UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

6-mercaptopurine induced cleft palate in the hamster : morphological and cellular aspects Burdett, David Norman 1985

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
UBC_1985_A6_7 B87.pdf [ 17.55MB ]
Metadata
JSON: 1.0096007.json
JSON-LD: 1.0096007+ld.json
RDF/XML (Pretty): 1.0096007.xml
RDF/JSON: 1.0096007+rdf.json
Turtle: 1.0096007+rdf-turtle.txt
N-Triples: 1.0096007+rdf-ntriples.txt
Original Record: 1.0096007 +original-record.json
Full Text
1.0096007.txt
Citation
1.0096007.ris

Full Text

6-MERCAPTOPURINE INDUCED CLEFT PALATE IN THE HAMSTER: MORPHOLOGICAL AND CELLULAR ASPECTS BY DAVID NORMAN BURDETT B.Sc, The University of British Columbia, 1975 D.M.D., The University of British Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard Dr. G. Scudder Dr. D. Kalousek Dr. W.L. Dunn (Dr. R. Shah (Supervisor) "Dr. J. Spouge The University of British Columbia April 1985 ©DAVID NORMAN BURDETT, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of \ aMvoto^ ^  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date trLly AM / I*; - ii -ABSTRACT A study on the pathogenesis of 6-mercaptopurine induced cleft palate was undertaken using light and electron microscopic, and enzyme acid phosphatase cytochemical techniques. Palatal development in control fetuses was observed in six stages at the gross level and five stages at the histological level. Between days 9:18 (9 days:18 hours) and 10:00 of gestation palatal primordia appeared from the roof of the oronasal cavity, and developed in the vertical direction until day 12:00 of gestation. Between days 12:00 and 13:00 of gestation the palatal shelves became horizontal and fused with one another. During closure the timely appearance of lysosomes was responsible for elimination of the intervening epithelia of the opposing palatal shelves through an intracellular process of autolysis. Gross observations showed that 6-mercaptopurine affected the vertical development of palatal shelves. In contrast to normal development, vertically developing palatal shelves on day 10:00 of gestation showed sublethal injury of the mesenchymal cells characterized by swelling of the perinuclear space and lysosomal development. Subsequently the epithelial cells were damaged, and the basal lamina fragmented and disappeared. The epithelial and mesenchymal cells communicated with one another. Eventually, however, the epithelial and mesenchymal cells recovered and the basal lamina re-established its continuity. - iii -It was concluded that sublethal injury of the mesenchymal and epithelial cells following 6-mercaptopurine treatment disturbed the controlled process of cytodifferentiation, and thus affected vertical development of the palatal shelves to develop a cleft palate. - IV -TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS '..*'iv LIST OF TABLES ' ' V1-LIST OF FIGURES * v^ ACKNOWLEDGEMENT xINTRODUCTION 1 Birth DefectsCleft Palate 2 Normal Development of the Secondary Palate 4 Abnormal Development of the Secondary Palate 10 6-Mercaptopurine 11 PURPOSE OF STUDY 2MATERIALS AND METHODSBreeding of Animals 21 Drug Treatment and Procurement of Tissues 2For Gross and Light Microscopic Studies 2 For Electron Microscopic Studies 2For Acid Phosphatase Enzyme Cytochemistry 23 Statistical Analysis 24 RESULTS 26 Morphogenesis of the Secondary Palate in Control Hamster FetusesMorphogenesis of the Secondary Palate in 6MP Treated Hamster Fetuses 32 Morphogenesis of the Secondary Palate in Control Hamster Fetuses in Relation to Fetal Weight 34 Morphogenesis of the Secondary Palate in 6MP Treated Hamster Fetuses in Relation to Fetal Weight 37 V -PAGE Morphogenesis of the Secondary Palate in Control Hamster Fetuses in Relation to Fetal Crown-Rump Length (CRL) 37 Morphogenesis of the Secondary Palate in 6MP Treated Hamster Fetuses in Relation to Fetal Crown-Rump Length 40 Light Microscopic Observations of the Developing Secondary Palate in Control Hamster Fetuses 43 Light Microscopic Observations of the Developing Secondary Palate in Hamster Fetuses Following 6MP Treatment 46 Electron Microscopic Observations of the Developing Secondary Palate in Control Hamster Fetuses 50 Electron Microscopic Observations of the Developing Secondary Palate Following 6MP Treatment 9 Light Microscopic Observations of Enzyme Acid Phosphatase in the Developing Secondary Palate of Control Hamster Fetuses 75 Light Microscopic Observations of Enzyme Acid Phosphatase in the Developing Secondary Palate of 6MP Treated Hamster Fetuses 78 DISCUSSION 9 Normal Palatal Development 76-Mercaptopurine Induced Cleft Palate 84 SUMMARY AND CONCLUSIONS 99 REFERENCES 101 APPENDIX 128 - vi -LIST OF TABLES PAGE Table I Factors Suspected to be Involved in Palatal Shelf Reorientation 6 Table II A Summary of Ultrastructural and Biochemical Studies of Teratogen Induced Cleft Palate Development 12 Table III Staged Development of the Secondary Palate in Control Hamster Fetuses 31 Table IV Staged Development of the Secondary Palate in 6-Mercaptopurine Treated Hamster Fetuses 33 Table Y The Mean Weights of Control and 6-Mercaptopurine (6MP) Treated Hamster Fetuses at Different Times During Gestation 35 Table VI Staged Development of the Secondary Palate in Relation to Control Hamster Fetal Weight 36 Table VII Staged Development of the Secondary Palate in Relation to 6-Mercaptopurine Treated Hamster Fetal Weight 38 Table VIII The Mean Crown-Rump Length (CRL) of Control and 6-Mercaptopurine (6MP) Treated Fetuses at Different Times During Gestation 39 Table IX Staged Development of the Secondary Palate in Relation to Control Fetal Hamster Crown-Rump Length (CRL) 41 Table X Staged Development of the Secondary Palate in Relation to 6-Mercaptopurine Treated Fetal Hamster Crown-Rump Length (CRL) 42 -vii -LIST OF FIGURES PAGE Figure 1 Catabolism of 6-mercaptopurine and hypoxanthine 15 Figure 2 Proposed sites of action of 6-mercaptopurine (6MP) and its anabolites on purine metabolism 16 Figure 3 Ventral view of the developing palate of a control hamster on day 10:00 of gestation 28 Figure 4 Frontal section through the secondary palate region in a control hamster fetus on day 10:00 of gestation ... 28 Figure 5 Ventral view of the developing palate of a control hamster on day 11:18 of gestation 28 Figure 6 Frontal section through the secondary palate region in a control hamster fetus on day 11:18 of gestation ... 28 Figure 7 Ventral view of the developing palate of a control hamster on day 12:00 of gestation 28 Figure 8 Frontal section through the secondary palate region in a control hamster fetus on day 12:00 of gestation ... 28 Figure 9 Ventral view of the developing palate of a control hamster on day 12:00 of gestation 28 Figure 10 Frontal section through the secondary palate region in a control hamster fetus on day 12:00 of gestation ... 28 Figure 11 Ventral view of the developing palate of a control hamster on day 12:18 of gestation 30 Figure 12 Ventral view of the developing palate of a control hamster on day 13:00 of gestation 30 Figure 13 Ventral view of the developing palate of a 6MP treated hamster on day 10:00 of gestation 30 Figure 14 Frontal section through the secondary palate region in a 6MP treated hamster fetus on day 10:00 of gestation 3Figure 15 Ventral view of the developing palate of a 6MP treated hamster on day 11:18 of gestation 30 Figure 16 Frontal section through the secondary palate in a 6MP treated hamster fetus on day 11:18 of gestation 30 - vi i i -PAGE Figure 17 Ventral view of the developing palate of a 6MP treated hamster on day 15:00 of gestation 30 Figure 18 Frontal section through the secondary palate in a 6MP treated hamster fetus on day 15:00 of gestation 30 Figure 19 Frontal section through the secondary palate region of a control hamster on day 9:06 of gestation 45 Figure 20 Frontal section through the secondary palate region of a control hamster fetus on day 10:00 of gestation showing the bilaminar epithelium of the palatal primordia 45 Figure 21 Frontal section through the secondary palate of a control hamster fetus on day 11:18 of gestation showing the two to three cell layered epithelium of the medial aspect of the vertical palatal shelf 45 Figure 22 Frontal section through the secondary palate of a control hamster fetus on day 12:00 of gestation 45 Figure 23 Frontal section through the secondary palate of a control hamster fetus on day 12:00 of gestation 45 Figure 24 Frontal section through the secondary palate of a control hamster fetus on day 12:06 of gestation showing a fragmenting (Stage V) epithelial seam 45 Figure 25 Frontal section through the secondary palate of a control hamster fetus on day 12:12 of gestation showing mesenchymal continuity between opposing palatal shelves 48 Figure 26 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 10:00 of gestation showing the primordial epithelium 48 Figure 27 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 10:06 of gestation 48 Figure 28 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 11:00 of gestation 48 Figure 29 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 12:18 of gestation 48 - ix -PAGE Figure 30 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 15:00 of gestation 48 Figure 31 Electron micrograph of the roof of the oronasal cavity in a control hamster fetus on day 9:12 of gestation 52 Figure 32 Electron micrograph of the secondary palate primordia in a control hamster fetus on day 10:00 of gestation showing a continuous basal lamina separating the bilaminar epithelium from the mesenchyme 52 Figure 33 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the epithelium of the horizontal palatal shelf 55 Figure 34 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the mesenchymal cells of the horizontal palatal shelf 55 Figure 35 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the epithelial seam 58 Figure 36 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing a fragment of an epithelial seam and a macrophage 58 Figure 37 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 9:06 of gestation 61 Figure 38 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation 61 Figure 39 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation 63 Figure 40 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation 63 Figure 41 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:06 of gestation 66 - X -PAGE Figure 42 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:12 of gestation showing cytoplasmic features of the epithelial cells 68 Figure 43 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:12 of gestation showing unaffected epithelial cells 68 Figure 44 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 11:18 of gestation showing a bilaminar epithelium 71 Figure 45 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 12:18 of gestation showing discontinuity in the basal lamina 71 Figure 46 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 12:18 of gestation showing a free cytoplasmic extension of the basal epithelial cell 74 Figure 47 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 13:18 of gestation 74 Figure 48 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 14:00 of gestation showing a stratified epithelium 77 » - xi -ACKNOWLEDGEMENTS I am very much obliged to Dr. R. Shah for his patience, suggestions, guidance, feedback, ideas, and for the use of his laboratory facilities and materials for the experimental work, and in the preparation of this thesis. My thanks to the members of my committee for their feedback and perceptions. My gratitude goes to Mr. Roger Suen and Mr. Andre Wong for their technical assistance in the experimental work. I must also thank Mr. Bruce MaCaughey for his photographic assistance and suggestions, Ms. Linda Skibo for her secretarial assistance, and the Oral Biology Department of the Faculty of Dentistry for the tolerant use of their facilities. The Medical Research Council of Canada must also be acknowledged for the funding of this research through Dr. Shah's grant. Finally, my sincerest thanks to my family for putting up with me. - 1 -INTRODUCTION Birth Defects Since the turn of the century it has been increasingly recognized that chemicals present in the environment, or administered therapeutically may be hazardous to the embryo/fetus. The most overtly identifiable adverse effect of exposure to a chemical in this fashion is intrauterine death. Alternatively the exposed fetus may develop anatomical defects or functional aberrations, including subtle behavioural or biochemical abnormalities, some of which may remain latent before being expressed at later stages of development. The recognition of such "teratogenic" effects (both anatomical and functional) gained further impetus through the tragic events of the early 1960's, when dramatic increases in the frequency of phocomelia in the newborn infants were traced to the use of thalidomide by pregnant women (Lenz, 1961; McBride, 1961). Since then both the scientist and society have been brought face to face with the devastating possibility of chemically induced birth defects in humans. The study of birth defects has assumed more significance now than in the past because mortality and morbidity due to congenital anomalies has declined far less than those for other causes of death such as infections and nutritional irregularities (Smithells, 1966; Saxen and Rapola, 1969; Persaud, 1979). It is estimated that approximately 40% of all pregnancies terminate as miscarriages, mainly due to faulty prenatal development (Saxen and Rapola, 1969). Approximately 20-40% of all still births and infant deaths are associated with severe congenital anomalies (Elwood and Rogers 1975; - 2 -Fairweather 1982). In addition, major birth defects are found in at least 2-7% of all