(Windholz, 1976), sparingly soluble in water and ethanol, and insoluble inchloroform, benzene, and diethyl ether (Rudy and Senkowski, 1973). Solubility of 5-FU in aqueous solution can be increased at alkaline pH and by increasing thetemperature. It has a molecular weight of 130.08. The drug is stable when exposedto air, hydrolyses under strongly basic conditions, and decomposes at about 280°-282°C.One of the most widely used anticancer drugs, 5-FU is used in the treatmentof a number of different malignancies, either alone or in combination with otherdrugs. For example, 5-FU has been shown to have a palliative activity in themanagement of cancer of the breast, gastrointestinal tract, colon and ovary, andcurative effect in the treatment of non-invasive basal cell carcinomas (Heidelbergeret al., 1957; 1962; DeVita et al., 1985).When administered parenterally to mice or rats, 5-FU rapidly enters all bodycompartments with large amounts reported in bone marrow, small intestine,kidney, liver and spleen (Liss and Chadwick, 1974; Chabner, 1982). As much as 80%of the administered drug is eliminated through metabolic degradation, while18approximately 5-20% by urinary excretion. In human, rapid intravenousadministration of 5-FU produces plasma concentrations of 0.1mM with a plasmahalf-life of 10-20 minutes (Chabner, 1982; Diasio and Harris, 1989). In hamster, thedrug crosses the placental barrier rapidly and reaches the fetuses within 30 minutes(Tuchmann-Duplessis, 1975). Oral absorption of 5-FU is variable, unpredictableand incomplete due to the significant variation in the bioavailability (0-80%) of thedrug (Diasio and Harris, 1989).Following parenteral administration, 5-FU is inactive , and requiresmetabolic activation to a nucleotide forms to be effective (Figure 1). The catabolismof 5-FU proceeds largely in the liver to a-fluoro-(3-alanine, urea, carbon dioxide andammonia (Chaudhuri et al., 1958; Mukherjee and Heidelberger, 1960; Miller, 1971;Chabner et al., 1975).Depending on the relative significance of deoxyuridine or uridinephosphorylase during metabolic activation, 5-FU may exert its effect on cells viatwo major mechanisms: (1) inhibition of DNA synthesis, and (2) alteration in theprocessing and function of RNA (Heidelberger et al., 1983; Carrico and Glazer,1979). The metabolic activation of the drug (Figure 1) can be achieved by threepossible pathways: (1) the reaction with ribose-l-phosphate catalysed by uridinephosphorylase to form 5-fluorouridine, followed by phosphorylation by uridinekinase; (2) the reaction with phosphoribosyl pyrophosphate catalysed bypyrimidine phosphoribosyl transferase directly to form 5-fluorouridine-5'-monophosphate; and (3) the reaction with deoxyribose-1-phosphate catalysed bythymidine phosphorylase to convert 5-FU to 5-fluoro-2'-deoxyuridine-5'-19monophosphate (FdUMP) by thymidine kinase (Kessel et al., 1966; Heidelberger etal., 1983). The deoxyuridine pathway leads to the formation of 5-fluorodeoxyuridine monophosphate, which is a potent inhibitor of thymidylatesynthetase (TS), an enzyme essential in the de novo synthesis of DNA. A covalentcomplex is formed between 5-fluorodeoxyuridine monophosphate, methylene-tetrahydrofolate and TS which, although reversible, has a sufficiently long half-lifeto prevent thymidylic acid synthesis (Chaudhuri et al., 1958; Mukherjee andHeidelberger, 1960; Miller, 1971; Chabner et al., 1975).Most mammalian cells use TS to make the thymidine-5'-monophosphate(dTMP) needed for DNA synthesis from 2'-deoxyuridine-5'-monophosphate(dUMP), obtaining the methyl group for the "5" position of the pyrimidine ringfrom 5,10 methylene tetrahydrofolate (Danenberg and Danenberg, 1978). BecauseFdUMP has a greater affinity for TS than the natural substrate dUMP, it canprevent the formation of dTMP (Danenberg and Danenberg, 1978) Consequentinhibition of DNA synthesis, combined with continued RNA and proteinsynthesis, it is suggested, may produce an imbalance in the cell that is incompatiblewith its survival (Chaudhuri et al., 1958; Mukherjee and Heidelberger, 1960;Miller, 1971; Chabner et al., 1975; Stevens et al., 1984; Uchida et a1.,1989; Prior et al.,1990).While some of the actions of 5-FU are explained by the inhibition of DNAsynthesis through the thymidylate pathway, the drug can also alter RNAmetabolism. For example, Chaudhuri et al (1958) identified 5-fluorouridine-5'-monophosphate (FUMP) in a hydrolysate of RNA from Ehrlich ascites cells andsarcoma-180 cells treated with 14C-FU. Mandel (1969) demonstrated that another20metabolite, 5-fluorouridine-5'-triphosphate (FUTP) could be incorporated intoRNA in place of uracil. Additional experimental evidence from in vivo studies alsosupport the contention that the action of 5-FU is, at least, partially independent ofits effect on TS. For example, co-administration of 5-FU and thymidine preventsthe early inhibition of DNA synthesis, but increases 5-FU incorporation into theRNA of normal and malignant cells (Carrico and Glazer, 1979). Maybaum andcolleagues (1980) observed that mouse lymphoma cells experienced two phases ofdrug effect when exposed to 5-FU. During an early phase (1-24 hours) theinhibition of cell growth was reversed by addition of thymidine to the culturemedium, but during the later phase (after 24 hours) the inhibition was notreversible. The authors suggested that the second phase of inhibition was probablycaused by progressive incorporation of 5-FU into RNA. Evans and co-workers(1980) noted that a three-hour incubation with 5-FU at low concentrations (5-20pM) produced a thymidine reversible toxicity, whereas high concentrations (50-20011M) produced toxicity not reversible by thymidine. On the basis of these data itwas suggested that the RNA incorporation mechanism would probably be favoredby high concentrations, and longer durations of exposure to 5-FU, and would beenhanced by the presence of thymidine, whereas the opposite conditions wouldfavour TS depletion (Chabner, 1982).5-FU also affects other cellular activities. The drug decreases the surfacecharge and transmembrane potential in tumor cells (Ingraham et al., 1980),decreases fucose incorporation into membrane proteins (Kessel, 1980), anddecreases protein synthesis (Kessel, 1980).21Figure 1 Metabolic pathways of 5-fluorouracil. (Adapted from IARCmonographs, pp. 217-235, 1981)22OO3:1;O3aE^2V a1 : 1 g ■... 7,= .c .^ 3  I:e a I zg 0 8 g^z7.--.: I. a I^U.4). isO023The embryotoxic and teratological effects of 5-FU has been documented inmammals such as rats, mice, monkey, guinea pigs and hamsters (Dagg, 1960;Morris et al., 1967; Wilson, 1971; Kromka and Hoar, 1973; Forsthoefel et al., 1978;Shah and Mackay, 1978; Skalko and Jacobs, 1978; Imagawa et al., 1979) and birds(Karnofsky et al., 1958; Ruddick and Runner, 1974). In these species, the teratogeniceffects of 5-FU were characterized by malformations of limb, eye, beak, tail, palate,lower jaw, gut and brain.Stadler and Knowles (1971) reported a case of human pregnancy in whichthe mother received a total dose of 7.5g 5-FU during the second and thirdtrimesters. The newborn showed transient effects, characterized by mildrespiratory distress and petechiae. No gross malformations were described. In1980, Stephens and associates reported multiple congenital anomalies in a humanfetus from a mother exposed to 5-FU during the first trimester. The anomaliesincluded bilateral radial aplasia and absent thumbs and fingers, single umbilicalartery, hypoplastic aorta, pulmonary hypoplasia and renal dysplasia.In hamster, a single intramuscular injection of 81mg/kg 5-FU induces cleftpalate by delaying reorientation of the shelves (Shah and Wong, 1980; Shah et al.,1984, 1989c). Arvystas and Cohen (1971) also observed 5-FU-induced cleft palate inmice due to a delay in the reorientation of palatal shelves.Collagen:Collagen is an important extracellular matrix protein of the connectivetissue. It represent one of the principle structural elements of the connective tissueand the most widely distributed protein in the animal kingdom.24In 1942, Schmitt and his colleagues observed cross striations of collagenfibrils through the electron microscope. Since then numerous reviews haveappeared in the literature about the assembly, structure, synthesis and functions ofcollagen (Gross, 1974; Shoshan and Gross, 1974; Miller and Matukas, 1974;Weinstock and Leblond, 1974; Fessler and Fessler, 1978; Prockop et al., 1979a, 1979b;Jackson, 1979; Burgeson, 1988).The basic type I collagen molecule is composed of three continuous helicalpolypeptide a-chains (two identical ai chains and one a2 chain) (Gross, 1974;Miller and Matukas, 1974; Shoshan and Gross, 1974). Each chain has a molecularweight of approximately 100,000 and contains approximately 1,000 amino acidresidues along its length. These chains are coiled into a left-handed triple helixwith three amino acids per turn. The three helical chains are then twisted aroundeach other into a right-handed helix. This unusual conformation gives themolecule a rigid rod like shape with dimensions of approximately 15x3000.