liveborn (Fairweather, 1982; Bower and Stanley, 1983; Fabro, 1983; Hemminki et al., 1983; Levin, 1983; Tuchmann-Duplessis, 1983). As pain and suffering caused by birth defects to both the victim and his/her family are immeasurable, birth defects have been considered among the most serious of human health problems (Pruzanski, 1961; Paul and Piazza, 1979). Cleft Palate Cleft palate is one of the major birth defects of humans (Elwood and Rogers, 1975; Paul and Piazza, 1979; Bower and Stanley, 1983). As it is a structural defect in the roof of the oral cavity, a cleft palate also affects the functions of mastication, deglutition, respiration and phonation. A child born with cleft palate will also present associated dental, emotional and vocational problems. Some of these functional and associated problems may be partially alleviated by surgery and/or a prosthesis, but the economic and social burden for the rehabilitation of the cleft palate individual remains very high (Paul and Piazza, 1979). The frequency of cleft palate is reported to be, varied among different populations, from 0.05 to 1.87 per 1,000 births (Lowry and Trimble, 1977; Chung et al., 1980; Czeizel, 1980; Koguchi, 1980; Mel nick et al., 1980; Shields et al., 1981; Iregbulem, 1982; Bonaiti et al., 1982; Chapman, 1983). A recent survey by Shields et al. (1981) has indicated that heritable factors can be traced only in 10-20% of cases of cleft palate. It is plausible that environmental factors, including teratogens, may account either directly or - 3 -jointly for a large proportion of cleft palate (Czeizel, 1980; Koguchi, 1980; Mel nick et al., 1980; Shields, et al., 1981; Tyan, 1982). The role of environmental factors in cleft palate formation had been implicated to some degree by experimental studies in animals. A series of experiments by Hale (1933, 1935, 1937) and by Warkany and associates (1941, 1943, 1948) showed that numerous congenital malformations, including cleft palate, occurred in the offspring of rodents who were fed nutritionally deficient diets during pregnancy. Subsequently, however, when Baxter and Fraser (1950) observed that cortisone treatment of pregnant mice produced cleft palate in the embryo, an impetus was received in the scientific community to identify other agents which might induce cleft palate in animals and in man. During the past three decades numerous teratogenic agents which induce cleft palate in laboratory animals have been identified, and are summarized by Kalter and Warkany (1959), Dagg (1966), Schardein (1976), and Shah (1979d). While the search for environmental factors responsible for cleft palate formation continues, recent emphasis has been toward identification of abnormal mechanisms by which cleft palate may develop in an embryo. The use of teratogenic agents as a tool for the study of cleft palate development has begun to contribute to a better understanding of normal developmental mechanisms, and their failures. More significantly, their use in laboratory animals has helped to define and improve the understanding of the factors involved in both normal and cleft palate development. - 4 -In the ensuing literature analysis both the issues involved in normal and abnormal palate development, and various experimental approaches to evaluate them, are reviewed. Normal Development of the Secondary Palate The first descriptive study on the normal development of secondary palate was published by Dursy in 1869. On the basis of observations in human embryos, he indicated that the secondary palate develops as two vertical projections (shelves) from the roof of the oronasal cavity lateral to the tongue. The vertical shelves then move to a horizontal position over the dorsal surface of the tongue. Subsequently the medial edges of the horizontal shelves unite with one another to separate the oral and nasal cavities. Following publication of Dursy's work, numerous studies have been reported in the literature analyzing both in vivo and in vitro morphological, cellular and biochemical aspects of mammalian secondary palate development (Walker and Fraser, 1956; Fulton, 1957; Asling et al., 1960; Wood and Kraus, 1962; Larsson, 1962a; Coleman, 1965; Pourtois, 1966, 19/2; Ma to et al., 1966; Burdi and Faist, 1967; Andersen and Matthiessen, 1967; Farbman, 1968, 1969; De Angelis and Nalbandian, 1968; Walker, 1969, 1971; Brusati, 1969; Hayward, 1969; Humphrey, 1969; Dostal and Jelnick, 1970; Smiley, 1970, Bollert and Hendrickx, 1971; Andrew and Zimmerman, 1971; Smiley and Koch, 1971, 1975; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974a, b; Tyler and Koch, 1975, 1977a, b; Goss, 1975; Babiarz et al., 19/5; Nanda and Romeo, 19/5; Luke, 1976; Shah and Travill, 1976a; Cleaton-Jones, 19/6; Greene and Pratt, - 5 -1976; Holmstedt and Bagwell, 1977; Ferguson, 1977; Innes, 1978, 1981; Wee et al., 1979; Zimmerman, 1979; Shah, 1979a, b, 1980, 1984; Meller et al., 1980; Tassin and Weill, 1980; Zimmerman et al., 1980; Greene et al., 1981, 1982; Gulamhusein and England, 1982; Greene, 1983; Schupbach, 1983; Schupbach and Schroeder, 1983; Schupbach et al., 1983; Brinkley, 1984). One may infer from these studies that at least three critical events must occur for normal closure of the secondary palate: 1. a change in the direction of palatal shelf development from a vertical position on the side of the tongue, to a horizontal position dorsal to it, i.e., reorientation of the palatal shelf; 2. union between the epithelia of the opposing horizontal palatal shelves to form a seam; and 3. removal of the epithelial seam and establishment of mesenchymal continuity. On the basis of observations in mammalian embryos several factors have been suggested in the literature to be responsible for the reorientation of palatal shelf. For the sake of simplicity and brevity the proposed factors may be grouped under the headings "extrinsic" and "intrinsic", and are summarized in Table I. Recent reviews indicate that although extrinsic factors may play a role in fostering an intraoral environment for palatal shelves to undergo reorientation, they are probably not responsible for the actual movement of the shelves (Babiarz et al., 1975; Ferguson, 1978; Wee et al., 1979; Shah, 1979a; Zimmerman, 1979; Zimmerman et al., 1980; Greene, 1983). The - 6 -Table I. Factors Suspected to be Involved In Palatal Shelf Reorientation (References) Extrinsic A. Involving descent, depression or movement of the tongue (Asling et al., 1960; Coleman, 1965; Humphrey, 1969; Burdi and Silvey, 1969; Walker, 1969, 1971; Walker and Ross, 1972; Wragg et al., 1972; Diewert, 1974; Taylor, 1978). B. Involving changes in cranial base angulation (Verrusio, 1970; Brinkley and Vickerman, 1978). ' Intrinsic A. Cell proliferation (Wood and Kraus, 1962; Mott et al., 1969; Jelnick and Dostal, 1973; Nanda and Romeo, 1975). B. Synthesis of extracellular matrix (Walker, 1961; Larsson, 1962a; Jacobs, 1964a, b, 1966; Anderson and Matthiessen, 1967; Andrew and Zimmerman, 1971; Ferguson, 1977, 1978; Brinkley, 1980; Jacobson, 1982). C. Cell movement (Babiarz et al., 1975; Wee et al., 1979; Shah, 1979a; Innes, 1981). - 7 -reorientation of the palatal shelf in mammals seems to occur via a remodelling process involving development of a bulge from the medial aspect of the vertical shelf over the dorsal surface of the tongue, with a simultaneous retraction of the vertical shelf (Walker and Fraser, 1956; Kochhar and Johnson, 1965; Greene and Kochhar, 1973; Shah and Travill, 1976a; Shah, 1979a), rather than by rotation (Coleman, 1965; Walker and Ross, 1972; Diewert, 1974; Babiarz et al., 1975). In 1956, Walker and Fraser suggested that an "intrinsic force" may be responsible for the reorientation of the palatal shelves via a remodelling process. Since then, using both in vivo and i_n vitro techniques, several intrinsic cellular and biochemical events have been analyzed (Table I) to determine the nature of "intrinsic force" (Larsson, 1962a; Anderson and Matthiessen, 1967; Mott et al., 1969; Babiarz et al., 1975; Nanda and Romeo, 1975; Ferguson, 1978; Shah, 1979a; Wee et al., 1979; Brinkley, 1980). The precise nature of the intrinsic force during palatal shelf reorientation, however, still remains obscure. As the palatal shelves reorient from a vertical to a horizontal plane, and then approximate, the bilaminar epithelium of their prospective medial edges undergoes cytodifferentiation. The medial edge epithelial cells (MEE) (1) stop proliferating, as confirmed by an absence of 3H-thymidine incorporation (Hudson and Shapiro, 1973; Pratt and Martin, 1975; Shah et al., 1985); (2) form increasing numbers of lysosomes as palatogenesis progresses (Mato et al., 1966; Brusatl, 1969; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974a, b; Lorente et al., 1974; Hoist and Mills, 1975; Im and Mulliken, 1983), and (3) show an increasing concentration of cyclic AMP with - 8 -progressive differentiation (Pratt and Martin, 19/5; Olson and Massaro, 1977; Greene and Pratt, 1979; Greene et al., 1980; Shah et al., 1985). Simultaneously the mesenchymal cells elongate and develop intracytoplasmic filaments (Babiarz et al., 19/5; Shah, 19/9a; Innes, 1981). The filaments appear to be contractile in nature (Krawczyk and Gil Ion, 1976), and thus may allow cells to move during reorientation. Also there is a concurrent increase in the synthesis of glycosaminoglycans, especially hyaluronic acid, in the extracellular matrix of the mesenchyme (Larsson, 1962a; Jacobs, 1964a, b; 1966; Pratt et al., 1973; Ferguson, 19/8; Brinkley, 1980; Jacobson and Shah, 1981; Jacobson, 1982). It has been implied that glycosaminoglycans facilitate the movement of cells during palatal shelf reorientation (Shah, 19/9a, c; Jacobson, 1982; Brinkley and Vickerman, 1982). While the debate on mechanisms of palatal shelf reorientation still continues, it is only recently that some attention has been directed towards characterization of the process by which the opposing palatal shelves unite with one another. The union between the opposing palatal shelves is a multistep process which initially involves "adhesion" between opposing epithelia, followed by their fusion to form a seam, and finally replacement of the seam by mesenchyme (Barry, 1961; Pourtois, 1972). Originally Farbman (1968, 1969), Hayward (1969), Greene and Kochhar (1974) , Hinrichsen and Stevens (19/4), Souchon (19/5), Pratt and Hassel1 (1975) , De Paola et al., (19/5), Meller and Barton (19/8) alluded to the possibility that a sticky substance, probably of a complex carbohydrate nature, may be responsible for the formation of the epithelial seam. Recent - 9 -experimental evidence, however, suggests that carbohydrates are probably only involved in the initial adhesion (Shah, 1979b; Shah and Crawford, 1980; Heinen et al., 1982; Baeckeland et al., 1982; Pratt, 1983; Greene, 1983). Fusion of the shelves to form a seam may involve elimination of the superficial epithelial cells via an autolysosomal process or desquamation (Pourtois, 1966; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974a, b; Shah, 1979b, Baeckeland et al., 1982; Heinen et al., 1982; Schupbach et al., 1983; Schupbach and Schroeder, 1983), followed by the formation of desmosomes between deeper epithelial cells of the opposing palatal shelves (De Angel is and Nalbandian, 1968; Brusati, 1969; Smiley and Koch, 1971; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974b; Shah, 1979b; Pratt, 1983; Greene, 1983). Once the seam is formed, then the remaining epithelial cells must be eliminated to achieve a mesenchymal union. It is generally accepted in the literature that elimination of cells from the epithelial seam can occur through two pathways, i.e., intracellular lysosomal autophagy, and/or exfoliation of degenerating cells into the amniotic fluid or into mesenchyme (Mato et al., 1966, 1967a, b; De Angel is and Nalbandian, 1968; Brusati, 1969; Hayward, 1969; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974a, b; Greene and Pratt, 1976; Greene, 1983). In the latter instance, the debris of degenerated epithelial cells are phagocytosed by macrophages. - 10 -Abnormal Development of the Secondary Palate It is generally accepted in the literature that cleft palate following teratogenic treatment can be induced in laboratory animals by affecting any one of the three critical events of normal palate morphogenesis. For example, studies in mice, rats and hamsters have shown that cortisone, triamcinolone, radiation, high doses of vitamin A, folic acid deficiency, diazo-oxo-norleucine, 5-fluorouraci1, and b-fluoro-2-desoxyuridine induce cleft palate by delaying reorientation of the palatal shelves from a vertical to a horizontal plane (Walker and Fraser, 195/; Walker and Grain, 1960; Asling et al., 1960; Callas and Walker, 1963; Kochhar and Johnson, 1965; Coleman, 1965; Ross and Walker, 196/; Dostal and Jelinek, 19/2; Ferguson, 197/; Shah, 19/9e; Diewert, 1979; Diewert and Pratt, 1979; Shah and Wong, 1980). Hydrocortisone treatment of pregnant hamsters affects the fusion process in fetuses (Shah and Travill, 19/6a, b). Indirect evidence has been presented to suggest that a cleft palate may develop by the rupture of a previously formed epithelial seam, or by the rupture of a mesenchymally united palate (Kitamura, 1966; Angelici, 1968). While studies on analysing morphological aspects of palate development following teratogenic treatment continue, efforts have also been directed toward exploring cellular and biochemical alterations associated with cleft palate development. Considerable efforts in cellular and biochemical aspects of cleft palate development have been directed toward understanding the mechanism of glucocorticoid induced cleft palate. The suggestion that glucocorticoid - 11 -treatment induced cleft palate by inhibiting the synthesis of glycosaminoglycans, mainly hyaluronic acid (Walker, 1961; Larsson, 1962b; Jacobs, 1964a, b; Pratt et al., 1973; Ferguson, 1978) has been disputed (Nanda, 1971; Andrew and Zimmerman, 1971). Recently inconclusive evidence has indicated that glucocorticoid action may be mediated via its binding to receptors in the palatal tissue (Goldman et al., 1977, 1978; Bekhor et al., 1978; Salomon and Pratt, 1979; Shah and Burton, 1980), which may subsequently lead to inhibition of intracellular protein synthesis (Zimmerman et al., 1970) and eventually to premature necrosis of palatal cells (Shah and Travill, 1976b; Shah, 1980; Kurisu et al., 1981). Mechanisms regulating the binding of glucocorticoid molecules to the receptors and the inhibition of protein synthesis, however, have not yet been clarified. In recent years attempts have been made at analyzing the ultrastructural and biochemical effects of teratogens other than glucocorticoids on palatal tissues during development (Table II). One may deduce from Table II that depending on the chemical nature of the teratogenic agent, different aspects of growth and differentiation of either epithelial, or mesenchymal or both tissues may be affected. 