Ä. Thechains are composed of repeating triplet structures, X-Y-Gly in which every thirdamino acid residue is glycine with the Y position often occupied by hydroxyproline(HYP) or hydroxylysine and the X position often occupied by proline (Miller, 1976;Prockop et al., 1979a; Burgeson and Morris, 1987). The occurrence of HYP incollagen is unique since this amino acid has been found in only a few otherproteins of vertebrates like elastin (Miller, 1976). In addition to the triple helicalregion there are short non-helical domains present at both the amino and carboxyterminals (Fessler and Fessler, 1978; Prockop et al., 1979a, b).25In recent years, it has become increasingly apparent that different tissues ofthe same organism are characterized by different collagen types. According toBurgeson and Morris (1987), collagen can be grouped into two main categories(Table 2) : (a) those that form periodically banded fibers, and (b) those that do notform banded fiber systems.A. Collagen that form fibers with uniform periodic cross striations:This group of collagens are capable of forming large fibers. These fibersrepresent the most common fiber form in connective tissue and are largelyresponsible for the rigidity of connective tissue matrices. These banded fibers canbe formed from three major types of collagen, type I, II, and III. This group is oftenknown as interstitial collagens.TYPE I. Type I is the most ubiquitous of all collagen species. It has beenisolated from the connective tissues of various structures and organs, includingskin, bone, tendon, dentin, cornea, fascia, periodontal ligament, gingiva and hardpalate (Bornstein and Sage, 1980). This type of collagen is largely responsible forthe rigidity of connective tissue matrices. It has been described in cell culture,tumors and in rapidly growing tissues (Bornstein and Sage, 1980). Thetropocollagen molecule of type I collagen is composed of [al(I)]2a2(I)]. Also thereis a variant of type I collagen of chain organization [al(I)]3 called type I trimer.TYPE II. The tropocollagen molecule of type II collagen is composed of threeidentical al chains designated as [al(II)]3. It is mainly seen in tissues such ashyaline cartilage, vitreous humor, the notochord and the sclera of the eye (Mayneand Von der Mark, 1983). It has been suggested that type II collagen may be capable26of forming an association between cartilage collagens and cartilage proteoglycans(Burgeson and Morris, 1987) through type IX collagen.TYPE III. The second most prevalent collagen species is type III. Type IIIcollagen is generally found in close association with type I (Burgeson, 1982). Thetype III tropocollagen molecule is composed of three identical al-chains [oclatO]3(Miller et al., 1971; Chung and Miller, 1974). It appears to form small fibrils inwhich the aminopropeptide of the collagen form of the molecule remainsuncleaved (Fleishmajer et al., 1981). This type of collagen is prevalent in tissueswhich require elasticity for normal function, eg, skin, blood vessels, gut andchorioamniotic membranes. It may play a role in development since it occurs invarious tissues of a developing fetus (Bhatnagar and Rapaka, 1975).B. Collagens that do not form uniformally banded fiber system:The most consistent feature distinguishing these collagens from theinterstitial collagens is their inability to form broad banded collagen fibers in vivoand in vitro. The genetically distinct collagen species of this group are summarizedin Table 2.TYPE IV. This collagen form contains two a chains, a1(IV) and a2(IV).This type of collagen is present in all structurally defined basement membrane and,for the most part secreted, by epithelial/endothelial cells (Timpl and Martin, 1982;Kleinman et al., 1986). Type IV collagen is present at the interface betweenepithelia and mesenchymal structures and it may play a direct role inmorphogenesis (Bornstein and Sage, 1980). The molecule is characterized by bothcollagenous and noncollagenous domains, and there are also interruptions in27morphogenesis (Bornstein and Sage, 1980). The molecule is characterized by bothcollagenous and noncollagenous domains, and there are also interruptions in28Table 2 Genetically Distinct Collagens [Adapted from Burgeson and Morris (1987)]A. The interstitial collagens-Collagens that form broad-banded fibersType I^[al (I)]2a2(I)^Found in most tissues exceptcartilage; major component of bone,tendon, skin, and dentin.Type I "trimer"^[a 1 (I)] 3^Some fetal tissues; product of certainmalignant and normal cell lines.Type IIM^[al (BM)]3^Cartilaginous tissues.Type IIm [al (11m)[3 Cartilagenous tissues.Type III^[al (III)]3^Found together with type I; inrelatively high concentrations inextensible tissues such as bloodvessels, skin, and gut.B. The minor collagens-less abundant collagens that do not form broad- banded fibersType IV^[al (IV)2a2(IV)^Major component of all basal laminae.(basement membrane collagen)al(IX)a2(IX)a3(IX)Type VType VIType IX("HMW-LMW")Type X("G collagen")Minor components of most tissuesexcept cartilage; fiber forms unknown.First identified in aortic intima, but nowthought to have a broad, but yetundefined, tissue distribution.Identified in amniotic membrane andskin; believed to be associated with allstratified epithelia; may beanchoring fibril protein.Identified as product of a variety ofcell types.Cartilaginous tissues.Product of cartilage hypertrophiccells[al(V)]2a2(V)[al(V)]3[a3(V)]3al(V)a2(V)a3(V)fal(VI)]2a2(VI)Type VII^[al(VII)]3"long-chain collagen"Type VIII^[al(VIII)]3"endothelial cell collagen"C. Minor cartilage collagens with unknown subunit compositionsla^ Compositionally similar to type V;2a structures, fiber forms, and functionsunknown.3a^ Compositionally similar to type II.29amino acid sequence, gly-X-Y, making this domain to be susceptible to a largenumber of proteolytic enzymes (Bornstein and Sage, 1980).TYPE V. This term refers to a family of several molecules of geneticallydistinct chains with similar structure (Burgeson et al, 1976). The chains aredesignated as a1(V), a2(V), a3(V) and a4(V) with different chain organization.Mayne and Von der Mark (1983) suggested that type V collagen is probably presentin all connective tissues with the exception of hyaline cartilage. Gay and associates(1981) observed type V collagen in association with cell surfaces, and, for thisreason, it was also described as cytoskeletal collagen. It is suggested that type Vcollagen may somehow be associated with larger fibrils of types I and II collagens,and may either hold them in position or may have a role in determining theirorganization (Mayne and Von der Mark, 1983).C. Minor cartilage collagen with unknown subunit compositions:Details of their structure and distribution are summarized in Table 2.Collagen Involvement in Organogenesis:Because collagen undergoes turnover at significant rates duringdevelopment, it has been implicated to play a role during development of manytissues and organs. Kleinman et al (1981) suggested that collagen is essential forcell adhesion and migration. The effect of collagen on adhesion could be direct ormediated via collagen-bound factors such as fibronectin and proteoglycans (Klebe,1974; Pearlstein, 1976; Engvall et al, 1978; Heine et al, 1990). Folkman and Tucker(1980) indicated that collagen influences cell growth via increasing adhesion. In30lung and salivary gland, collagen was implicated in allowing epithelial-mesenchymal interaction and subsequently the branching morphogenesis duringdevelopment (Grobstein and Cohen, 1965; Wessels and Cohen, 1968; Nakanishi etal., 1986a,b). Fukuda and associates (1988) observed that interstitial collagenasefrom bovine dental pulp, which degrades types I and III collagen, but not IV or V,inhibited in vitro branching of developing mouse submandibular gland epithelium.This observation led them to suggest that the collagen required for cleft initiationcould be type I and/or III. Similarly, Nakanishi and his colleagues (1988) suggestedthat collagen type III may be a key substance for either in vitro or in vivo cleftinitiation of the developing mouse submandibular epithelium. Chen and Little(1987) used anticollagen type IV antibodies on embryonic lung sections to showthat type IV collagen may be the extracellular scaffold within which earlybranching morphogenesis of lung epithelial cells may be accomplished. Recentlythe role of collagen type I was investigated in the developing mouse cornea by Bardet al (1988) who compared the stromal morphogenesis in normal corneas withthose of homozygous Mov13 mice which do not make collagen type I. Their datasuggested that collagen type I plays only a structural role in the stromalmorphogenesis and that its absence is not compensated for. It is clear from thesedata that synthesis of different types of collagen may play a significant role inmorphogenesis of an organ or structure.Collagen involvement in the secondary palate development:Over the past thirty years, considerable efforts have been focused on thepossible mechanism of mammalian secondary palate reorientation with a specialemphasis on the role of ECM. As already noted above, the involvement of31collagen during the mammalian secondary palate formation was studied only byfew investigators (Shapira, 1969; Pratt and King, 1971; Shapira and Shoshan, 1972;Uitto and Thesleff, 1979; Silver et al., 1984). In some of these studies, drugs such asglucocorticoids, which affects collagen synthesis (Shapira and Shoshan, 1972; Uittoand Thesleff, 1979), or P-aminoproprionitrile, which interfere with collagen fibreformation (Steffek et al, 1972; Pratt and King 1972) were used to observe their effectson palate development. Whether collagen played any critical role during thereorientation of mammalian palate, however, was unclear.32on palate development. Whether collagen played any critical role during thereorientation of mammalian palate, however, was unclear.33PURPOSE OF THE STUDYThe preceding analysis of the literature indicates that considerable effortshave been directed toward studying the cellular and biochemical sequence ofevents and their possible molecular regulation during morphogenesis of secondarypalate in mammals. Among the issues that have been discussed in these studies isthe mechanism responsible for the reorientation of palatal shelves, a phenomenonunique to mammals. Although an increase in collagen synthesis has beenobserved during the morphogenesis of mammalian palate, its contribution to thereorientation of shelves remains unclear. Earlier studies were performed either invitro, where shelf reorientation could not be studied (Uitto and Thesleff, 1979), or invivo where collagen synthesis was studied at an interval of 12-24 hours (Shapira,1969; Pratt and King, 1971; Shapira and Shoshan, 1972; Silver et al., 1984). In thelater instance, the interpretation of data after such a prolonged intervals are post-hoc since reorientation of palatal shelf in rodents (mice, rat, hamster, etc.) is rapidoccurring in 2-3 hours (Walker and Fraser, 1956; Coleman, 1965; Shah andChaudhry, 1974a). Also extensive intervals in the experimental design would notallow one to characterize subtle alterations which may occur at specific stages ofpalate development. Hence, the first purpose of this study is to investigatecollagen synthesis during the reorientation stage of the secondary palatemorphogenesis in hamster. Since, during avian palatogenesis, shelf reorientationstage is absent, and if the collagen synthesis is involved in regulating thereorientation of palatal shelf in mammals, one would expect that the profile of34collagen synthesis in birds would be different from that seen during mammalianpalatogenesis. Therefore, the second purpose of this study is to investigate thehitherto unknown aspect of collagen synthesis during quail palate morphogenesis.Cleft palate is one of the major malformations induced by 5-FU in mammals(Shah and Wong, 1980; Shah et al., 1984), and bird (Karnofsky et al., 1958; Ruddickand Runner, 1974). Since, unlike mammals, the morphogenesis of 5-FU-inducedcleft palate is not studied in birds, the third purpose of the current study is toanalyse the effect of 5-FU on palate development in quail. Finally, since 5-FUdelays the shelf reorientation stage in hamster (Shah and Wong, 1980; Shah et al.,1984, 1989c), a stage which is absent in birds, the collagen synthesis will be analysedboth in hamster and in quail following treatment with 5-FU to further analyse,from a comparative biology viewpoint, the involvement of collagen synthesisduring vertebrate palate development.35according to HH staging method and observed a high correlation between the ageof quail embryo and the stage of its development. Also, subsequently, this stagingmethod was employed successfully in studying both in vivo and in vitropalatogenesis in quail by Shah et al (1985a) and Shah and Cheng (1988).The quail embryonic secondary palate was dissected, and grouped accordingto the HH stage. An average of 12 ± 2 staged palates were pooled. The pooledpalates were subjected to an incubation and post-incubation protocol similar to theone described above for hamster.The samples of both hamster and quail palates were then used for one of thefollowing assays:1. Collagen digestion assay.2. Total protein determination using Biuret method as described by Gornalland associates (1948).3. High performance liquid chromatography (HPLC).4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).C.^Collagen digestion assayThe solubilized samples (both hamster and quail, as described in B) weredialysed against 0.5 N acetic acid (0-4°C). During dialysis, acetic acid solution waschanged four times following a minimum duration of six hours. From eachdialysis bag 1m1 of sample was pipetted into an eppendorf vial for determination oftotal protein (described below in D). The remaining samples were dialysed furtheragainst 0.5 M Tris-HC1 buffer, pH 7.4 (0-4°C). The Tris-HCL buffer was changed fourtimes at six hours intervals.36Following dialysis, 0.4m1 of each radiolabelled samples were digested at 37°Cfor four hours with 100111 of 25 units (0.2mg/m1) bacterial collagenase(Clostridiopeptidase A; EC, from Clostridium histolyticum; type VII, SigmaChemicals, St. Louis, Missouri, USA, Catalog # C-0773, Lot # 79F6817) in thepresence of 0.1mM N-ethylmalemide. Then, to each sample, 10111 of 10% FCS and125111 of a mixture of 10% trichloroacetic acid (TCA) and 1% tannic acid (TA) wereadded, and left overnight at 0-4°C to precipitate the proteins. Subsequently, thesamples were spun in a microcentrifuge (Biofuge A, Heraeus-Christ Gmbh, W.Germany) at 13,000 rpm for 40 minutes. The pellets were discarded and 200111 ofthe supernatant from each sample was counted in a liquid scintillation counter(Phillips, Holland, Model PW 4700). The measurement was corrected for thecounting efficiency, and disintegration per minute (DPM) was determined.D.^Measurement of total proteinThe total protein was determined by Biuret method described by Gornalland associates (1948). To the acetic acid-dialysed samples in eppendorf vials(described above in C) 2501.1.1 of 10% TCA-1%TA mixture was added. The vials werevortexed and left overnight at 0-4°C. The samples were spun in a microcentrifugefor 30 minutes and the supernatant discarded. To the precipitate 1m1 of 0.05NNaOH was added, vortexed and left overnight at 0-4°C. Subsequently, 0.1m1 ofdigest was used for the total protein determination using human albumin andglobulin in saline (Sigma Diagnostics, Catalog # 540-10, Lot: 48F-6087, St. Louis,Missouri, USA) as a protein standard.37Both the 14C-proline DPM and the total protein measurements werestandardized and expressed as DPM/mg protein. Each experiment was repeated 3-5times, and means ± SD calculated. Both the control and drug-treated data on therates of collagen synthesis were analyzed by the ANOVA method. The comparisonbetween treated and control groups were evaluated by Student's-t test at asignificance level of 0.05.E. High performance liquid chromatographyI. EquipmentThe HPLC system (Waters Associates, Millipore, Massachusetts, USA)consisted of a Model 730 Data processor, a Model 721 chromatography controlstation, two Model 510 solvent pumps, a Model 440 absorbance detector(wavelength 254 nm), and a Model 710B automatic sample processor. A Pico-Tagcolumn (Waters Associates, Millipore, Massachusetts, USA) was kept at atemperature of 38° ± 0.1°C. A Pico-Tag work station (Waters Associates, Millipore,Massachusetts, USA) was used for vapour hydrolysis of the dried specimens.Incorporation of proline into procollagen and its subsequent hydroxylationto HYP was measured by HPLC. HYP is an amino add which is virtually unique tocollagen (Miller, 1983). Thus analysis of the amount of HYP can be used toevaluate collagen biosynthesis (Miller, 1983; Svanberg, 1987).II. Sample preparation201.d aliquots of the acetic acid digested samples, as described above in B,were brought to dryness in 6 x 50 mm pyrex culture tubes (Corning glass works,Corning, New York, USA) using a Pico-Tag work station. (Up to 12 samples and38one standard in culture tubes can be processed per glass reaction vial). Then, 200121of 6 N hydrochloric acid containing 0.1% phenol was added to the bottom of thereaction vial. Oxygen was removed from the reaction vial by three successiveevacuations interposed by flushing with nitrogen. After the third evacuation, thereaction vials were sealed under vacuum and samples hydrolysed at 110°C for 20-24 hours. After cooling the reaction vial, each sample tube was wiped dry, thereaction vial cleaned and dried, and the sample tubes placed back into the reactionvial and then brought to dryness. Next, 20111 of fresh redrying solution(ethanol:water:triethylamine (TEA), 2:2:1 by volume) was added to each samplewhich was then vortexed and brought to dryness. Subsequently 50g1 of freshderivatization solution (ethanol:water:TEA:phenylisothiocyanate (PITC), 7:1:1:1, byvolume) was added to each sample, vortexed, left for 20 minutes at roomtemperature and brought to dryness using the Pico-Tag work station. Thereafter,200121 of sample diluent (0.05 M phosphate buffer) was added to each sample,vortexed for 10 seconds and then passed through Millex-HV4 filters into lowvolume inserts (Waters Associates, Millipore, Massachusetts, USA).III. ChromatographyFor chromatography, 25111 of the reconstituting sample, was injected onto aC18 Pico-Tag Column for separation and determination of PITC derivative of HYP.The flow program for the solvent gradient of buffer A and B are given in Table 3.(Buffer A consists of 19 g of sodium acetate trihydrate, 1000 ml water, 0.05 ml TEA;titrated to pH 6.4 with glacial acetic acid. Then 60 ml of acetonitrile were added to940 ml of this solution. Buffer B consisted of 60% acetonitrile in 40% water byvolume). Each sample was separated over a period of 12 minutes followed by a39gradient washing and re-equilibration for 8 minutes. Chromatographed profile ofthe sample was obtained (Figures 2 & 3) which was then compared with thestandard amino acid profile (Figure 4) to determine the location of HYP peak.40Table 3.^The flow program for the solvent gradient of buffer A and B forchromatography:Time^Flow (ml/min)(min.sec.)% of buffer A* % of buffer B* CurveInitial^1.010.0^1.010.5^1.011.5^1.012.0^1.5125^1.520.0^1.520.5^1.0100540000010010010004610010010000055666666*^For composition of buffer see text41Figure 2^Chromatographic profile of sample obtained from a control hamsterpalatal tissue on day 12:04 of gestation showing hydroxyproline peak.IC"5IFigure 2421,143Figure 3^Chromatographic profile of sample obtained from a control quailpalatal tissue on day 8 of incubation showing hydroxyproline peak..11  ■■■444ti.a.,Figure 3•245Figure 4 Chromatographic profile of standard amino acids showinghydroxyproline peak.11I146I$tF^in1I \.Figure 4I47From the profiles, the area of absorbance of HYP in correspondence with retentiontime of samples were calculated for both the control and 5-FU-treated palates. Thecontrol and 5-FU-treated data were compared by student's t-test at the 95% level ofsignificance.F. Collagen Extraction for SDS-PAGEThe isotypes of collagen were separated using a modified method of Chungand Miller (1974) as described by Narayanan and Page (1976). Because during theinitial efforts of identifying isotype of collagen in quail palate was not successful,the quail samples were double-labelled with 14C-proline and 14C-glycine (SpecificActivity 250p.Ci/mmole, New England Nuclear, Boston, Massachusetts, USA).Both the drug-treated and control samples, left for 24-48 hours in 2m1 of 0.5N aceticacid as described in B, were digested by 200pg pepsin (100n/m1) at 4°C for 24 hours.Lathyritic rat skin collagen carrier (5mg) was then added to the samples followed byslow addition of solid NaC1 to 1.7M, to help precipitate the collagen. The mixturewas allowed to stand overnight at 4°C, and centrifuged (Beckman Instruments,Model L8-70 Ultracentrifuge, Palo Alto, California, USA) at 27,000g for 25 minutesat 4°C to collect the collagen precipitate. The supernatants were discarded and thepellets dissolved in 0.5ml of 0.5N acetic acid containing 10mM EDTA for aminimum of 24 hours at 4°C. Subsequently, the samples were spun in amicrocentrifuge at 13,000 rpm for 10 minutes. The pellets were discarded,supernatant lyophilized, and then, reconstituted with 100111 of 0.5N acetic acid.G. SDS-Polyacrylamide Gel ElectrophoresisSodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)48was performed using 7.5% polyacrylamide in SDS Tris-glycine, pH 8.8 (Laemmli,1970). An aliquot (36p1) of extracted collagen, as described in F, was dissolved in9121 of "Laemmli" sample buffer (composed of 4m1 of distilled water, lml of 0.5MTris-HC1 pH 6.8, 0.8m1 glycerol, 0.4m1 of 2-13 mercaptoethanol, 1.6m1 of 10% SDSand 0.2m1 of 0.05% bromophenol blue), boiled for 5 minutes, and loaded into theSDS-PAGE as described by Laemmli (1970). The electrophoresis was performed at125V, 75mA for 15 minutes. At this stage, the gel was interrupted by switching offthe current and the sample wells filled with 5% v/v 2-mercaptoethanol in samplebuffer to separate collagen type I from collagen type III. Mercaptoethanol wasallowed to diffuse into the gel for 15 minutes before the current was again switchedon to resume electrophoresis for a further period of one hour (Sykes, et al., 1976).Subsequently, the gels were dehydrated in dimethyl sulphoxide and soaked in asolution of 2,5-diphenyloxazol in dimethylsulphoxide. The gels were then driedand each gel was exposed on 8"x10" Cronex X-ray (E. I. DuPont de Nemours,Willington, Delaware, USA) film at -70°C for 15 days. Radiolabelled bands,representing different types of collagen, were identified using 14C-labelled rainbowprotein molecular weight marker (Specific Activity 37KBq, 112Ci, Amersham, U.K.,Batch 13, Lot 4).H.