6-Mercaptopurine In 1952 El ion and associates reported the synthesis of a purine analogue, 6-mercaptopurine (6MP). The chemical is a yellowish, odourless, crystalline powder, insoluble in water, acetone, chloroform and diethyl - 12 -Table II. A Summary of Ultrastructural and Biochemical Studies of Teratogen Induced Cleft Palate Development Agent (species) Ultrastructural Studies Vincristine Sulfate & acridine orange (rat) Meclozine (rat) 3-aminopropri oni tri 1 e (rat) Hydrocortisone (hamster) Diazo-oxo-norleucine (rat) Triamcinolone (hamster) (mouse) Phenylbutazone (mouse) 5-fluorouracil (hamster) Mechanism Inhibition of cell death Effect on the epithelial cell piasmamembrane permeability Factors extrinsic to shelves; did not observe any cellular effects Retarded differentiation in epithelium Premature necrosis of the basal epithelial cells Alteration in intracellular distribution of lysosomal enzymes Alterations at the epithelial-mesenchymal interface Inhibition of cell proliferation Alterations at the epithelial-mesenchymal interface Premature, sublethal injury of the mesenchymal cells References Mato & Uchiyama, 1972 Mato et al., 1975a Mato & Uchiyama, 1975 Morgan, 1976 Mato et al., 1975b Shah & Travill, 1976b Greene & Pratt, 1978 Shah, 1980 Kurisu et al., 1981 Montenegro & Paz de la Vega, 1982 Shah et al., 1984a - 13 -Table II. (continued) Agent (species) Mechanism References Biochemical Studies Hypervitaminosis A (rat) Increased synthesis of DNA Lorente & Miller, and glycoprotein 1978 (rat) Inhibition of DNA, collagen Sauer & Evans, 1980 and glycosaminoglycan synthesis (rat) Inhibition of DNA synthesis Pick & Evans, 1981 B-aminoproprionitrile Inhibtion of collagen Pratt & King, 1972 (rat) synthesis (rat) Stimulation of Del Balso & Kauffman, glycosaminoglycan catabolism 1975 Diazo-oxo-norleucine Inhibition of Pratt et al., 1973 (rat) mucopolysaccharide synthesis (rat) Block glucosamine formation Pratt & Greene, 1976 (rat) Inhibit glycoprotein Greene & Pratt, 1977 synthesi s Salicylate (mouse) Inhibit mucopolysaccharide Larsson & Bostrom, synthesis 1965 2-deoxyglucose (rat) Decreased synthesis of Pratt & Greene, 1976 lysosomal enzymes Epidermal growth factor Reduced levels of cAMP Hassel & Pratt, 1977 (rat) Methylmercury (mouse) Impaired placental transfer Olson & Massaro, 1977 of aminoacids 5-fluoro-2-desoxyuridine Reduced synthesis of Ferguson, 1977 (rat) mucopolysaccharide Chlorcyclizine (rat) Degradation of Wilk et al., 1978 glycosaminoglycans Phenytoin (mouse) Reduced protein and RNA Sonawane & Goldman, synthesis 1981 - 14 -ether, but soluble in boiling water, alkaline solutions, and warm ethanol (Windholz, 1976). It has a molecular weight of 152.19 and a melting point of 313° C. When administered intraperitoneally to mammals a large proportion (21.4%) of 6MP is excreted unchanged in the urine during the following 24 hours, and the remainder as catabolites (Fig. 1), mainly 6-thiouric acid (18.9%) and inorganic sulphate (29.5%) (Elion et al., 1963). After intravenous administration, the drug concentration rapidly becomes high within many organs. In liver the concentration reaches four times that of plasma within minutes, but falls rapidly thereafter in both the organs and plasma (Tterlikkis et al., 1977). The half life of 6MP appears to be very short. After intravenous administration of 6MP, the plasma half life of the drug was 14 minutes in mice, 9 minutes in rats (Donelli et al., 1972), and 1.5 hours in humans (Hamilton and Elion, 1954). Although the precise mechanism by which 6MP exerts its effect is uncertain, it is generally agreed that the drug eventually inhibits the synthesis of purine. Several possible biochemical sites of action for inhibition of purine synthesis by 6MP have been suggested (Elion, 1967; Paterson and Tidd, 1975; Tidd and Dedhar, 1978; Gringauz, 1978; Ding and Benet, 1979; Breter and Zahn, 1979; Plagemann et al., 1981; Lennard and Maddocks, 1983), but the exact mechanism of its action is not yet clear. Acting in vitro as a free base, the chemical competitively inhibits xanthine oxidase and hypoxanthine-guanine phosphoribosyl transferase which convert hypoxanthine to xanthine and inosinate respectively (Fig. 2). However, in vivo the drug is anabolised to 6-methylthioinosinate and thioinosinate, which 1 Catabolism of 6-Mercaptopurine and Hypoxanthine Co 6-Mercaptopurine N Hypoxanthine Uric Acid Adapted from Gringauz (1978) Fig. 2 Proposed Sites of Action of 6-Mercaptopurine (6MP) and its Anabolites on •H Purine Metabolism 6MP, Xanthine 1) Tidd and Dedhar (1978) 2) Plagemann et al. (1981) 3) Ding and Benet (1979) 4) Breter and Zahn (1979) 5) Paterson and Tidd (1975) 6) Gringauz (1978) 7) Leonard and Maddocks (1982) Purine De novo synthesis Ufg) Xanthylate (XMP) GMP I GDP GTP AMP ADP ATP Nucleic acid 6-Thioinosine Thioinosinate 6-Methylthioinosinate *•* 6-Thioxanthylate (4> i! I 6-Methylthioinosine 6-Thioguanylate (1A4) Thio GDP (4) Thionucieic acids - 17 -may act to inhibit de novo synthesis of purines (Paterson and Tidd, 1975; Breter and Zahn, 1979). Paterson and Tidd (1975), Gringauz (1978), and Breter and Zahn (1979) have indicated that thioinosinate may also act to inhibit purine ribonucleotide metabolism and its transconversions (Fig. 2). Others (Tidd and Dedhar, 1978; Ding and Benet, 1979; Breter and Zahn, 1979; Lennard and Maddocks, 1983), however, stress that the drug acts via the conversion of 6-thioinosinate into 6-thioxanthylate, leading to the formation of thionucleic acids (Fig. 2). Several researchers have also indicated that the effect of 6MP may be mediated via directly affecting mRNA synthesis (Roy-Burman, 1970; Neubert et al., 1970; Mewes et al., 1971). Recently Kawahata et al. (1980) suggested that 6MP may inhibit DNA-dependant RNA polymerase activity which in turn reduces RNA synthesis. It appears that eventually the interference with one or more of these aforementioned pathways is perhaps responsible for the growth suppressive effect which has made 6MP useful in the treatment of various forms of leukemia (Calabresi and Parks, 1975). The chemical also has immunosupressive properties, and is considered useful in the treatment of ulcerative colitis (IARC, 1981). The embryotoxic and teratological effects of 6MP are well documented in echinoderms and vertebrates (tadpole, chick, rat, mouse, rabbit, hare and hamster) (Bieber et al., 1952; Thiersch, 1954; Zunin and Borrone, 1955; Didcock et al., 1956; Tuchmann-Duplessis and Mercier-Parot, 1958, 1966, 1968; Karnofsky, 1960; Murphy, 1960, 1962; Blattner et al., 1960; Adams et al., 1961; Mercier-Parot and Tuchmann-Duplessis, 1967; VTshniakov, 1968, 1969; Bragonier and Carver, 1968; Kury et al., 1968; Adhamti and Noack, 1975; Asisi and Merker, 1975; Merker et al., 1975; Puget et al., 1975; Grubb and - 18 -Montiegel, 1975; Neubert et al., 1977; Adhami, 1979; Shah and Burdett, 1979). In mammals the embryotoxic and teratological effects are manifested only after the 6MP has crossed the placenta to the fetus, where it is then anabolized by the fetus into an active form (Neubert et al., 1980). The teratogenic effects were characterized by malformations of limbs, beak, eye, brain, palate, teeth, salivary glands, tongue, mandible, respiratory tract, alimentary tract, liver, kidney and tail. In addition, sterility has been observed during postnatal life in both male and female animals which were exposed to 'sub-teratological' doses of 6MP during gestation (Reimers and Sluss, 1978; Reimers et al., 1980). Review of the literature indicates that in at least 21 human pregnancies 6MP was administered during the first trimester. Of the twenty-one, three mothers died undelivered (Raichs, 1962; Nicholson, 1968a), eight aborted fetuses (Thiersch, 1956; Rothberg et al., 19b9; Boggs et al., 1962; Raichs, 1962; Bilski-Pasquier et al., 1962; Hoover and Schumacher, 1966), one delivered a 'dysmature' infant who died shortly after birth (Merskey and Rigal, 1956), one delivered an anaemic infant (McConnell and Bhoola, 1973), seven delivered normal infants (Diamond et al., 1960; Frenkel and Meyers, 1960; Mangiameli, 1961; Sinykin and Kaplan, 1962; Bi1ski-Pasquier et al., 1962; Ravenna and Stein, 1963; Nicholson, 1968a), and one delivered a malformed infant who died at 10 weeks of age (Diamond et al., 1960). The malformed infant had multiple defects including a low birth weight, cloudy cornea, poorly developed gentalia, and a cleft of the soft and posterior hard palate. The mother of the malformed infant had chronic granulocytic leukeumia. For the treatment of her disease she received 100 mg bMP per day - 19 -orally during the first six weeks of gestation, and a single treatment of 200 r of radiation during the first month of gestation. After six weeks 6MP administration was stopped in favour of 4 to 6 mg Busulfan per day for an additional thirty weeks of gestation, followed by 125 mg 6MP per day till term. In addition, twenty-eight mothers have been reported to have received 6MP treatment during the second and third trimesters of pregnancy. Of these twenty-eight cases, three mothers died undelivered (Nicholson, 1968a,b), one aborted (O'Leary and Bepko, 1963), one had a still birth (Parekh et al., 1959), and twenty-three delivered normal infants (Schumacher, 195/; Hill, 1958; Rothberg et al., 1959; Frenkel and Meyers, 1960; Hill and Loeb, 1960; Sandberg, I960; Loyd, 1961,; Lee et al., 1962; Elizarova and Stupnitskaya, 1962; Olmer and Carcassonne, 1962; Raichs, 1962; Neu, 1962; Bi1ski-Pasquier et al., 1962; Stewart, 1964; Rigby et al., 1964; Rezende et al., 1965; Nicholson, 1968b). A high incidence of fetal loss and abnormalities (11/21) following 6MP administration during the first trimester suggests that perhaps the drug may also be fetotoxic and teratogenic in humans. - 20 -PURPOSE OF THE STUDY The aforegoing analysis of the literature indicates that the sequence of events during morphogenesis and histogenesis of the secondary palate have been repeatedly studied in different mammals. These studies were mainly concerned with events and issues involved during the reorientation of palatal shelves from a vertical to a horizontal position, and their subsequent fusion. No attention, however, was paid to the events leading to the development of palatal shelves in the vertical direction in any species. One of the purposes of this investigation, therefore, was to describe the vertical development of hamster palatal shelves using both light and electron microscopy. Cleft palate is one of the major malformations induced by 6MP in mammals. When 70 mg/kg body weight of 6MP was administered intraperitoneally into hamsters on day 9:00 of gestation, all the fetuses at term showed cleft palate without an appreciable embryotoxic effect (Shah and Burdett, 1979). Another purpose of the present study was, therefore, to analyze hitherto unreported aspects of the pathogenesis of 6MP induced cleft palate using light and electron microscopy, and enzyme cytochemistry. - 21 -MATERIALS AND METHODS Breeding of Animals Seven-week-ol d male and female Golden Syrian hamsters were obtained from Simonsen Laboratories, California, U.S.A. The animals were caged singly and maintained in an environment of temperature (24 ± 1°C), humidity (50 ± 5%), and an alternate cycle of light (6 a.m. - 6 p.m.) and dark. Food and water were available ad libitum. In the present study 200 female hamsters were utilized. Following at least one week of environmental acclimatization, each female (80 ± 5 grams) was placed with a male in the breeding cage, and copulation was observed. The mating was allowed to continue from 7 p.m. to 9 p.m. The midpoint of the mating period, 8 p.m., was considered as day 0 (0 days : 0 hours) of gestation. Drug Treatment and Procurement of Tissues On day 9:00 of gestation, each female was given an intraperitoneal injection of either 70 mg/kg 6MP (Sigma Chemicals, St. Louis, M0, Calalog #M7000, Lot #82C-1130) suspended in 1 ml distilled water, or 1 ml distilled water. The latter animals served as controls. A group of drug treated and control animals were killed at six hour intervals between days 9:00 and 14:00, and then on day 15:00 of gestation. The fetuses were delivered by caesarian section and immediately immersed in the appropriate fixative as described be!ow. - 22 -For Gross and Light Microscopic Studies For macroscopic and light microscopic studies the fetuses were immersed in Bouin's solution for 48 hours. They were then dehydrated through an ascending concentration of ethanol, starting at 30%. Upon reaching a 70% concentration of ethanol, each fetus was