^Light microscopyThree control and three 5-FU-treated hamster embryos were obtainedrandomly from different litters at various gestational times as described in49Materials and Methods, Section B. Three control and three 5-FU-treated quailembryos were also obtained between days 5-9 of incubation. The embryos wereimmersed in Bouin's solution for 72 hours. They were then dehydrated throughan ascending concentration of ethanol, starting at 30%. Upon reaching 100%concentration, the embryos were processed through three changes of chloroform,and embedded in paraffin to procure frontal sections. Seven micron serial sectionswere obtained and stained with Haematoxylin and Eosin for light microscopicexamination of the status of palatal development in hamster and quail embryos.50RESULTS5-FU Dose-Time Response in QuailThe effects of different concentrations of 5-FU, administered on differentdays of incubation, on palate development in quail is outlined in Table 4. One maydeduce from the table that 5-FU treatment on different days of incubation did notaffect the mean fetal weight. Drug treatment on day 4 of incubation caused highmortality rate (P<0.05) following a dose of 75-150gg 5-FU. However, only 100-150gg5-FU increased the width between the palatal shelves (P< 0.05).The width between the palatal shelves remained unaffected following 5-FUtreatment on day 5 of incubation, although there was an increase in the mortalityrate at a dose range of 125-150pg,. After 5-FU administration on day 6 of incubationneither the mortality rate nor the width between the palatal shelves has affected.On the basis of this analysis, it is clear that an administration of 100gg 5-FU on day4 of incubation to quail eggs affords the best dose-time regimen to increase thewidth between the palatal shelves, i.e., cleft palate. Hence, this dose-time regimenwas used in the further studies on the effect of 5-FU on quail secondary palatedevelopment.Light Microscopic Observations of the Developing Secondary PalateHamsterControlLight microscopic observations indicated that the individual morphologicevents of normal palatal development in hamster can be seen to fall within one of51the following four stages:Stage 1:^The palatal shelves, composed of mesenchyme covered by anepithelium, are vertical alongside the tongue (Figure 5).Stage 2:^The palatal shelves are changing from a vertical position, on the sideof the tongue, to a horizontal position above the tongue (Figure 6).Stage 3:^The palatal shelves are horizontal above the tongue (Figure 7).Stage 4:^The horizontal palatal shelves are united with one another thusseparating the oral and nasal cavities (Figure 8). The union of thehorizontal shelves results in the formation of an epithelial seam(Figure 8), which eventually fragments to establish a mesenchymalcontinuity between the united palatal shelves (see Figure 13 in Shahand Chaudhry, 1974a).Between days 11:00 and 12:00 of gestation, the palatal shelves are vertical(Stage 1). During the next four hours, i.e., until day 12:04 of gestation, the palatalshelves reoriented (Stage 2) to a horizontal position (Stage 3) and united (Stage 4).5-FU-treatedFollowing 5-FU treatment, only the first three stages of palate developmentare observed. The palatal shelves are vertical (Stage 1) between days 11:00 and 12:16of gestation (Figure 9). The reorientation of 5-FU treated shelves, however, isdelayed by 12-20 hours. Unlike controls, which reunit between days 12:00 and12:04 of gestation, the treated shelves reorient (Stage 2) from a vertical to ahorizontal position (Stage 3) between days 12:16 and 13:00 of gestation (Figures 10 &11). Stage 4 (fusion) seen in controls, is not observed in 5-FU-treated fetuses.52Table 4. Effects of 5-fluorouracil on mortality, fetal weight and the palate gap inquail.Incubation Dose No. of Mortality Mean weight of^Man palateday of (gg) eggs (%) live fetuses^gap*injection ±SD (gm) ±SD (mm)4 Control 31 0 7.73±0.45^0.59±0.0275 15 13** 7.86±0.41^0.63±0.031100 31 13** 6.80±0.75^0.71±0.036**125 34 13** 7.33±0.59^0.78±0.070**150 34 15** 7.79±0.40^0.71±0.054**5 Control 30 0 7.35±0.47^0.36±0.01575 34 0 7.92±1.20^0.36±0.031100 28 0 7.90±0.65^0.40±0.012125 31 6** 7.81±0.73^0.41±0.026150 39 8** 6.91±0.97^0.44±0.0256 Control 30 6 7.35±0.40^0.40±0.11075 36 0 7.76±0.42^0.41±0.130100 32 6 6.66±0.72^0.51±0.119125 35 5 6.96±0.61^0.49±0.112150 32 0 7.68±0.61^0.55±0.108* Distance between palatal shelves measured on day 15 of incubation.** P< 0.05; Student's Rest.53QuailControlThe quail palatal shelves develop as horizontal ridges from the beginning ofday 5 of incubation (HH stages 29-30). The horizontal ridges form intraorally fromthe medial aspect of the maxillary processes, dorsal to the tongue (Figure 12). Eachridge (shelf) consist of a core of mesenchyme enveloped by an epithelium.Between days 6-8 of incubation (HH stages 31-34), the palatal shelvescontinue to grow horizontally toward one another until they approximate withone another on day 9 (HH stages 35-36; Figures 13, 14 & 15). A distinct osteogenicsite is present in the mesenchyme (Figure 14).On day 9 (HH stages 35-36), when the opposing horizontal shelvesapproximate, several small epithelial invagination from the oral side extend intothe mesenchyme (Figure 15). They represent precursors of minor salivary glands(Shah et al., 1985a). In addition, a distinct, well differentiated osteogenic site ispresent in the mesenchyme (Figure 15).5-FU-treatedFollowing 5-FU administration, the palatal ridges extend horizontally(Figure 16) from the maxillary processes on day 5 of incubation (HH stages 29-30).The structure of a 5-FU-treated palatal ridge resemble that seen in the control.Subsequently, between days 6-7 of incubation (HH stages 31-33), thehorizontal shelves grow toward one another (Figure 17).On days 8-9 of incubation (HH stages 34-36), although the palatal shelvescontinue their growth toward one another they never approximate (Figure 18 &19). Unlike their control counterparts, a wide gap persist between the medial edges54Figure 5 Frontal section through the secondary palate in a control hamsterembryo on day 11:18 of gestation. The palatal shelves (P) are vertical(Stage 1) on the sides of the tongue (T). ONC-oronasal cavity.Hematoxylin and Eosin stain. 125X.Figure 6 Frontal section through the secondary palate in a control hamsterembryo on day 12:00 of gestation. The palatal shelves (P) arereorienting (Stage 2) above the tongue (T). ONC-oronasal cavity.Hematoxylin and Eosin stain. 125X.Figure 7 Frontal section through the secondary palate in a control hamsterembryo on day 12:02 of gestation. The palatal shelves (P) arehorizontal (Stage 3) above the dorsal surface of the tongue (T).Hematoxylin and Eosin stain. 125X.Figure 8 Frontal section through the secondary palate in a control hamsterembryo on day 12:04 of gestation. The opposing palatal shelves (P) areunited with one another (Stage 4) separating the nasal cavity (NC)from the oral cavity (OC). Hematoxylin and Eosin stain. 125X.Figure 9 Frontal section through the secondary palate in a 5-FU-treatedhamster embryo on day 12:00 of gestation. The palatal shelves (P) arevertical (Stage 1) on the sides of the tongue (T). ONC-oronasal cavity.Hematoxylin and Eosin stain. 125X.Figure 10 Frontal section through the secondary palate in a 5-FU-treatedhamster embryo on day 12:16 of gestation. The palatal shelves (P) arereorienting (Stage 2) above the tongue (T). ONC-oronasal cavity.Hematoxylin and Eosin stain. 125X.Figure 11 Frontal section through the secondary palate in a 5-FU-treatedhamster embryo on day 12:20 of gestation. The palatal shelves (P)arehorizontal (Stage 3) above the surface of the tongue (T). Hematoxylinand Eosin stain. 125X.5556of 5-FU treated horizontal palatal shelves (cf. Figure 14 & 18, and 15 & 19). Inaddition, the precursors of minor salivary glands, seen in control palates on day 9of incubation, are absent in 5-FU-treated palates (cf. Figures 15 & 19). Also, acondensation of mesenchyme is seen in the treated palatal shelves, but sites ofosteogenesis observed in control palates, are absent in 5-FU-treated ones (Figures 18& 19).Measurement of Incorporation of 14C-proline into CollagenHamsterControlIn hamster, between days 11:00 and 12:00 of gestation, i.e., the period whenpalatal shelves are growing vertically (Figure 20), the rate of collagen synthesis,remains unchanged (Figure 20). During the next four hours, i.e., between days12:00 and 12:04 of gestation, when the palatal shelves reorient from a vertical to ahorizontal plane and fuse, the rate of collagen synthesis doubles (P<0.05). In theensuing four hours, when the epithelial seam is fragmenting (Shah and Chaudhry,1974a), the rate decreases. On day 12:08 of gestation, the rate of collagen synthesis isapproximately 40% of that seen four hours earlier (P<0.05). Subsequently, whenthe palate closed completely through its length, the rate of collagen synthesisincrease approximately 6-fold.5-FU-treatedFollowing 5-FU treatment between days 11:04 and 12:02 of gestation, the rateof colagen synthesis in the developing secondary palate of hamster remainunchanged (Figure 20). During this period, however, the rate is lower than theFigureFigureFigureFigure5712 Frontal section through the secondary palate in a control quailembryo on day 5 of incubation. The palatal primordia (arrows)develop horizontally from the maxillary process (M) above thetongue (T). Hematoxylin and Eosin stain. 