weighed, and measured for crown-rump length (CRL). The status of palate development was ascertained and recorded. The fetuses were then further processed through higher concentrations of ethanol (80%-100%), and then through three changes of chloroform, and embedded in paraffin to procure frontal sections. Six micron serial sections were stained with Hematoxylin and Eosin. For Electron Microscopic Studies For electron microscopic studies the fetal heads were immersed in 0.1 M phosphate buffered 2.5% gluteraldehyde (pH 7.3) at 0-4°C (Sabatini et al., 1963) for 8-12 hours. They were then given four 10 minute rinses of 0.1 M phosphate buffer (pH 7.3) at 0-4°C. The tongue and mandible were dissected from the fetal heads and discarded. The fetal heads were then immersed in 1% phosphate buffered osmium tetroxide for 90-120 minutes in the dark at 0-4°C. The heads were then quickly washed with 0.1 M phosphate buffer (pH 7.3; 0-4°C) followed by a rinse with distilled water (0-4°C). Subsequently the tissues were dehydrated through ascending concentrations of ethanol starting from 30%. Upon reaching 100% ethanol, the tissues were processed through two changes of propylene oxide. The heads were then immersed in a 3:1, and then a 1:1 mixture of propylene oxide:Epon-Araldite for at least 30 minutes each, and left overnight in 1:3 propylene oxide:Epon-Araldite mixture for - 23 -infiltration. The next morning the tissues were transferred into freshly prepared Epon-Araldite embedding medium and placed under 25 lb. vacuum for thirty minutes. Prior to embedding dimethyl aminomethyl phenol (DMP-30) was added as an accelerator to the Epon-Araldite embedding medium to achieve a 2% concentration and then thoroughly mixed (Mollenbauer, 1964). The palates were trimmed by removing extraneous tissues and then cut into anterior and posterior halves. The cut palates were placed in the Beem capsule to procure frontal sections, embedded in Epon-Araldite containing 2% accelerator, and placed in a dry heat oven at 37°C for 2 hours to eliminate gases and to permit initial polymerization. Subsequently the capsules were transferred to a 60°C dry heat oven for 3-4 days. Fran the polymerized blocks one micron sections were stained with IX Toluidine Blue, and examined for identification and orientation purposes. Thin sections (60 nm) were obtained from appropriate blocks and transferred to 300 mesh copper grids. They were then stained with methanolic uranyl acetate (Stempak and Ward, 1964) for fifteen minutes, followed by lead citrate (Reynolds, 1963) for five minutes. The grids were examined in a Philips 300 Electron Microscope operating at an accelerating potential of 60 KV. For Acid Phosphatase Enzyme Cytochemistry For acid phosphatase enzyme cytochemistry, fetal heads were immersed in formol-calcium (0-4°C) for 24-36 hours. The heads were placed on a cryostat tissue holder (International Harris Cryostat model CTD, International Equipment Corp., Needham Heights, Mass.) in a pool of Tissue Tek (Lab-Tek Products, Naperville, 111.) and quickly frozen in a -30°C cryostat. Eight - 24 -micron serial sections were cut and transferred to cold pre-cleaned glass slides. The sections were air dried for one hour, and then incubated in a mixture (Appendix I) containing naphthol AS-TR phosphoric acid, pararosaniline hydrochloride, sodium nitrite, and veronal acetate buffer at 37°C for 90 minutes in a dry heat oven (Barka and Anderson, 1962). After incubation the slides were quickly washed in distilled water, stained for 30 seconds in methyl green (pH 4.0), and washed again in distilled water. They were then quickly dehydrated by immersion in 50%, 70%, 95%, and absolute ethanol, cleared in xylene, and mounted in DPX (BDH chemicals, Vancouver, Product Code 3303, Lot # 505309). The enzyme activity was indicated by a vivid red azo dye. Two controls were used for the incubation reaction: for a negative control sections from each fetal head were incubated in a substrate-free medium, which was otherwise identical; for a positive control sections of normal rat liver were included in each incubation jar with the fetal head sections. Statistical Analysis For statistical analysis the data were subjected to the following methods. The correlations between gestational age, CRL and fetal weight were evaluated by a linear regression and exponential regression (Sokal and Rohlf, 1969). The regression lines for treated and control data were compared to assess significance. The palatal staging was tabulated against gestational age, CRL and fetal weight and evaluated by contingency table analysis (Zar, - 25 -1974). Standard curves illustrating the probability of each palatal stage in relation to gestational age, CRL and fetal weight were obtained and evaluated for significance by the chi-square method (Berksoh, 1957). - 26 -RESULTS Morphogenesis of the Secondary Palate 1n Control Hamster Fetuses Based on gross observations, which were later confirmed histologically, the morphogenesis of control secondary palate can be categorized within one or another of the following stages: Stage 1. The appearance of the palatal primordia from the roof of the oronasal cavity toward its floor (Figs. 3,4). The tongue is absent (Fig. 4). Stage 2. The palatal shelves are vertical on the sides of the tongue, which originates from the floor of the oronasal cavity (Figs, b, 6). Stage 3. The palatal. shelves are horizontal above the tongue (Figs. 7, 8). Stage 4. The palatal shelves unite in the middle third, but remain open in the anterior and posterior thirds of the secondary palate (Figs. 9, 10). Stage 5. The palatal shelves unite in the posterior third of the secondary palate. The anterior third remains open (Fig. 11). Stage 6. The palatal shelves unite in the anterior third of the secondary palate, and with this event the closure of the palate is complete (Fig. 12). The data on gross observations of normal palate development in relation to gestational age of the fetus are presented in Table III. One may deduce from the table that: - 28 -F1g. 3 Ventral view of the developing palate of a control hamster on day 10:00 of gestation. The mandible has been removed. Stage 1. The palatal primordia (asterisk) appear from the roof of the oronasal cavity. 16.5X. F1g. 4 Frontal section through the secondary palate region in a control hamster fetus on day 10:00 of gestation. Stage 1. The palatal primordia (arrows) develop from the roof of the oronasal cavity towards the floor (F). The tongue is absent. Paraffin section. Hematoxylin and Eosin stain. 58X. F1g. 5 Ventral view of the developing palate of a control hamster on day 11:18 of gestation. The mandible and tongue have been removed. Stage 2. The palatal shelves (P) are in vertical position. 8.5X. F1g. 6 Frontal section through the secondary palate region in a control hamster fetus on day 11:18 of gestation. Stage 2. The palatal shelves (P) are vertical on the sides of the tongue (T). Paraffin section. Hematoxylin and Eosin stain. 75X. F1g. 7 Ventral view of the developing palate of a control hamster on day 12:00 of gestation. The mandible and tongue have been removed. Stage 3. The palatal shelves (P) are horizontal. 8.5X. Fig. 8 Frontal section through the secondary palate region in a control hamster fetus on day 12:00 of gestation. Stage 3. The palatal shelves (P) are horizontal above the tongue (T). Paraffin section. Hematoxylin and Eosin stain. 258X. F1g. 9 Ventral view of the developing palate of a control hamster on day 12:00 of gestation. The mandible and tongue have been removed. Stage 4. The palatal shelves unite in the middle third (arrow head), but remain open in the anterior (a) and posterior (b) thirds of the secondary palate. 8.5X. Fig. 10 Frontal section through the secondary palate region in a control hamster fetus on day 12:00 of gestation. Stage 4. The opposing palatal shelves (P) are fused with one another. NC - nasal cavity. OC - oral cavity. Paraffin section. Hematoxylin and Eosin stain. 255X. - 30 -Fig. 11 Ventral view of the developing palate of a control hamster on day 12:18 of gestation. The mandible and tongue have been removed. Stage 5. The palatal shelves unite in the posterior third (b) of the secondary palate. The anterior third (arrowhead) remains open. 6X. F1g. 12 Ventral view of the developing palate of a control hamster on day 13:00 of gestation. The mandible and tongue have been removed. Stage 6. The palatal shelves are united in the anterior third, thus marking a complete closure of the secondary palate. 6X. Fig. 13 Ventral view of the developing palate of a 6MP treated hamster on day 10:00 of gestation. The mandible has been removed. Stage 1. The palatal primordia (asterisk) appear from the roof of the oronasal cavity. 16.5X. F1g. 14 Frontal section through the secondary palate region in a 6MP treated hamster fetus on day 10:00 of gestation. Stage 1. The palatal primordia (arrows) develop from the roof of the oronasal cavity towards the floor (F). Epon-araldite section. Methylene blue stain. 92X. F1g. 15 Ventral view of the developing palate of a 6MP treated hamster on day 11:18 of gestation. The mandible and tongue have been removed. Stage 2. The palatal shelves (P) are vertical. 9X. F1g. 16 Frontal section through the secondary palate in a 6MP treated hamster fetus on day 11:18 of gestation. Stage 2. The palatal shelves (P) are vertical on the sides of the tongue (T). Epon-araldite section. Methylene blue stain. 75X. F1g. 17 Ventral view of the developing palate of a 6MP treated hamster on day 15:00 of gestation. The mandible and tongue have been removed. Stage 2. The palatal shelves (P) are vertical. 5X. Fig. 18 Frontal section through the secondary palate in a 6MP treated hamster fetus on day 15:00 of gestation. Stage 2. The palatal shelves (P) are rudimentary. T - tongue. Bone formation (B) is present within the shelves. Paraffin section. Hematoxylin and Eosin stain. 40X. - 31 -Table III. Staged Development of the Secondary Palate In Control Hamster Fetuses Gestational Number Number of Fetuses at Each Stage of Age of Palatal Development lys: hours) Utters 1 2 3 4 5 6 9:06 5 51* 9:12 5 61* 9:18 5 47* 10:00 6 65 10:06 5 44 10:12 6 68 10:18 5 55 11:00 5 55 11:06 5 41 11:12 5 5/ 11:18 5 58 1 12:00 5 23 13 15 12:06 5 2 10 33 1 12:12 5 5 50 8 12:18 4 32 11 13:00 4 8 38 14:00 3 41 15:00 4 45 *Palatal primordia are absent - 32 -1. The initiation of the palatal primordia occur between days 9:18 and 10:00 of gestation (Stage 1). 2. The vertical development of the palatal shelves (Stage 2) is accomplished in all fetuses between days 10:06 and 12:00 of gestation. 3. In almost 95% of the fetuses Stages 3 (horizontal shelves), and Stage 4 (closure of the shelves in the middle third of the secondary palate) are achieved between days 12:00 and 12:06 of gestation. 4. In 92% of the fetuses Stage 5 (closure of the shelves in the posterior third of the secondary palate) is completed by day 12:12 of gestation. 5. In over 83% of fetuses Stage 6 (complete closure of the secondary palate) is accomplished by day 13:00 of gestation. Morphogenesis of the Secondary Palate In 6MP Treated Hamster Fetuses The observations of 6MP treated fetal palatal development are summarized in Table IV. From the table, one may suggest that, like the controls, the initiation of the drug treated palatal primordia (Stage 1) occurs between days 9:18 and 10:00 of gestation (Figs. 13, 14). The palatal shelves are oriented vertically on the sides of the tongue (Stage 2) between days 10:06 and 12:00 of gestation (Figs. 15, 16). Subsequently, however, approximately 98% of the drug treated fetuses remained in Stage 2 of palatal development. In these fetuses Stages 3-6 were never accomplished. At term, the palatal - 33 -Table IV. Staged Development of the Secondary Palate 1n 6-Mercaptopurine Treated Hamster Fetuses Gestational Number Number of Fetuses at Each Stage of Age of Palatal lys: hours) Litters 1 2 3 9:06 3 29* 9:12 3 24* 9:18 3 27* 10:00 5 49 10:06 3 30 10:12 3 32 10:18 3 30 11:00 4 36 11:06 5 56 11:12 3 31 11:18 3 26 12:00 4 34 12:06 4 39 4 12:12 5 41 12:18 5 45 6 13:00 6 48 3 14:00 4 36 15:00 4 35 *Palatal primordia are absent - 34 -shelves are rudimentary and oriented vertically on the sides of the tongue (Figs. 17, 18). Morphogenesis of the Secondary Palate in Control Hamster Fetuses in Relation  to Fetal Weight The mean weight of control fetuses increases gradually from 11.7 mg to 345.7 mg between days 9:18 and 13:00 of gestation (Table V). Thereafter the fetal weight increases rapidly and at day 15:00 of gestation the mean fetal weight was 1901.6 mg. Since at different times during gestation the fetuses varied in their stage of palatal development (Table III), and in weight (Table V), an attempt was made to see if there was a relationship between the fetal weight and the stage of palatal development. Fetuses were grouped by weight as shown in Table VI. The palates were examined for their morphologic stage of development. The observations in the control group revealed: 1. Morphologic Stage 2 is achieved in all fetuses weighing 51 mg. 2. Stages 3-4 are completed in all fetuses weighing 251-300 mg. 3. Stage 5 is reached in 90% fetuses weighing 301 mg. 4. In fetuses weighing 551 mg, morphologic Stage 6 of palatal development is completed. - 35 -Table V. The Mean Weights of Control and 6-Mercaptopur1ne (6MP) Treated Hamster Fetuses at Different Times During Gestation Gestational Mean Fetal Weight In Milligrams +_ Standard Deviation Age days:hours Control 6MP Treated 9:06 7.8 + 2.6 8.9 + 4.1 9:12 12.6 + 2.8 11.6 + 3.7 9:18 12.0 + 2.7 15.7 + 5.5 10:00 21.2 + 6.5 22.7 + 6.5 10:06 31.4 + 9.0 26.0 + 7.6 10:12 35.4 + 4.1 32.7 + 5.6 10:18 52.1 + 10.8 31.2 + 5.1 11:00 75.1 + 14.4 60.6 + 14.0 11:06 80.5 + 20.1 67.8 + 26.2 11:12 96.2 + 14.2 54.3 + 13.6 11:18 138.5 + 21.0 75.0 + 26.5 12:00 142.3 + 21.9 90.2 + 32.0 12:06 261.3 + 39.5 128.3 + 35.6 12:12 271.1 + 29.5 135.1 + 41.9 12:18 300.0 + 44.4 182.3 + 67.6 13:00 345.7 + 108.5 181.0 + 115.4 14:00 1061.4 + 320.3 583.8 + 210.7 15:00 1901.6 + 470.6 732.1 + 309.9 - 36 -Table VI. Staged Development of the Secondary Palate 1n Relation to Control Hamster Fetal Weight Fetal Weight Number of Fetuses at Each Stage of Palatal Development (mg) 1 2 3 4 5 6 1 - 50 224* 101 51 - 100 161 101 - 150 85 1 151 - 200 46 19 3 2 201 - 250 10 4 30 17 1 251 - 300 12 33 2 301 - 350 3 21 6 351 - 400 9 18 401 - 450 5 14 451 - 500 3 13 501 - 550 1 4 551 -2750 85 *For 159 fetuses the palatal primordia were absent - 37 -Morphogenesis of the Secondary Palate in 6HP Treated Hamster Fetuses in  Relation to Fetal Height Following 6MP treatment, the mean fetal weight increased from 18.5 mg at day 9:18 to 181 mg at day 13:00 of gestation (Table V). On day 15:00 of gestation, the average fetal weight was 732.1 mg. Statistical evaluation of the data from Table V indicate that there was no difference in the mean fetal weight between control and 6MP treated fetuses from day 9:06 to 10:00 of gestation, i.e., during the period of initial shelf development. In the ensuing 12 hours the mean weight of 6MP treated fetuses was lower than that of the controls. Subsequently on day 10:18 of gestation, however, a pronounced difference in the mean fetal weight was observed between the control and treated groups, and it persisted until day 15:00 (PO.001). In Table VII the data on staged palatal development following 6MP treatment are arranged in relation to the fetal weight. One may infer that, as in the controls, the morphologic Stage 2 of palate development is reached in 6MP treated fetuses weighing 51 mg. Thereafter, even though the fetal weight continues to increase, the palatal shelves remained vertical. Morphogenesis of the Secondary Palate in Control Hamster Fetuses in Relation  to Fetal Crown-Rump Length (CRL) During normal development, the mean fetal CRL increases progressively from 5.1 mm to 14.1 mm between days 9:18 and 13:00 of gestation (Table VIII). Subsequent increase in the fetal CRL is rapid and at day 15:00 of gestation it was 26.8 mm. - 38 -Table VII. Staged Development of the Secondary Palate 1n Relation to 6-Mercaptopur1ne Treated Hamster Fetal Weight Fetal Weight Number of Fetuses at Each Stage of Palatal Development 4 5 6 (mg) 1 2 3 1 - 50 129* 142 51 - 100 146 101 - 150 • 86 151 - 200 43 3 201 - 250 25 1 251 - 300 10 4 301 - 350 5 * 2 351 - 400 3 1 401 - 450 9 1 451 - 500 5 1 501 - 550 3 551 -2750 42 *For 80 fetuses the palatal primordia were absent - 39 -Table VIII. The Mean Crown-Rump Length (CRL) of Control and 6-Mercaptopurine (6MP) Treated Fetuses at Different Times During Gestation Gestational Mean CRL In Millimeters + Standard Deviation Age days:hours Control 6MP Treated 9:06 4.1 + 0.6 4.0 + 0.6 9:12 5.2 + 0.4 4.9 + 0.6 9:18 5.1 + 0.4 5.5 + 0.7 10:00 5.7 + 0.6 6.0 + 0.6 10:06 7.3 + 0.4 6.7 + 0.5 10:12 7.1 + 0.8 7.2 + 0.3 10:18 7.9 + 0.5 6.8 + 0.3 11:00 8.5 + 0.6 8.1 + 0.7 11:06 9.6 + 0.7 8.1 + 0.9 11:12 9.2 + 0.4 8.0 + 0.6 11:18 10.6 + 0.6 8.8 + 1.0 12:00 10.7 + 0.7 9.5 + 0.8 12:06 12.4 + 0.6 10.6 + 1.1 12:12 12.7 + 0.6 10.7 + 0.9 12:18 13.6 + 0.8 11.5 + 1.4 13:00 14.1 + 1.7 11.5 + 2.5 14:00 21.5 + 1.8 16.5 + 2.5 15:00 26.8 + 2.2 19.3 + 3.0 - 40 -Table IX shows grouping of fetuses by CRL. The palates were examined for morphologic stage of development. One may deduce from the table that: 1. Stage 2 of palate development is reached in all fetuses measuring 7 mm CRL, and is completed in all fetuses measuring 12 mm CRL. 2. Stages 3-4 are accomplished in all fetuses measuring 13 mm CRL. 3. Stage 5 is achieved in all fetuses measuring 15 mm, and stage 6 by 16 mm CRL. Morphogenesis of the Secondary Palate in 6MP Treated Hamster Fetuses 1n  Relation to Fetal Crown-Rump Length The mean fetal CRL increased from 5.8 mm to 11.5 mm between days 9:18 and 13:00 of gestation (Table VIII). Subsequently on day 15:00 the average fetal CRL was 19.3 mm. Statistical analyses of the data from Table VIII indicate that the mean fetal CRL of both the treated and control groups were similar between days 9:06 and 11:00 of gestation. During next 48 hours, i.e., until day 13:00 of gestation, the mean CRL in the treated group remained lower than that in the controls. Thereafter, a pronounced difference in the mean fetal CRL was observed between the control and treated groups which persisted on day 15:00 of gestation (P<0.001). In Table X the data on staged palatal development are arranged in relation to the 6MP treated mean fetal CRL. The observations suggest that, as in the controls, Stage 2 of palate development is reached in all fetuses measuring 7 mm CRL. Thereafter, even though the fetal CRL continued to increase, the palatal shelves remained vertical. - 41 -Table IX. Staged Development of the Secondary Palate 1n Relation to Control Fetal Hamster Crown-Rump Length (CRL) CRL Number of Fetuses at Each Stage of Palatal Development (mm) 1 2 3 4 5 6 0 - 1.9 2.0 - 2.9 2* 3.0 - 3.9 6* 4.0 - 4.9 65* 5.0 - 5.9 105** 6.0 - 6.9 46 31 7.0 - 7.9 89 8.0 - 8.9 83 9.0 - 9.9 69 10.0 - 10.9 71 1 11.0 - 11.9 •48 20 16 4 12.0 - 12.9 12 3 23 26 3 13.0 - 13.9 9 31 3 14.0 - 14.9 5 23 17 15.0 - 15.9 7 19 16.0 - 16.9 15 17.0 - 30.0 86 * Palatal primordia are absent **For 86 fetuses the palatal primordia are absent - 42 -Table X. Staged Development of the Secondary Palate 1n Relation to 6-Mercaptopurine Treated Fetal Hamster Crown-Rump Length (CRL) CRL Number of Fetuses at Each Stage of Palatal Development (mm) 1 2 3 4 5 6 0 - 1.9 2.0 - 2.9 3.0 - 3.9 8* 4.0 - 4.9 37* 5.0 - 5.9 41** 6.0 - 6.9 43 49 7.0 - 7.9 102 8.0 - 8.9 81 9.0 - 9.9 62 10.0 - 10.9 73 11.0 - 11.9 46 2 12.0 - 12.9 24 2 13.0 - 13.9 11 3 14.0 - 14.9 7 4 15.0 - 15.9 16 16.0 - 16.9 6 2 17.0 30.0 42 * Palatal primordia are absent **For 35 fetuses the palatal primordia are absent - 43 -Light Microscopic Observations of the Developing Secondary Palate In Control  Hamster Fetuses On day 9:06 of gestation the palatal primordia are not formed. The roof of the oronasal cavity is flat and made of loose mesenchyme covered by a single layer of epithelial cells (Fig. 19). The epithelial cells are cuboidal, and contain a large spherical nucleus surrounded by a thin rim of cytoplasm. The mesenchymal cells are stellate and contain a large nucleus. Mitosis is occasionally observed in cells of both the epithelium and mesenchyme. During next 18 hours, i.e., until day 10:00 of gestation, the morphology of both the epithelial and mesenchymal cells remains unchanged. Histologically, from day 10:00 onward, however, the normal palatal development can be observed in five stages. Stage I. The palatal primordia appear from the roof of the oronasal cavity. A palatal primordium is composed of mesenchymal tissue covered by a bilaminar epithelium (Fig. 20). The mesenchymal cells are stellate and closely packed. The superficial epithelial cells are flat and contain an elongated nucleus. The basal epithelial cells are cuboidal and contain a large spherical nucleus. Stage II. The epithelium on the medial aspect of the vertical palatal shelf, the prospective fusion epithelium (Shah, 1979a), is two to three cell layers thick (Fig. 21). The appearance of both the epithelial and mesenchymal cells, however, remained unaltered. Few cells undergoing mitosis are observed in the epithelium and mesenchyme. In the epithelial cells, mitosis is seen only in the basal layer. - 45 -F1g. 19 Frontal section through the secondary palate region of a control hamster on day 9:06 of gestation. The roof (R) of the oronasal cavity is covered by a single layer of cuboidal epithelial (E) cells. The mesenchymal cells are stellate. Epon-araldite section. Methylene blue stain. 920X. Fig. 20 Frontal section through the secondary palate region of a control hamster fetus on day 10:00 of gestation showing the bilaminar epithelium of the palatal primordia (Stage I). The bilaminar epithelium is made of flat superficial (S) and cuboidal basal (B) cells. The mesenchymal cells are stellate. Epon-araldite section. Methylene blue stain. 980X. F1g. 21 Frontal section through the secondary palate of a control hamster fetus on day 11:18 of gestation showing the two to three cell layered epithelium (arrow) of the medial aspect of the vertical palatal (P) shelf (Stage II). 1 - tongue. Epon-araldite section. Methylene blue stain. 360X. F1g. 22 Frontal section through the secondary palate of a control hamster fetus on day 12:00 of gestation. The medial edge epithelium (arrow heads) of the horizontal palatal shelves (P) is bilaminar (Stage III). The mesenchymal cells are stellate or elongated. Epon-araldite section. Methylene blue stain. 410X. F1g. 23 Frontal section through the secondary palate of a control hamster fetus on day 12:00 of gestation. The medial edge epithelia of the opposing horizontal palatal shelves (P) have contacted to form a seam (arrow) (Stage IV). Paraffin section. Hematoxylin and Eosin stain. 410X. F1g. 24 Frontal section through the secondary palate of a control hamster fetus on day 12:06 of gestation showing a fragmenting (arrows) (Stage V) epithelial seam. Epon-araldite section. Methylene blue stain. 504X. - 46 -Stage III. The palatal shelves are horizontal above the tongue (Fig. 22). The prospective fusion epithelium on the medial edge (MEE) of the horizontal shelf shows no histological change from the previous stage. The mesenchymal cells are stellate or elongated in shape (Fig. 22). Mitosis is absent in the MEE, but is occasionally seen in the mesenchymal cells. Stage IV. The MEE of the opposing horizontal shelves are in contact with one another. The epithelial contact results in the formation of a seam (Fig. 23). The seam is two to three cell layers thick. The cells are irregular in shape and contain an oval nucleus. The mesenchymal cells did not show any alteration in their morphology. Stage V. The epithelial seam is broken into fragments (Fig. 24). The space between the fragments is occupied by mesenchymal cells. The epithelial fragments eventually disappear, and mesenchyme of the two palatal shelves becomes continuous (Fig. 25). Light Microscopic Observations of the Developing Secondary Palate In Hamster  Fetuses Following 6MP Treatment During first 18 hours, i.e., between days 9:06 and 10:00 of gestation, the histological appearance of both the epithelial and mesenchymal cells in the roof of the oronasal cavity of 6MP treated fetuses is similar to that of the controls. Stage I. The palatal primordia develop from the roof of the oronasal cavity on day 10:00 of gestation. During the first six hours of primordial development the histological appearance of the bilaminar epithelial cells resembles that of the controls. An occasional mesenchymal cell shows a small - 48 -Fig. 25 Frontal section through the secondary palate of a control hamster fetus on day 12:12 of gestation showing mesenchymal continuity between opposing palatal shelves (P). 0 - oral epithelium. N -nasal epithelium. Paraffin section. Hematoxylin and Eosin stain. 410X. F1g. 26 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 10:00 of gestation showing the primordial epithelium (Stage I). The epithelium is bilaminar with flat superficial (S ) cells and cuboidal basal (B) cells. An occasional mesenchymal cell shows a small darkly stained dense body (arrow heads). Epon-araldite section. Methylene blue stain. 980X. Fig. 27 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 10:06 of gestation. The histological appearance of the epithelial and mesenchymal cells of the vertically developing shelf (Stage II). Dense bodies are present in the mesenchymal cells (arrow heads), epithelial cells (arrows) and in the cell fragments (f). Epon-araldite section. Methylene blue stain. 980X. Fig. 28 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 11:00 of gestation. The dense bodies are absent in the mesenchymal and epithelial cells of the vertical palatal shelf (Stage II). Epon-araldite section. Methylene blue stain. 510X. Fig. 29 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 12:18 of gestation. The basal epithelial cells are light in appearance (arrow head). Epon-araldite section. Methylene blue stain. 280X. F1g. 30 Frontal section through the secondary palate of a 6MP treated hamster fetus on day 15:00 of gestation. The epithelium of the stunted palatal shelf is stratified squamous (arrow head). Bone formation (B) is observed in the mesenchyme. Paraffin section. Hematoxylin and Eosin stain. 