100X.13 Frontal section through the secondary palate in a control quailembryo on day 7 of incubation. palatal shelves (P) are horizontalabove the tongue (T). Hematoxylin and Eosin stain. 100X.14 Frontal section through the secondary palate in a control quailembryo on day 8 of incubation. The approximated palatal shelves (P)are above the dorsal surface of the tongue (T). Site of osteogenesis (0)within the palate. Hematoxylin and Eosin stain. 100X.15 Frontal section through the secondary palate in a control quailembryo on day 9 of incubation. Showing approximated palatalshelves (13) above the tongue (T). Minor salivary gland (G) within theshelves Site of osteogenesis (0). Hematoxylin and Eosin stain. 100X.Frontal section through the secondary palate in a 5-FU-treated quailembryo on day 5 of incubation. The palatal primordia (arrows)develop horizontally from maxillary processes (M) above the tongue(T). Hematoxylin and Eosin stain. 100X.Frontal section through the secondary palate in a 5-FU-treated quailembryo on day 7 of incubation. The palatal shelves (P) are horizontalabove the tongue (T). Hematoxylin and Eosin stain. 100X.Frontal section through the secondary palate in a 5-FU-treated quailembryo on day 8 of incubation. The palatal shelves (P) are horizontalabove the dorsal surface of the tongue (T). Site of osteogenesis (0)within the shelves (cf. Figure 13). Hematoxylin and Eosin stain. 100X.Frontal section through the secondary palate in a 5-FU-treated quailembryo on day 9 of incubation. The palatal shelves (P) are above thetongue (T). Site of osteogenesis (0) (cf. Figure 15). Hematoxylin andEosin stain. 100X.Figure 16Figure 17Figure 18Figure 195859Figure 20 Rate of collagen synthesis in the control and 5-FU-treated developingsecondary palate of hamster embryos between days 11 and 13 ofgestation.Figure 21 Rate collagen synthesis in the control and 5-FU-teated developingsecondary palate of quail embryos between days 5 and 10 ofincubation.20000 - —411-- control---•--- treated01010000 -111^ 12Gestation Time (day:hour)13C.1cl) 1000-Hfu0 800o..1'toci 0. .....^600an cz43) ry,s g>, CL4 400cnrcs'3'ro4 24 2 00OC..4.)—21— control—40— treated7^8^9^10^11Incubation day560Figure 20Figure 2161controls (P<0.01). It then increases approximately 10-fold to peak at day 12:12 ofgestation (P<0.01). On day 12:16 of gestation, when reorientation begins in the drug-treated shelf, the rate drops 8-fold before showing a 4-fold increase on day 13:00 ofgestation (Figure 20).QuailControlThe rate of collagen synthesis in the developing secondary palate of quail,between days 5 and 6 of incubation, remains unchanged (Figure 21). It increasesapproximately 4-fold between days 6 and 8 of incubation (P<0.01). During the next24 hours, the rate drops 35% (P<0.01) before levelling off between days 9 and 10 ofincubation (Figure 21).5-FU-treatedThe rate of collagen synthesis in 5-FU-treated secondary palate of quailembryos, between days 5 and 7 of incubation, demonstrates the same pattern ofchange as that seen in the controls but overall uptake is significantly decreased. Itincreases 3-fold to peak on day 8 of incubation (P<0.05), but drops on day 9 beforelevelling off on day 10 of incubation (Figure 21).High Performance Liquid ChromatographyBecause in hamster controls the reorientation of palate occurs between days12:00 and 12:04 of gestation, and in 5-FU-treated between days 12:16 and 13:00 ofgestation the amount of HYP was determined at these times.In quail, because the profile of collagen synthesis changes between days 6 and9 of gestation, the amount of HYP was measured during these periods.62Figure 22 Hydroxyproline content in the control and 5-FU-treated developingsecondary palate of hamster embryos at their respective periods ofreorientation. (No comparable controls was obtained on days 12:16and 13:00 of gestation because in control the palate was already closed).Figure 23 Hydroxyproline content in the control and 5-FU-treated developingsecondary palate of quail embryos between days 6 and 9 of incubation.8 98   controlIla treated7Incubation day6Figure 22633- IN controlIli treated012:00^12:04^12:16^13:00Gestation time (days:hours)Figure 2364Figure 24 Total protein content in the control and 5-FU-treated developingsecondary palate of hamster embryos between days 11:04 and 13:00 ofgestationFigure 25 Total protein content in the control and 5-FU-treated developingsecondary palate of quail embryos between days 5 and 10 ofincubation.0.10 - • Control• TreatedZsrz^0.08-'71t:14--a)E^0.06-=.11"5^0.04-ra.740^0.02-E-10.005 6 7^8Incubation day9 1 0■ control® treated0.20-65Figure 2411:04 11:0811:1211:1611:2012:0012:02 12:0412:0812:1212:1612:2013:00Gestation time (days:hours)Figure 2566HamsterControlThe result on HYP content in the developing secondary palate of hamsterembryos are summarized in Figure 22. The results show that the amount of HYP,as represented by the area of absorption on the chromatogram, remains unchangedbetween days 12:00 and 12:04 of gestation (P>0.05). This observation is in contrastto the increase in the rate of collagen synthesis observed during this period in thecontrol palate of hamster (Figure 20), suggesting that only a portion of 14C-prolineis utilized for hydroxylation to HYP, and hence for collagen synthesis. The spurt in14C-proline uptake, thus may reflect synthesis of both collagenous and non-collagenous proteins.5-FU-treatedThe observation on HYP content in the 5-FU-treated developing secondarypalates of hamsters are outlined in Figure 22. The data indicates that in spite of asignificantly low uptake of 14C-proline between days 12:00 and 12:04 of gestation in5-FU-treated palates, the amount of HYP accumulated is comparable to thecontrols. This would indicate that, in comparison to controls, proportionally alarge amount of HYP is accumulated in 5-FU-treated palates, perhaps utilizingmuch of the 14C-proline for collagen synthesis. On day 12:16 of gestation, when thereorientation begin in 5-FU-treated palates, and when the rate of 14C- prolineuptake is comparable to that seen in control palates on day 12:00 of gestation, theHYP accumulation is five-fold higher possibly reflecting accumulated HYPfollowing high uptake of 14C-proline on day 12:12 of gestation. Subsequently,however, even though the 1 4C-proline uptake increase approximately four-fold,67the HYP level remains unchanged. This suggests that perhaps much of the 14C-proline may have been utilized for non-collagenous protein synthesis.QuailControlThe data on HYP content in the developing secondary palate of quailembryos are summarized in Figure 23. One may infer from the data that theamount of HYP in the control is 50% higher on day 8 than on day 6 of incubation(P<0.05). These data are consistent with the observations that the uptake of 14C-proline in the developing palate of quail is 4-fold higher on day 8 than on day 6 ofincubation (Figure 21). The data further show that although the rate of 14C-prolineuptake is higher on days 7 and 9 than on day 6 (Figure 21), the HYP content aresignificantly lower (P< 0.05) on days 7 and 9 than on day 6 of incubation. Thiswould indicate that, in comparison to day 6, much of the 14C-proline uptake ondays 7 and 9 is associated with the synthesis of non-collagenous, rather thancollagenous, proteins.5-FU-treatedThe results of HYP content in the 5-FU-exposed secondary palate of quailembryos are summarized in Figure 23. The data indicates that, in comparison tocontrols, 5-FU treatment of quail results in a reduction in the amount of HYP inthe developing palate on days 6 and 8 of incubation (P<0.05). Although thereduction in HYP content is more severe on day 8 (75%) than on day 6 (50%), thetotal amount of HYP on both days in 5-FU-treated quail palate is similar, perhapssuggesting that much of the 14C-proline uptake on day 6 may be related to thesynthesis of non-collagenous proteins. In addition, although the rate of 14C-68proline uptake in 5-FU treated palates is similar on days 6, 7 and 9 (Figure 21), theamount of HYP is lower on day 7 than on days 6 and 9 of incubation. This mayindicate a higher rate of accumulation of non-collagenous proteins on day 7 thanon days 6 and 9.Measurement of Total ProteinHamsterControlThe data on the total protein content of the developing secondary palate inhamster are outlined in Figure 24. One may infer from the figure that, betweendays 11:04 and 12:04 of gestation, i.e., during the period when the shelvescompleted their vertical growth and reoriented to a horizontal plane and fused, theamount of total protein in the developing secondary palate doubles (P<0.05). Inthe ensuing 20 hours the amount of total protein levell-off.5-FU-treatedThe results of the measurement of total protein content following 5-FUtreatment are summarized in Figure 24. In general, between days 11:04 and 13:00 ofgestation, the total protein contents in 5-FU treated palates is approximately 50-75%lower than control palates (P<0.05). Also, unlike controls, the amount of totalprotein in 5-FU treated palates is unchanged between days 11:04 and 13:00 ofgestation, i.e., the period during which the cleft palate was forming.QuailControlThe data on the amount of total protein in the developing secondary palate69of quail are summarized in Figure 25. The data show that in the control palates ofquail embryos there is a gradual increase in the amount of total protein betweendays 5 and 10 of incubation (P<0.05). Also, the amount of total protein