2U8X. - 49 -darkly stained dense body in its cytoplasm {Fig. 26). Such cells with dense bodies are not present at the corresponding stage during normal development. Stage II. In the next 12 hours, i.e., between days 10:00 and 10:12 of gestation, the histological appearance of both the mesenchyme and epithelial cells changes, and on day 10:12 of gestation they are strikingly different from that of the controls. The dense bodies are present in many mesenchymal cells (Fig. 27) of the treated palatal shelf. Also small fragments of cells containing dense bodies are present in the extracellular matrix of the mesenchyme. A few epithelial cells, in addition, show dense bodies in their cytoplasm (Fig. 27). Subsequently, during next the 12 hours, only a few epithelial and mesenchymal cells show dense bodies. On day 11:00 of gestation none of the epithelial and mesenchymal cells show dense bodies (Fig. 28). Histologically both the epithelial and mesenchymal cells of the treated vertical palatal shelf resemble those of the controls. Between days 11:00 and 12:18 of gestation the epithelial and mesenchymal cells of 6MP treated palatal shelves appear unaltered. Unlike those of the controls, some basal cells in the epithelium, on day 12:18 of gestation, are relatively light in appearance (Fig. 29). The mesenchymal cells, however, appear unchanged. The treated vertical palatal shelves appear smaller than those of the controls (cf. Figs. 6, 16). The light cells persist in the epithelium during the ensuing 24 hours, i.e., until day 13:18 of gestation. On day 14:00 of gestation, and thereafter, the light cells are absent. The epithelium on the stunted - 50 -vertical shelf undergoes stratification. On day 15:00 of gestation, the epithelium is stratified squamous (Fig. 30). The mesenchyme shows evidence of bone formation (Figs. 18, 30). Stages III-IV. Histologic Stages III (horizontal shelves), IV (formation of an epithelial seam), and V (fragmentation of epithelial seam) of the normal palatal development are not seen in the 6MP treated fetuses at any time during gestation. The palatal shelves instead remain vertical and appear stunted at term (Fig. 18). Electron Microscopic Observations of the Developing Secondary Palate in  Control Hamster Fetuses Prior to the appearance of palatal primordia, the roof of the oronasal cavity is lined by simple cuboidal epithelium. The epithelium is separated from the underlying mesenchyme by a continuous basal lamina (Fig. 31). Spaces are present between the epithelial cells which are otherwise attached to one another by desmosomes. The epithelial cells (Fig. 31) contain a large nucleus surrounded by polyribosomes, a few mitochondria, and occasional cisternae of rough endoplasmic reticulum. The mesenchymal cells (Fig. 31) contain a round to oval nucleus surrounded by organelles similar to the epithelial cells. With the appearance of palatal primordia histological Stages I through V of palatogenesis were studied at the ultrastructural level. - 52 -Fig. 31 Electron micrograph of the roof of the oronasal cavity in a control hamster fetus on day 9:12 of gestation. The epithelium is separated from the mesenchyme by a continuous basal lamina (BL). The mesenchymal cell (NIC) contains a round to oval nucleus surrounded by organelles similar to the epithelial cells. ICS -intercellular space. N - nucleus. M - mitochondrion. D -desmosome. RER - rough endoplasmic reticulum. 8664X. F1g. 32 Electron micrograph of the secondary palate primordia in a control hamster fetus on day 10:00 of gestation showing a continuous basal lamina (BL) separating the bilaminar epithelium from the mesenchyme. A small golgi complex (GC) appears in the mesenchymal cell. B - basal cell. S - superficial cell. Nl - mitochondrion. ICS - intercellular space. N - nucleus. RER - rough endoplasmic reticulum. D - desmosome. 10180X. - 53 -Stage I. The palatal primordium is covered by a bilaminar epithelium (Fig. 32). The basal cells are cuboidal and the superficial cells flat. Both cell types contain a large nucleus surrounded by polyribosomes, mitochondria, cisternae of rough endoplasmic reticulum and a small Golgi complex. Numerous large spaces are present between cells of the epithelium. Both the superficial and basal cells are attached to one another by desmosomes. The basal lamina separating the epithelium from the mesenchyme is continuous. The mesenchymal cells of the vertically developing palatal primordia (Fig. 32) contain more organelles than those observed in the roof of the oronasal cavity. In addition, a small Golgi complex appears in the mesenchymal cells of the vertically developing palatal primordia (Fig. 32). Stage II. The morphology and contents of both the epithelial and mesenchymal cells seen during Stage I remain unchanged in the vertical palatal shelf. Stage III. The epithelium of the horizontal palatal shelf is separated from the mesenchyme by a continuous basal lamina (Figs. 33, 34). In comparison to large intercellular spaces seen at Stages I and II, the intercellular spaces in the epithelium of the horizontal palatal shelf are smaller. The cytoplasm of the epithelial cells show membrane-bound and membrane-free areas containing aggregations of electron dense granules, and dense bodies (Figs. 33, 34). On a morphological basis, these areas are interpreted as lysosomes (DeDuve, 1963; Erickson, 1969). Other cytoplasmic contents of the epithelial cells remain unchanged from Stage II. - 55 -Fig. 33 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the epithelium of the horizontal palatal shelf, lysosomes (L) are present in both the superficial and basal epithelial cells. BL - basal lamina. N -nucleus. 6412X. F1g. 34 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the mesenchymal cells of the horizontal palatal shelf. H - nucleus. M - mitochondrion. BL - basal lamina, L - lysosome. 8000X. - 56 -With the exception of lysosomes, the mesenchymal cells contain organelles (Fig. 34) similar to those of the epithelial cells. Stage IV. During this stage the epithelia of the opposing shelves come into contact with one another thus forming a seam (Figs. 23, 24). The epithelial seam is two to four layers in width (Fig. 35). The cells in the seam are attached to one another by desmosomes. A continuous basal lamina separates the epithelial seam from the surrounding mesenchyme. The cells of the epithelial seam show considerable variation in size, shape and contents (Fig. 35). They are cuboidal to irregular in shape and contain an oval or irregular nucleus surrounded by numerous polyribosomes, Golgi complex, and a few cisternae of rough endoplasmic reticulum. The mitochondria show disruption in their cristae. Lysosomes are more numerous than those observed at Stage III. The mesenchymal cells are stellate and resemble those of Stage III. Stage V. In contrast to previous stages, the basal lamina separating the fragments of epithelial seam from mesenchyme is not continuous (Fig. 36). The cells of fragmented seam contain large lysosomes which appear to dissolve the cytoplasmic content. The mesenchymal cells, near the epithelial fragments, contain a few vacuoles and lysosomes (Fig. 36). These cells are interpreted as macrophages. They appear to carry away the debris of autolysed epithelial cells, thus clearing the area for mesenchymal union. - 58 -F1g. 35 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing the epithelial seam. The cells of the seam are attached to one another by desmosomes (D). A basal lamina (BL) separates the epithelial cells from the surrounding mesenchyme on both sides of the seam. N - nucleus. L - lysosomes. 8330X. Fig. 36 Electron micrograph of the secondary palate in a control hamster fetus on day 12:06 of gestation showing a fragment of an epithelial seam (e) and a macrophage (m). A discontinuous basal lamina (BL) surrounds the cells of the epithelial fragment. Lysosomes (L) are present in both the epithelial cells and the macrophage. 6125X. - 59 -Electron Microscopic Observations of the Developing Secondary Palate  Following 6MP Treatment During first 18 hours following drug administration, i.e., until day 9:18 of gestation, the morphology and contents of both the epithelial and mesenchymal cells of the roof of the oronasal cavity (Fig. 37) resemble those of the controls. The epithelium is simple and made of cuboidal cells which are separated from the subjacent stellate mesenchymal cells by a continous basal lamina (Fig. 37). The cytoplasm of both the epithelial and mesenchymal cells contains a nucleus surrounded by polyribosomes, mitochondria, and rough endoplasmic reticulum. Subsequently, histological Stages I and II of palatal development following 6MP treatment were examined at the ultrastructural level. Stage I. On day 10:00 of gestation, the ultrastructural appearance of the epithelial cells of the 6MP treated palatal primordia remains identical to that of the control. The basal lamina separating the epithelium and mesenchyme is continuous (Fig. 38). Alterations are, however, seen in the mesenchyme of the 6MP treated, palatal primordia. Some of the mesenchymal cells (Fig. 38) appear irregular, and contain an oval nucleus surrounded by polyribosomes, few mitochondria, and an occasional dense body. The perinuclear space of the nuclear envelope is swollen, and the nucleoplasm appears clear due to clumping of chromatin (Fig. 38). Other mesenchymal cells (Fig. 39) are stellate. They contain an oval or indented nucleus surrounded by polyribosomes, mitochondria, a few cisternae of rough endoplasmic reticulum, and a well developed Golgi complex. An - 61 -Fig. 37 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 9:06 of gestation. The epithelium is separated from the mesenchyme by a continuous basal lamina (BL). N - nucleus. M - mitochondrion. RER - rough endoplasmic reticulum. 5897X. F1g. 38 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation. The basal lamina (BL) separating the epithelium and mesenchyme is continuous. The perinuclear space of the mesenchymal cell nuclear envelope is swollen (arrow heads), and the nucleoplasm appears clear due to clumping of chromatin. N - nucleus. M - mitochondrion. db -dense body. E - epithelium. 5783X. - 62 -- 63 -Fig. 39 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation. The mesenchymal cells show a nucleus (N) surrounded by polyribosomes, mitochondria (M), a few cisternae of rough endoplasmic reticulum (RER), and a well developed Golgi complex (GC). BL - basal lamina. 5880X. F1g. 40 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:00 of gestation. The mesenchymal cell shows a membrane bound lysosome (L). N - nucleus. M -mitochondrion. 5927X. - 64 -occasional cell also shows, in addition, membrane-bound and membrane-free structures of varying electron densities containing granular material and cytoplasmic organelles such as mitochondria (Fig. 40). On a morphological basis, these structures were interpreted as lysosomes (DeDuve, 1963; Erickson, 1969). The lysosomes were absent at a comparable time during normal development. Stage 11. On day 10:06 of gestation, a few mesenchymal cells show massive disruption of their cytoplasmic morphology (Fig. 41). The cytoplasm appears vacuolated, and shows numerous lysosomes. The lysosomes vary in size, and contain dense granules, dense bodies and vacuoles. The nucleus is indented, and, along with other cytoplasmic organelles, pushed to the side. These mesenchymal cells are interpreted as macrophages. Small membrane-bound structures containing dense bodies and cytoplasmic organelles (Fig. 41) are also found in the intercellular space near the macrophages. Presumably these structures represent fragments of expelled material from the mesenchymal cells, and are in the process of being phagocytosed by the macrophages. Subsequently, on day 10:12 of gestation, the mesenchymal cells continue to show disruptive changes in their cytoplasm. In addition, alterations are also seen for the first time in the epithelium. Some cells of the bilaminar epithelia are altered, and show a nucleus surrounded by polyribosomes, a few mitochondria, cisternae of rough endoplasmic reticulum, lipid droplets, and lysosomes (Fig. 42). Other epithelial cells are not altered (Fig. 43), and resemble their control counterpart. The basal lamina beneath both the - 66 -F1g. 41 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:06 of gestation. A mesenchymal cell showing massive disruption of its cytoplasmic morphology, and interpretted as a macrophage. The cytoplasm appears vacuolated, and shows numerous lysosomes (L). The nucleus (N) is indented and is pushed to the side. Small membrane bound structures (arrow heads) containing dense bodies and cytoplasmic organelles are present near the macrophage. M - mitochondria. 7594X. - 68 -Fig. 42 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:12 of gestation showing cytoplasmic features of the epithelial cells. The nucleus (N) is surrounded by polyribosomes, mitochondria (M), cisternae of rough endoplasmic reticulum (RER), lipid droplets (1), and lysosomes (l). The basal lamina (BL) is continuous. 5789X. Fig. 43 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 10:12 of gestation showing unaffected epithelial cells (cf. Fig. 42). The basal lamina (BL) is continuous. N - nucleus. M - mitochondrion. 5894X. - 69 -altered and unaltered epithelial cells, however, is continuous (Figs. 42, 43). In the ensuing 24 hours, i.e., between days 10:18 and 11:18 of gestation, the epithelial and mesenchymal changes in the 6MP treated palate are gradually reduced. On day 11:18 of gestation, the epithelium on the medial aspect of the vertical palatal shelf is bilaminar (Fig. 44). Both the superficial and basal epithelial cells are irregular in appearance. They contain an irregular nucleus surrounded by polyribosomes, a few mitochondria, an occasional cisterna of rough endoplasmic reticulum and lipid droplets. The basal cells are attached with one another, and with the superficial cells by desmosomes. Numerous spaces otherwise intervene between cells of the epithelium. The basal lamina separating the epithelium and mesenchyme is continuous. The mesenchymal cells are stellate and contain organelles similar to those described for epithelial cells (Fig. 44). Subsequently, unlike those of the control palates, the epithelial cells of 6MP treated did not show any appreciable alterations between days 11:18 and 12:18 of gestation. On day 12:18 of gestation, the epithelium on the medial aspect of the vertical palatal shelf is bilaminar (Fig. 45). Both the superficial and basal cells contain a large nucleus surrounded by few cytoplasmic organelles. In comparison to 6MP treated palatal epithelium on day 11:18, however, the epithelial cells are closely packed and intercellular spaces are small or absent on day 12:18 of gestation (Fig. 45). The mesenchymal cells on day 12:18 of gestation are irregular in appearance (Fig. 45). Their cytoplasmic contents remain unchanged. - 71 -F1g. 44 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 11:18 of gestation showing a bilaminar epithelium. Both the superficial and basal epithelial cells contain a nucleus (N) surrounded by polyribosomes, mitochondria (M), and rough endoplasmic reticulum (RER). The epithelial cells are attached to one another by desmosomes (D). A mesenchymal cell shows a nucleus (N) surrounded by polyribosomes, mitochondria (M) and rough endoplasmic reticulum (RER). ICS - intercellular space. BL - basal lamina. 6892X. F1g. 45 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 12:18 of gestation showing discontinuity in the basal lamina (BL). A thin cytoplasmic extension (arrow head) of the basal epithelial cell extends through the basal lamina and contacts a mesenchyml cell (MC). Intercellular spaces are missing (cf. Fig. 44). N - nucleus. M - mitochondrion. RER - rough endoplasmic reticulum. 6105X. - 72 -At the epithelial-mesenchymal interface, striking changes are visible on day 12:18 of gestation. At various places the basal lamina is discontinuous (Fig. 45). A thin cytoplasmic extension of the basal epithelial cell extends through the basal lamina, and either contacts a mesenchymal cell (Fig. 45), or remains free (Fig. 46). During the ensuing 24 hours, i.e., between days 12:18 and 13:18 of gestation, the basal lamina changes become progressively more severe in the 6MP treated palates. On day 13:18 of gestation, when palates of control fetuses are already closed, changes are observed in the epithelium, the mesenchyme and the basal lamina of the 6MP treated vertical palatal shelf. In comparison to the superficial epithelial and mesenchymal cells, the basal epithelial cells appear light due to reduced polyribosome content (Fig. 47). In addition, their cytoplasm contains a few mitochondria, cisternae of rough endoplasmic reticulum, and a small Golgi Complex (Fig. 47). In contrast to those observed at day 12:18 of gestation, the mesenchymal cells on day 13:18 of gestation vary in shape from stellate to elongated (Fig. 47). They contain a flat nucleus surrounded by polyribosomes, numerous cisternae of rough endoplasmic reticulum, few mitochondria and a well developed Golgi Complex. The basal lamina at the interface of the epithelium and mesenchyme is almost missing (Fig. 47). In many instances a cytoplasmic extension of the mesenchymal cell appears to have penetrated the disappearing basal lamina to contact the epithelial cell (Fig. 47). Eventually, however, the alterations in the epithelial cells become less severe. Between days 14:00 and 15:00 of gestation, the epithelium on the 6MP - 74 -Fig. 46 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 12:18 of gestation showing a cytoplasmic extension (arrow head) of the basal epithelial cell. BL - basal lamina. 42325X. F1g. 47 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 13:18 of gestation showing a light basal cell (BC). The cytoplasm of the light basal cell contains a reduced polyribosome content, mitochondria (M), rough endoplasmic reticulum (RER), and a Golgi complex (GC). The elongated mesenchymal cells contain a nucleus (N) surrounded by polyribosomes, rough endoplasmic reticulum (RER), mitochondria (M) and a Golgi complex (GC). The basal lamina (BL) is almost missing. A cytoplasmic extension (arrow head) of the mesenchymal cell contacts the epithelial cell. N - nucleus. D - desmosome. 7565X. - 75 -treated cleft palatal shelf is made of 3-5 cell layers (Fig. 48). The basal cells are roughly cuboidal, and contain an irregular nucleus surrounded by polyribosomes, mitochondria, few cisternae of rough endoplasmic reticulum, and tonofilaments. The outer 2-4 layers of cells are flat, and contain organelles similar to those in the basal epithelial cells. Spaces are numerous between the basal, and between the basal and suprabasal epithelial cells (Fig. 48). Both the basal and outer flat cells are attached to their neighbours by desmosomes. The basal lamina separating the epithelium from the mesenchyme is continuous (Fig. 48). Numerous hemidesmosomes are seen for the first time between the plasma membrane of the basal cell and the basal lamina (Fig. 48). The mesenchymal cells of the cleft palatal shelves remain unaltered. Light Microscopic Observations of Enzyme Acid Phosphatase in the Developing  Secondary Palate of Control Hamster Fetuses Prior to day 12:00 of gestation, i.e., histological Stages I and II of palatal development, the reaction product of enzyme acid phosphatase is absent in both the epithelial and mesenchymal cells. A mild reaction product of the enzyme is observable in cells of the medial edge epithelium of the horizontal palatal shelves (Stage III), and epithelial seam (Stage IV). At histological Stage V, the acid phosphatase reaction product is seen in the epithelial cells of the fragmented seam, and in the nearby mesenchymal cells. - 77 -F1g. 48 Electron micrograph of the secondary palate in a 6MP treated hamster fetus on day 14:00 of gestation showing a stratified epithelium. The basal cells contain irregular nuclei (N) surrounded by polyribosomes, mitochondria (M), cisternae of rough endoplasmic reticulum (RER), and tonofilaments (tf). The outer 3 to 4 layers of cells are flatter, and contain organelles similar to those in the basal epithelial cells. Numerous spaces (ICS) intervene between epithelial cells. D - desmosome. HD -hemidesmosome. BL - basal lamina. 8373X. - 78 -Light Microscopic Observations of Enzyme Acid Phosphatase in the Developing  Secondary Palate of 6MP Treated Hamster Fetuses Unlike controls, the enzyme acid phosphatase reaction product in the 6MP treated palate is first visible in the mesenchymal cells on day 10:06, and then in both the epithelial and mesenchymal cells on day 10:12 of gestation (histological Stage II). Subsequently, the enzyme activity is not seen at any time in 6MP treated palates during development. The appearance of intracytoplasmic areas containing dense granules and dense bodies at ultrastructural level, and corresponding localization of enzyme acid phosphatase reaction product at light microscopic levels, in both the control and 6MP treated palatal tissues, confirms the lysosomal nature of the dense granules and dense, bodies (Novikoff, 1963; Erickson, 1969). - 79 -DISCUSSION Normal Palatal Development Results of the present study have shown that the normal palatal development in hamster is completed between days 10:00 and 13:00 of gestation. During this period the palatal shelves initiate vertically from the roof of the oronasal cavity toward its floor. The shelves then change the direction of their development from vertical to horizontal. Subsequently, opposing epithelia of the horizontal shelves fuse with one another to form a seam. The epithelial seam, thereafter, undergoes fragmentation to allow the mesenchyme of the two shelves to become continuous. These sequential events of hamster palatal development, with the exception of the initial formation of palatal shelves (Stage 1), have also been described in other mammals such as mice (Walker and Fraser, 1956; Dostal and Jelnick, 1970; Waterman et al., 1973), rats (Asling et al., 1960; Zeiler et al., 1964; Coleman, 1965; Ferguson, 1978; Schupbach et al., 1983), monkeys (Asling and Van Wagenen, 1967; Steffek et al., 1968; Bollert and Hendrickx, 1971), gerbils (Holmstedt and Bagwell, 1977), rabbits (Walker, 1971; Meller et al., 1980), ferrets (Gulamhusein and England, 1982), and human (Fulton, 1957; Wood and Kraus, 1962; Kitamura, 1966; Burdi and Faist, 1967; Anderson and Matthiessen, 1967; Luke, 1976). Furthermore, observations between days 12:00 and 13:00 of gestation have shown that the reorientation of the palatal shelves from vertical to horizontal plane was completed in all fetuses by day 12:06 of pregnancy. Subsequently, the closure of the middle third of the secondary palate was - 80 -completed in all fetuses by day 12:12 and that of the posterior third and anterior third by days 12:18 and 13:00 of gestation, respectively. Similar chronological observations on palatal closure in hamster have been made in other studies (Shah and Chaudhry, 1974a; Shah and Travill, 1976a; Shah, 1979a, b; Shah and Wong 1980). In addition, the present study also reconfirmed the observations made in these studies that not only the age, but also the weight and CRL of the fetus are reliable indicators of stages of normal palatal development in hamster. In their studies on mouse palatogenesis Walker and Crain (1960) and Dostal and Jelnick (1972) also noted a good correlation between the stages of palatal development and fetal weight, but not between the stages of palatal development and fetal age. The reliability of chronologic • age as an indicator of the stages of normal palatal development in hamster may be due to a relatively shorter period of organogenesis and a corresponding smaller period of palatogenesis in hamster as compared with mice. It is anticipated that the reproducibility of the accurate timings, fetal weights, and fetal CRL for the important events of palatogenesis in hamster should strengthen its usefulness as a biological system for further embryological and teratological studies. In contrast to mammals, the palatal shelves in the other vertebrates such as chick (Shah and Crawford, 1980; Koch and Smiley, 1981), quail (Shah et al., 1984b), and alligator (Ferguson, 1981) originate horizontally over the dorsal surface of the tongue. Subsequently the horizontal shelves never fuse. One may, therefore, suggest that Class differences exist among vertebrates with regard to the sequence of palatal development. Further investigation of difference in palatogenesis between various vertebrate - 81 -classes may prove useful in understanding normal and abnormal biology of the palate. There is a paucity of literature on the issue of why the mammalian palatal shelves first develop in a vertical direction when their eventual position is horizontal. It has been presumed that a large tongue occupies the oronasal cavity before the appearance of the palatal primordia. A lack of room over the tongue would consequently direct the palatal shelves to grow vertically along the sides of the tongue (Keibel and Mall, 1912; Fraser, 1947; Hayward and Avery, 1957; Patten, 1971; Tuchmann-Duplessis and Haegel, 1974; Dryden, 1977). The results of the present study, however, show that the palatal shelves appear prior to the development of the tongue. By the time a large tongue occupies the space in the oronasal cavity, the vertical palatal shelves are already well developed. One may, therefore, suggest that initial formation of palatal primordia is not related to the development of the tongue. Further credence to this suggestion comes from an analysis of human cleft palate cases where it was noted that in many instances of aglossia associated with cleft palate in embryos, the shelves were vertical (Shah, 1977b). Recently Flint (1980) observed the initiation of mouse palatal primordia from the roof of the oronasal cavity, but he did not indicate the status of tongue development at the time of palatal initiation. However, observations on embryonic palatal development in chick (Shah and Crawford, 1980; Koch and Smiley, 1981), alligator (Ferguson, 1981) and quail (Shah et al., 1984b) have shown that, despite the presence of a large tongue, the palatal shelves appear horizontal from the beginning. Thus these observations in lower - 82 -vertebrates would also argue against an active role of tongue in guiding the initial development of the palate in a vertical direction. Earlier Shah (1977b) and Ferguson (1981) had hypothesized that the initiation of mammalian embryonic palate in the vertical direction may be determined genetically, and is probably not dependant on the physical presence of surrounding structures such as the tongue. The observations of the present study, along with those made from lower vertebrates, lends support to this hypothesis. It seems that although the basic sequence of palatal development in mammals is similar, there are species differences in the pattern of palatal closure. For example, in mice (Walker and Fraser, 1956), rats (Zeiler et al., 1964; Coleman, 1965; Schupbach et al., 1983), gerbils (Holmstedt and Bagwell, 1977), ferrets (Gulamhusein and England, 1982), and humans (Fulton, 1957; Kitamura, 1966; Burdi and Faist, 1967; Waterman and Meller, 1974; Luke, 1976) the closure starts in the middle third of the palate and then simultaneously extends anteriorly and posteriorly, whereas in rabbits (Walker, 1971; Meller et al., 1980) and monkeys (Asling and van Wagenen, 1967; Steffek et al., 1968; Bollert and Hendrickx, 1971) the closure begins anteriorly and proceeds posteriorly. The present investigation showed that in hamsters closure starts in the middle third of the secondary palate, extends posteriorly, and finally anteriorly in that order. Thus it seems that even though the basic sequence of mammalian palatogenesis is similar, species differences in the patterns of palatal closure may be reflected during abnormal palatogenesis and result in the morphologically different types of palatal clefts seen in humans (Veau, 1931; Lynch et al., 1966; Fara, - 83 -1971; Smiley, 1972; Mitts et al., 1981), and in laboratory animals after experimental manipulation (Giroud and Martinet, 1956; Buresh and Urban, 1964; Walker, 1967; Dostal and Jelinek, 1971; Shah and Travill, 1976a; Shah and Kilistoff, 1976; Shah, 1977a, b; Shah and Mackay, 1978; Shah and Burdett, 1979; Shah et al., 1979; Kusanagi, 1983; Schupbach, 1983). Lysosomes were observed in the epithelial cells and met the morphologic features of secondary lysosomes and autophagic vacuoles or cytosegresomes (DeDuve, 1963; Erickson, 1969). The lysosomes were first seen in the medial edge epithelial cells of the horizontal palatal shelves. After the formation of the epithelial seam, the number and size of lysosomes had increased in the cells of the seam until they were lost. These observations are in accordance with those reported earlier by Chaudhry and Shah (1973, 1979) and Shah and Chaudhry (1974a, b). However, De Angel is and Nalbandian (1968) , Farbman (1968, 1969) and Brusati (1969) did not observe any lysosomes in the prefused epithelia. Mato et al. (1966, 1967a), Hayward (1969), Morgan (1969) and Smiley (1970) observed them in the basal cells prior to fusion but did not attach any significance to the timings of lysosomal appearance. In their studies Angelici and Pourtois (1968), Koziol and Steffek (1969), Vargas et al. (1972), Shah and Chaudhry (1974a), Lorente et al. (1974), Hoist and Mills (1975), and Im and Mulliken (1983) have noted that during and after the fusion of opposing epithel ia there is a significant increase in the acid hydrolytic activity which reaches its maximum when the epithelial seam starts to undergo fragmentation. As a corresponding increase in the number and size of lysosomes was observed during the present investigation, the observations give further credence to the proposition that the timely appearance and - 84 -subsequent increase in number of lysosomes play an important role in the autophagic degeneration of cells (Mato et al., 1966; Smiley, 1970; Chaudhry and Shah, 1973, 1979; Shah and Chaudhry, 1974a, b; Greene, 1983; Shah, 1984). With regard to the role of macrophages during palatal development, the observations in the present study are in harmony with those reported by Mato et al. (1967a), Chaudhry and Shah (1973, 1979) and Shah and Chaudhry (1974b). At no time was one able to observe the macrophage penetrating the basal lamina to destroy the epithelial cells, as has been previously suggested by Anderson and Matthiessen (1967). It would, therefore, appear that macrophages are mainly concerned with the removal of cellular debris, rather than the destruction of cells during palatal closure. 