on day 10 istwice the amount seen on day 5 of incubation.5-FU-treatedThe results of total protein content in 5-FU treated palates are outlined inFigure 25. One may deduce from the figure that the total protein contents in 5-FUtreated palates is 50-70% lower than their control counterparts between days 5 and10 of incubation (P<0.05). Furthermore, as observed in the 5-FU-treateddeveloping palate of hamster, the amount of total protein content remainsunchanged during the period of observation.SDS-Polyacrylamide Gel ElectrophoresisHamsterThe results on SDS-Polyacrylamide Gel Electrophoresis to separate differentcollagen isotypes in the developing palate of hamster embryos are presented inFigure 26. Collagen isotypes were determined in both the control and drug-treatedanimals at the time of shelf reorientation. In control hamster embryos on day12:02 of gestation only type I collagen is detected by SDS-PAGE. Similarly after 5-FUadministration, only type I collagen is seen on SDS-PAGE of embryonic palates atthe time of delayed shelf reorientation, i.e., on day 12:16 of gestation. In bothcontrols and drug-treated palates, a delayed reduction was performed using 2-mercaptoethanol to separate type I isomers from type III. The results indicated thateither type III collagen is present in such a small quantity that it was not possible to70detect by SDS-PAGE method in the present study or that it does not exist in vivo inthe developing secondary palate of hamster.QuailThe observations on SDS-Polyacrylamide Gel Electrophoresis in developingquail secondary palate are documented in Figure 27. In both the control and drug-treated quail palates collagen types were determined on day 8 of incubationbecause there is an increase in collagen synthesis at this time. Like hamster, inboth the control and 5-FU treated quail palates only collagen type I is detected onSDS-PAGE electrophoresis. When delayed reduction was performed using 2-mercaptoethanol to separate type I from type III, type I was the only isomer ofcollagen identified.71Figure 26 SDS-PAGE showing type I collagen in the control and 5-FU-treateddeveloping secondary palate of hamster embryos on days 12:02 and12:16 of gestation.Track 1. Molecular weight Marker (MW 14300-200000).Track 2. Standard type I collagen showing al and a2 chains.Track 3. Control secondary palate of hamster embryo (12:02).Track 4. 5-FU-treated secondary palate of hamster embryo (12:02).Track 5. Control secondary palate of hamster embryo (12:16).Track 6. 5-FU-treated secondary palate of hamster embryo (12:16)721^2^3^4^5^6rit200K 4_0/1`Ce 2prop-Aued92 . 5 K Ole69K as46K up30K INV 2673Figure 27 SDS-PAGE showing type I collagen in the control and 5-FU-treateddeveloping secondary palate of quail embryos on day 8 of incubation.Track 1. Molecular weight Marker (MW 14300-200000).Track 2. Standard type I collagen showing al and a2 chains.Track 3. Control secondary palate of quail embryo (Day 8).Track 4. 5-FU-treated secondary palate of quail embryo (Day 8).741^2^3^475DISCUSSIONIn the initial study, a dose-time relationship of the effect of 5-FU on palatedevelopment in quail was determined because this information was not availablein the literature, and was crucial for subsequent studies on the effect of 5-FU oncollagen synthesis in the developing palate of quail. The data of the present studyshow that following 5-FU treatment of quail eggs on day 4 of incubation, the rate ofmortility in embryos increased. This may be due to a variability in the embryonicgrowth rates during early phases of development or it could be due to a possiblegenetic variability of susceptibility of embryos to 5-FU as well. During the first fourdays of incubation, the variability in HH stages, implying variabilities in growthrates of quail embryos is greater than at the later times of incubation (Pedgett andIvey, 1960; Sato et al., 1971). Thus, developmentally older embryos may berelatively less vulnerable to the 5-FU assault than younger embryos on day 4 ofincubation. Following various experimental manipulations during early phases ofdevelopment, an increased rate of mortility of avian embryos has also been notedin other studies (Landauer, 1954, 1976; Karnofsky et al., 1958; Singh and Gupta,1972; Ruddick and Runner, 1974; Grubb and Montiegel, 1975; Mann and Persaud,1978; Jelinek and Kistler, 1981; Gilani and Chatzinoff, 1983). It has been indicatedthat during the early phases of development, embryos are generally highlyvulnerable to the effects of environmental agents because rapidly dividingembryonic cells are more susceptible than non-dividing cells to an assault by anagent (Saxën, 1976; Scott, 1979). Perhaps this may explain an increase in mortility76rate following 5-FU administration on day 4 of incubation observed in the presentstudy.Results of the present study further showed that the treatment of quail eggswith 1001.1,g 5-FU/egg on day 4 of incubation caused an increase in the distancebetween palatal shelves, eg, increased clefting of the palate. The drug showed littleor no adverse effect on fetal weight or distance between palatal shelves wheninjected on day 5 or 6 of incubation. These observations are consistent withfindings by Karnofsky et al (1958) and Ruddick and Runner (1974) who alsoobserved increased cleft palate in chick embryos following 5-FU treatment. Hence,injection of 10011g/egg 5-FU on day 4 of incubation was used in the present studyon subsequent experiments on quail.The light microscopic observations of palate development in controlhamster showed that the palatal shelves were vertical until day 12:00 of gestation.They reoriented and fused between days 12:00 and 12:04 of gestation. Theseobservations of the development of hamster secondary palate in the present studyare similar to those described earlier in normal hamster by Shah and Chaudhry(1974a), Shah and Travill (1976a), Shah (1979b), Shah and Wong (1980) and Kiso etal (1984). Thus, the temporal reproducibility of events of normal palatedevelopment is once again reconfirmed.In the present study, like controls, the 5-FU-treated palatal shelves werevertical on day 12:00 of gestation. Subsequently, the reorientation of hamsterembryonic palatal shelves occurred between days 12:16 and 13:00 of gestation, i.e., itwas delayed by approximately 16-20 hours. Fusion between the shelves neveroccurred and a cleft palate formed. These observations are consistent with earlier77data on 5-FU induced cleft palate in hamster reported by Shah and Wong (1980)and Shah et al (1984). Also, on the basis of their study in mice, Arvystas and Cohen(1971) showed that 5-FU affects reorientation of shelves to induce cleft palate. Amorphological mechanism of cleft palate formation by delayed reorientation ofshelves has been observed following administration of different environmentalagents in various mammalian species i.e., mice, rats, hamster, etc., (Table 1). Onthe other hand the morphological mechanism by which 5-FU-induces cleft palatein hamster differs from the mechanism of cleft palate development in hamsterfollowing treatment with other agents such as hydrocortisone, which preventfusion between the palatal shelves (Shah and Travill, 1976a), and 6-mercaptopurine, hadacidine and bromodeoxyuridine, which affect verticaldevelopment of the shelves (Burdett and Shah, 1988; Burdett et al., 1988; Shah etal., 1990; 1991). Finally, treatment of mice with cortisone over a four day period(Walker and Fraser, 1957), or of hamster with a single dose of 5-FU (present study)produces cleft palate by delaying reorientation of palatal shelves whileadministration of a single dose of hydrocortisone to pregnant hamsters affects thefusion between shelves in embryos (Shah and Travill, 1976a, b). These analysessuggest that differences amongst species, the nature of teratogenic chemicals, andtreatment schedule are some of the factors which may affect the morphologicalmechanism of cleft palate development in mammals.The light microscopic observation of the development of secondary palate incontrol quail in the present study showed that, unlike hamster, the quail palatalshelves develop horizontally ad initium on day 5 of incubation, and grow towardone another until approximation on day 8 of incubation. Thereafter, the shelves78never fuse with one another. These observations of quail secondary palatedevelopment are similar to those described earlier by Shah et al (1985a). A similarmode of palate development was also observed in other birds, e.g., chick (Shah andCrawford, 1980; Koch and Smiley, 1981), duck (Shah et al., 1987), and pigeon (Shahet al., 1988).Treatment of quail eggs with 5-FU resulted in an increased gap between thetwo horizontal palatal shelves. Numerous teratogenic agents which increasespalate clefting in birds, have been identified (Landauer, 1954; Karnofsky et al., 1958;Ruddick and Runner, 1974; Verrett et al., 1980; Tamarin et al.,1984). There are,however, no studies in the literature which analyzed the morphologicalmechanism of teratogen-induced cleft of secondary palate in any species of bird.Consequently, the light microscopic morphological results of the present study on5-FU-induced cleft palate in quail are difficult to compare. Nevertheless, it is clearthat 5-FU affects growth of the shelves towards one another leading to widening ofthe palate cleft.In the present study, a changing profile of uptake of 14C-proline, reflectingcollagen synthesis (discussed below), in the normally developing secondary palateof hamster embryos was observed. Specifically, at the time of shelf reorientation inhamster, the rate of 14C- proline uptake showed a two-fold increase. EarlierShapira (1969) and Shapira and Shoshan (1972) observed a two-fold increase of 3H-proline uptake in the normally developing palatal shelves of mice before shelfreorientation. In their study on rats, Pratt and King (1971), noted that the amountof collagen doubled at the time of palate closure. Hassell and Orkin (1976)observed 82% increase in the rate of incorporation of radiolabelled proline in the79vertical shelf, prior to shelf reorientation. One of the drawbacks of these earlierstudies on mice and rats is that the collagen synthesis was analyzed at 12-24 hourintervals. In such experimental circumstances, subtle changes in the rates ofradiolabelled proline uptake, during the shelf reorientation (which in mammalsoccur very rapidly), are not localized. Nevertheless these observations suggest that,at least during mammalian palatogenesis, a spurt in the uptake of radiolabelledproline may be related to the reorientation of the palatal shelves.Because collagen synthesis is generally measured by incorporation of prolineinto procollagen, and its subsequent hydroxylation to HYP, an amino acid which isvirtually unique to collagen (Bornstein and Traub, 1979), determination of levelsof HYP is considered to be an useful measure of collagen synthesis (Svenberg,1987). Also, it is generally accepted that both synthesis and degradation of collagenin a tissue can be monitored by the release of HYP (Svenberg, 1987). One may notethat in interpreting the experimental results of a study on collagen turnover,under normal circumstances, the actual amount of collagen present in anystructure at a given time is the result of its synthetic and degradative rates(Neblock and Berg, 1987). In the steady state circumstances, the rate of synthesisequals the rate of degradation (Laurent, 1986). Degradation of collagen moleculescan occur at both intracellular and extracellular sites (Laurent, 1986) and can beassessed from the appearance of radiolabelled HYP (Laurent, 1986; McAnulty andLaurent, 1987). Neblock and Berg (1987) suggested that 10-50% of all newlysynthesized collagen could be degraded by cells within a short period of time.Thus, it is clear that the amount of HYP could be used as a measure of degradationand/or synthesis of collagen.80In the present study, HPLC was used to determine HYP content in thedeveloping secondary palate. The results on control hamster palate showed thatthe amount of HYP between days 12:00 and 12:04 of gestation, eg., during the periodof palatal shelf reorientation remained unchanged. Since, the uptake of 14C-proline also doubles during the period of shelf reorientation, the simultaneousdoubling in the HYP content could be interpreted to reflect the hydroxylation ofproline to HYP at the time of reorientation thereby suggesting, in line of thepreviously held view (Svenberg, 1987; Neblock and Berg, 1987), an increasesynthesis of new collagen. Earlier Shapira and Shoshan (1972) and Hassell andOrkin (1976), also suggested a synthesis of new collagen in mice and rats prior toachieving the reorientation of palatal shelf.The quantitative observations of the present study on the normal palatedevelopment in quail showed a 4-fold increase in the rates of collagen synthesisbetween days 6 and 8 of incubation. This increase in the rate of collagen synthesisoccurred in spite of the fact that the reorientation stage is absent during thedevelopment of palate in quail, as well as in other birds (Shah and Crawford, 1980;Koch and Smiley, 1981; Greene et al., 1983; Shah et al., 1985a, 1987, 1988).HPLC data obtained from the developing secondary palate of quail indicatedthat, in contrast to the 4-fold increase observed in the rate of collagen synthesis,HYP content increased by only two-fold between days 6 and 8 of incubation. Thismay indicate that part of the synthesized collagen is rapidly degraded. No effortswere made during the course of present investigation to either determine the typesand amounts of non-collagenous proteins or analyze their roles in quail palatedevelopment. It is, however, clear that, at the molecular level, there are81substantial differences in the types and amounts of proteins synthesized duringavian and mammalian palate morphogenesis which, in turn, may relate to theshelf reorientation in mammals, or its lack in birds. This issue needs furtherinvestigation. Also, it is interesting that during palate development in bothmammals and birds the increase in the amount of HYP was similar, eg., two-fold.This could, perhaps, suggest that proportionally, the actual amount of collagensynthesized in the developing palate of birds and mammals may be similar.The foregoing analysis, along with the issue of palatal shelf reorientationdiscussed in the INTRODUCTION segment of this thesis, raises an interestingquestion: Does a spurt in the synthesis of new collagen in the developingsecondary palate of mammals causes the shelf to reorient?In the present study, this question was evaluated using a teratologicalapproach. In the hamster, 5-FU delays the reorientation of shelves to induce a cleftpalate (Shah and Wong, 1980; Shah et al., 1984; present study). It was thought that,if 5-FU affected collagen synthesis and if the collagen synthesis is critical for shelfreorientation, then a spurt in the collagen synthesis would be delayed tocorrespond with the 5-FU-induced delayed reorientation of the shelves.Like controls, a changing profile of collagen synthesis was also observed in 5-FU-treated developing palate of hamster. Specifically, in the drug treated palatesinitially the proline uptake was suppressed. Subsequently, however, a 10-foldincrease (cf. control 2-fold increase) in the rate of proline uptake was observed onday 12:12 of gestation (Figure 20), eg., at least four hours prior to the period ofdelayed reorientation. In an earlier study, it was shown that within six hours ofdrug administration, 5-FU injured a significant proportion of cells in the vertically82developing palate of hamster (Shah et al., 1984). In addition, following drugtreatment, the expansion of the shelf volume was delayed (Shah et al., 1989c). Thereorientation of shelves occurred only after the resolution of cellular damage onday 12:06 and subsequent restoration of shelf volume on day 12:12 of gestation. Intheir recent work, Young et al (1991b) have shown that following 5-FUadministration, initial alteration in the synthesis of glycosaminoglycans wasquickly restored prior to the time of normal shelf reorientation in hamster, andthus it did not appear to be related to the delayed reorientation. Hence, it is likelythat altered cytodifferentiation due to 5-FU-induced cell injury (Shah et al., 1984)would in turn, affect collagen synthesis (present study), and consequently theexpansion of the shelf volume (Shah et al., 1989c). A delay in the spurt of collagensynthesis following 5-FU treatment may thus be one of the event of the recoveryphase of palate morphogenesis. During such a phase, a "catch-up" growth in therate of collagen synthesis could contribute to procuring the volume of thedeveloping shelf and prepare it for reorientation in the 5-FU assaulted embryos.There are no reports in the literature on the effects of 5-FU on collagen synthesis inany developing system. Also there is a paucity of studies analyzing drug-inducedalterations in collagen synthesis during palate development. In the only in vivostudy (Shapira and Shoshan, 1972), where drug (cortisone)-induced alterations inthe rate of collagen synthesis were observed, the data showed a two-fold increase incollagen synthesis prior to the period of delayed reorientation. In the presence ofglucocorticoids, an increased rate of collagen synthesis observed in cultured palataltissue explants (Uitto and Thesleff, 1979), but the rate was suppressed when palatalcells were isolated and cultured (Sasaki and Kurisu, 1983). Notwithstanding the83different observations, which may be attributed to variations in in vivo and in vitroexperimental approaches, one may suggest, on the basis of limited evidenceavailable in the literature and along with the data of the present study, thatperhaps a certain minimum amount of collagen accumulation in the developingpalate may be necessary before reorientation of the palatal shelf could occur.An accumulation of HYP was also observed in 5-FU-treated hamster palate.Interestingly, between days 12:00 and 12:04 of gestation, even though collagensynthesis in 5-FU-treated palate was depressed and the shelf reorientation delayed,the HYP levels, indicative of collagen content, were comparable to that of thecontrols. Subsequent spurt in collagen synthesis preceeded HYP accumulation andinitiation of shelf reorientation. Indeed, in the drug treated palates, the spurt inHYP content corresponded with decrease in collagen synthesis. A high level ofHYP thus could imply either an increased rate of synthesis of new collagen, or adecreased degradation of collagen (Laurent, 1986; Neblock and Berg 1987) due todrug treatment during palate development. In such circumstances, although acertain amount of collagen synthesis could still occur further morphogenesis ofpalate may be thwarted due to degradation of proteins reflected in high HYPcontent.In comparison to that seen in the hamster, the profile of collagen synthesisin 5-FU-treated developing quail palate showed less dramatic changes than theircontrol counterpart. Following drug treatment, a three-fold increase in the rate ofcollagen synthesis was observed in quail palate between days 7 and 8 of incubation.Also, the overall rate of collagen synthesis was lower in 5-FU-treated than in thecontrol quail palates. Unlike recovery in the rate of collagen synthesis within 3684hours of 5-FU assault of hamster palate, however, the recovery in collagensynthesis, over a 5 day period, in the drug-treated quail palate was low. This couldbe due to a lack of placenta in birds. In the absence of placenta, since the quailembryo was directly exposed to the drug the resultant cellular response wasprobably severe and hence the recovery less pronounced than observed in hamster.Nevertheless, when the data on the rate of collagen synthesis and that of HYPaccumulation in quail are analyzed together, it is clear that in spite of a significantreduction in the incorporation of 14C-proline in the drug-treated quail palates therelative amount of HYP remained high in 5-FU exposed quail palates. As indicatedfor the hamster, the high level of HYP, in light of reduced 14C-prolineincorporation in the 5-FU-treated quail palate, may be interpreted to reflectincreased degradation of collagen (Laurent, 1986, 1987; Neblock and Berg, 1987).This, however, need to be varified.Since proline is an amino acid ubiquitous to most proteins (Miller, 1983),and since in developing tissues a great amount of proline gets incorporated intocollagen via hydroxylation to HYP, a question, whether the profile of total proteinsaccumulation would follow that of collagenous protein, was examined. In thepresent study, we evaluated only the accumulation of total protein in both thecontrol and 5-FU-treated developing palate of quail and hamster. The resultsindicated that although in the control palates of both species the proteinaccumulation increased during palate morphogenesis, it remained suppressedfollowing 5-FU treatment in both vertebrates. Using a dose-time regimen for 5-FUtreatment similar to the one employed in the present study, Ruddick and Runner(1974) also observed a suppression of protein synthesis in homogenates of chick85embryo. Unlike the observation of the present study, in which 5-FU treated palatesdid not show an increase in the protein accumulation, Ruddick and Runner (1974)observed a continuous but slow accumulation of protein in chick embryohomogenates. Clearly, following 5-FU treatment, individual structures, such asthe palate, may show a different profile of protein accumulation in comparison tothe whole embryo. Indeed, our results are consistent with the previously notedeffect of 5-RJ on reduced protein accumulation in various individual organs andtissues (Anand and Han, 1975; Cohen and Glazer, 1984; Sandborg and Siegel, 1990).Also, it is interesting that a recovery in collagen synthesis occurred in both thereorienting palate of hamster and non-reorienting palate of quail. This wouldreinforce the proposition made earlier that an accumulation of certain minimumamount of collagen in the developing palate may be one of the features preceedingshelf reorientation.The mechanism by which 5-FU induces suppression of protein synthesis, orenhance its degradation is, however, not clear. At least during palate developmentin hamster 5-FU does not affect DNA synthesis (Shah et al., 1989c). A series ofrecent observations on various tissues, however, have shown that 5-FU mayinhibit production of cytoplasmic ribosomal RNA (rRNA) which in turn couldaffect the protein synthesis (Hadjiolova et al., 1981; Cohen and Glazer, 1984; Iwata,1986; Greenhalgh and Parish, 1989; Sandborg and Siegel, 1990). Hence, it would beinteresting to examine if 5-FU assault of the developing secondary palate ofvertebrates (a) suppresses the synthesis of rRNA and consequently of proteins tosubsequently induce the cellular injury observed by Shah et al (1984), and (b)whether a recovery in rRNA synthesis precedes the "catch-up" growth observed86during hamster and quail palate morphogenesis. In such circumstances post-translational accumulation of newly synthesized collagen, as well other proteins,would be crucial for the advancement of palate morphogenesis.The analysis of the results of the present study, along with the data from theliterature, clearly suggest that since (a) in birds, a spurt in collagen synthesis occursin the absence of shelf reorientation, (b) following 5-FU assault a recovery ofcollagen synthesis occurs four hours prior to initiation of shelf reorientation, and areduction occurs at the time of reorientation, a spurt in collagen synthesis may notbe critical for causing mammalian palatal shelf reorientation. On the other hand,it is plausible that an increasing collagen synthesis during vertebrates' palatemorphogenesis may play a role hitherto not considered in the literature.Although collagen has been implicated to play a role in the development ofmany structures and organs, its precise role during morphogenesis has begun toemerge only during the recent years. For example, it has been indicated that bothtypes I and III collagen acts as stabilizing molecules during initiation andmaintenance of the branching pattern of the salivary glands (Kallman andGrobstein, 1966; Spooner and Faubion, 1980; Nakanishi et al., 1988). On the otherhand, Kratochwil et al (1986) and Chen and Little (1987) suggested that collagentype I has no role in the branching morphogenesis since type I can be fully replacedby type III, IV and V collagen to act as an extracellular scaffold within which aglandular parenchyme may branch. Also, during initial healing of wound type IIIcollagen is synthesized which, as the subsequent maturation process of woundprogresses, is replaced by type I collagen (Gay et al., 1978; Alvarez, 1987). In thiscircumstances, type I collagen provides a lattice network to stabilize the wound and87thus could facilitate proliferation and migration of endothelial and fibroblastic cells(Gay et al., 1978; Maciag et al., 1982; Kramer et al., 1983; Alvarez, 1987). During themorphogenesis of the limb, a structure which, like the secondary palate, growsoutward from the body surface, increasing synthesis of type I collagen has beenrelated to cell orientation and arrangement (Trelstad and Hayshi, 1979; Hurle et al.,1989) Synthesis of type II collagen on the other hand appears to be related tocartilage development (Devlin et al., 1988). As observed in the present study, aswell as noted by previous investigators (Shapira, 1969; Pratt and King, 1971; Shapiraand Shoshan, 1972; Silver et al., 1984) the developing secondary palates ofmammals show type I collagen during their vertical growth and reorientationstages. Type III collagen was observed in vivo only in the vertical shelf (Silver et al.,1981), or in in vitro circumstances (Uitto and Thesleff, 1979; Sasaki and Kurisu,1983). It thus appears that type I is the predominent collagen isoform synthesizedin the developing palate of both mammals and bird (present study). Since type Icollagen is important for providing rigidity to the ECM (Bornstein and Sage, 1980),and in the developing structures such as salivary gland, limb, lung (referencescited above) and during wound healing for providing a stabilizing network onwhich cells can proliferate, migrate and rearrange themselves, it is plausible thatduring the formation of vertebrate secondary palate, the type I collagen maycontribute both to the shelf volume, as well as to the rigidity of the substratum onwhich cells may perform their various functions. In this manner, a continuedsynthesis of collagen would serve an important biological function during themorphogenesis of the vertebrates' secondary palate.88SUMMARY AND CONCLUSION1. A single injection of 5-FU in 0.1m1 saline into the air sac of quail eggson day 4 of incubation increased the gap between the two palatal shelves toincrease palatal clefting2. A single intramuscular injection of 81mg/kg 5-FU in 1m1 saline on day 11:00of gestation in hamster delayed the reorientation of palatal shelves by 16-20hours and induced cleft palate.3. The rate of collagen synthesis changed during vertebrate palatogenesis.During morphogenesis of the palate in hamster control, the rate of collagensynthesis peaked between days 12:00 and 12:04 of gestation, i.e., during shelfreorientation. 5-FU treatment initially suppressed the collagen synthesis.Later, during the recovery phase, it peaked in the drug treated palates on day12:12 of gestation, eg., just before reorientation. The shelf reorientation in 5-FU-treated palates began on day 12:16 of gestation at which time the collagensynthesis was low.4. During morphogenesis of palate in control quail, in spite of an absence ofthe reorientation stage, the rate of collagen synthesis peaked on day 8 ofincubation. Also, following 5-FU treatment, the rate of collagen synthesispeaked on day 8 of incubation but, unlike hamster, the recovery wasincomplete.5.^HPLC results showed that the rates of conversion of proline to HYP,indicating the content of collagen in both the control and 5-FU-treatedhamster palates were similar and remained unchanged between days 12:0089and 12:04 of gestation. However, in 5-FU-treated palates it increasedsubstantially on day 12:16 and 13:00 of gestation, when shelves werereorienting.6. In control palate of quail the amount of HYP was higher on day 8 than onday 6 of incubation which was consistent with the rate of collagen content.Low accumulation of HYP on days 7 and 9 of incubation, relative to the rateof collagen synthesis, may suggest significant synthesis of non-collagenousproteins during quail palate development. In 5-FU treated quail palates, theamount of HYP is reduced on both day 6 and 8, but not on days 7 and 9 ofincubation.7. The amount of total protein in the developing secondary palate in hamsterdoubled between days 11:04 and 12:04 of gestation, i.e., during the periodwhen shelf completed their vertical growth and reoriented to a horizontalplane and fused. The total protein content in 5-FU treated hamsterembryonic palates were approximately 50-75% lower than control palates .8. In the control palates of quail embryos, between days 5 and 10 of incubationthere was a gradual increase in the amount of total protein. In comparison,the total protein content in 5-FU treated palates was reduced by 50-70%.9. Using SDS-PAGE, type I collagen was identified in the developing secondarypalates of both control and 5-FU-treated hamster and quail.10. The analysis of the results of the present study, along with the data from theliterature, indicates that since (a) in birds, a spurt in collagen synthesis occursin the absence of shelf reorientation, (b) an equal amount of new collagenwas synthesized in both the control and 5-FU-treated hamster embryos90during the period of normal reorientation, and (c) in 5-FU-treated hamsterembryos, a recovery in collagen synthesis occurs prior to, and a reductionoccurs at the time of delayed shelf reorientations, the results suggest thatcollagen synthesis may not cause shelf reorientation in mammals.11. Synthesis of type I collagen during vertebrate's palatogenesis may contributetoward the volume of the developing shelf and may provide structuralrigidity to maintain the shelf for further morphogenesis.91REFERENCESAds, A.H., Piddington, R., Goldman, A.S., and Herold, R., 1983. Cortisol inhibitionof development of various lysosomal enzymes in cultured palatal shelves frommouse embryos. Archs. 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