6-Mercaptopurine Induced Cleft Palate In 1979, Shah and Burdett reported that 6MP is a potent teratogen when injected into pregnant hamsters, and that it produces cleft palate in the fetuses. An extensive review of the literature indicates that this is the first comprehensive investigation which shows that a teratogen, 6MP, induces cleft palate by affecting vertical development of the palatal shelves. Flint (1980) observed that in the CBA/101 mutant mouse, cleft palate arises due to a failure of the primordia to grow from the beginning. Morphological observations of palatogenesis in mice and rats following teratogenic treatment have shown that cortisone, high doses of vitamin A, radiation, folic acid deficiency, 5-fluoro-2-desoxyuridine, and - 85 -diazo-oxo-norleucine delay reorientation of the palatal shelves from a vertical to horizontal plane (Walker and Fraser, 1957; Walker and Crain, 1960; Asling et al., 1960; Callas and Walker, 1963; Kochhar and Johnson, 1965; Coleman, 1965; Ross and Walker, 1967; Nanda, 1971; Dostal and Jelnick, 1972; Ferguson, 1977; Diewert, 1979; Diewert and Pratt, 1979). Similar observations in hamster have shown that triamcinolone and 5-fluorouracil treatment retard the palatal shelf reorientation (Shah, 1979e; Shah and Wong, 1980), whereas hydrocortisone treatment prevents the fusion between opposing horizontal shelves (Shah and Travill, 1976a, b). During the present investigation the timing of the initial appearance of palatal primordia in the treated fetuses resembled that of the controls. Thereafter, the vertical development of the palatal shelves was affected. The reorientation of the palatal shelves from vertical to horizontal, and their subsequent fusion never occured. One must be cautious in comparing the results reported in the present study with those noted earlier in the paragraph. The caution is in part due to the species differences involved, and partly due to the chemical natures of teratogens and different schedules of treatment used. For example, triamcinolone, 5-fluorouracil and hydrocortisone were administered intramuscularly on day 11:00 of gestation into the pregnant hamster (Shah and Travill, 1974; Shah, 1979e; Shah and Wong, 1980) as compared to intraperitoneal injection of 6MP given to the hamsters on day 9:00 of gestation in the present study. It was shown during the present study that both fetal weight and CRL provide reliable indicators for the stages of normal palatal development. Following 6MP treatment, however, only the first stage of palatal development - 86 -could be predicted on the basis of both fetal weight and CRL. The palatal closure was completed in normal fetuses weighing 551 mg and measuring 16 mm (Tables VI, IX), but in the treated ones with similar weight and CRL (Tables VII, X), the palatal shelves were vertical. At birth, however, the treated fetuses were significantly lighter than the controls. One may, therefore, suggest that following 6MP treatment (a) both fetal weight and CRL are poor indicators to determine the stages of palatal development, and (b) there is an association between the cleft palate and the reduction in fetal CRL, and in fetal weight. Similar observations were made earlier by Fraser and Fainstat (1951), Chaudhry et al. (1967), Shah and Travill (1974) and Shah and Wong (1980) following treatment of mice and hamsters with different teratogens. At cellular and subcellular levels, four main differences were observed during palatal development between 6MP treated fetuses and controls. These were (a) nuclear alterations, (b) premature appearance of lysosomes, initially in the mesenchymal cells and then in the epithelial cells, (c) subsequent changes in the epithelial cells, and (d) alterations in the basal lamina. Following 6MP treatment in the present study, the initial ultrastructural alteration observed was swelling of the perinuclear space in the mesenchymal cells of the developing palatal primordia. Similar alterations have been observed in the neural epithelium of developing rats and mice fetuses exposed to trypan blue (Peters et al., 1979), high doses of vitamin A (Morriss, 1973; Theodosis and Fraser, 1978), and fluoro-deoxyuridine (Langman and Cardell, 1978), human epithelial cells - 87 -maintained in vitro after cadmium exposure (Ree et al., 1982), rabbit erythrocyte (Glauert, 1963) and rat dermal fibroblast (Daniel et al., 1966) after retinol exposure, leukemic monocytes (Smetana and Hermansky, 1966), cat muscle fibres degenerating after long term immobilization (Cooper, 1972), and in porcine muscle affected with congenital myofibrillar hypoplasia (Zelena et al., 1978). The swelling of the perinuclear space is, however, not reported in other ultrastructural studies of cleft palate (Table II), or following 6MP induced limb malformations in rats (Merker et al., 1975). The precise nature of the defect of the nuclear envelope is not clear. Most investigators did not attach any significance to the observation. It has been suggested that the swollen nuclear envelope may reflect its altered permeability as a sequela of primary injury to the membrane system of the cell (Morriss, 1973), or as part of a sequela of cell death (Peters et al., 1979). Normally the perinuclear space contains several enzymes (Whaley et al., 1971) which regulate the interaction of chromosomes with the nuclear envelope (Franke et al., 1981), and subsequently allow nuclear-cytoplasmic interaction (Baud, 1963; Goldstein, 1974; Franke, 1974). It is plausible that following 6MP assault of the mesenchymal cells, the enzyme systems of the nuclear membrane may be affected, thereby altering its permeability (Morriss, 1973), and subsequently disrupting nuclear-cytoplasmic interaction and thus the cytoplasmic homeostasis. At no time in the present study was degeneration of mesenchymal cells observed to follow swelling of the perinuclear space, as was observed by Peters et al. (1979). In the treated embryos lysosomes were first recognized in the mesenchymal cells of the vertically developing palatal primordia at a very - 88 -early age (i.e., day 10:00 of gestation). The number of affected mesenchymal cells increased for approximately 12 hours, and then decreased. On day 10:12 of gestation, lysosomes also appeared in the superficial and basal epithelial cells of the vertically developing palatal shelves. Lysosomally affected cells in the mesenchyme, however, were always more than in the epithelium. On day 11:00 of gestation, and thereafter, lysosomes were absent in both the epithelial and mesenchymal cells of the cleft palatal shelves. The appearance of lysosomes in the epithelial cells of developing vertical palatal shelves is temporally abnormal, and thus not related to the physiological cell death observed during normal palatal development. On the other hand, during normal palatogenesis lysosomes do not appear in differentiating mesenchymal .cells, except in macrophages. The appearance of lysosomes in the mesenchymal cells following 6MP treatment is, therefore, abnormal. A similar abnormal lysosomal response in the mesenchymal cells following 6MP treatment has been observed in developing limb bud (Merker et al., 1975), and in cleft palate induced by other chemicals (Ferguson, 1978; Shah et al., 1984a). Several investigators have observed a transient appearance of lysosomes in a variety of embryonic tissues following treatment with numerous teratogens (Jurand, 1966, 1968; Crawford et al., 1972; Schweichel and Merker, 1973; Morriss, 1973; Merker et al., 1975; Sadler and Cordell, 1977; Kochhar et al., 1978; Herken et al., 1978; Langman and Cardell, 1978; Ferguson, 1978; Merchant-Larios and Coello, 1978; Peters et al., 1979; Abramovici et al., 1980; Desesso, 1981). They assumed that the appearance of lysosomes and their morphological variants was an expression of cell death. - 89 -It has been, however, repeatedly suggested that autophagocytosis will be followed by cell death, only if the processes of sequestration and subsequent digestion of the damaged portions of the cell cannot cope with the toxic stress (Swift and Hruban, 1964; Erickson, 1969; Kerr, 1971; Wyllie, 1981; Trump et al., 1983). Since, in the present study, the cytoplasmic features of the mesenchymal cells containing lysosomes appeared otherwise normal, one may suggest that such cells were able to overcome the effects of 6MP. A similar deduction was also reached by Theodosis and Fraser (1978) in their study on hypervitaminosis A induced limb defects in mice and by Shah et al. (1984a) in their study on 5-fluorouracil induced cleft palate development in hamster. It seems that the differentiating palatal mesenchymal cells are capable of either neutralizing and/or detoxifying the exogenous substrate, 6MP, or its metabolic product through the synthesis of lysosomal enzymes (Kerr, 1971; Arstila et al., 1974; Trump et al., 1983; Gedigk and Totovic, 1983). Thus the premature appearance of lysosomes in the present study may be interpreted as a sublethal, and protective response. The interpretation is further strengthened by observations that, unlike a dying cell where cytoplasmic organelles undergo profound changes (Morriss, 1973; Peters et al., 1979; Trump et al., 1983; Constantinidis, 1984), the general appearance of cytoplasmic organelles in the mesenchymal cells remained healthy. The sublethal injury to the mesenchymal cells, however, seems to have irreversibly altered their path of differentiation. Data on the rate of mesenchymal cell proliferation, and the synthesis of extracellular matrix molecules by mesenchymal cells at the time of development of palatal - 90 -primordia is lacking. These issues are, however, considered to be important during subsequent palatal development (Table II). It is not clear whether the stunted vertical palatal shelves seen at term reflect a reduction in the mesenchymal cell population, or synthesis of extracellular matrix, or both. The premature appearance of lysosomes in the palatal epithelial cells may be the result of either a direct sublethal response to 6MP treatment or its metabolic product, or an indirect response to the preceding mesenchymal cellular injury. There is no direct evidence in the present study to support either view. As discussed for the mesenchymal cells in the earlier paragraphs, premature lysosomal formation in the epithelial cells may also indicate a sublethal response to neutralize and/or detoxify the harmful cytotoxic effects of 6MP or its metabolic product. The plausibility of this explanation is further enhanced by the fact that the morphology of cytoplasmic organelles of the epithelial cells appeared healthy. On the other hand, although an epithelial-mesenchymal interaction is considered to be important for terminal differentiation of the palatal epithelial cells (Pourtois, 1972; Tyler and Koch, 1975, 1977a, b; Shah et al., 1983; Shah, 1984), there is no evidence in the present study or in the literature, that the mesenchyme may affect differentiation of epithelial cells at the time of palatal primordial development. Furthermore, the basal lamina separating the injured epithelial and mesenchymal cells was intact (Fig. 42). An exchange of information, if any, between them may still occur at the molecular level through the basal lamina. Under such circumstances the appearance of lysosomes in the epithelial cell may reflect a response to injury in the mesenchymal cell. Thus this possibility cannot be ruled out. - 91 -The injury to epithelial cells, like the mesenchymal cells, also appears to have irreversibly altered their path of differentiation. Consequently between days 12:00 and 13:00 of gestation the 6MP treated palatal epithelia do not exhibit any signs of lysosomally mediated programmed cell death observed during normal development (Mato et al., 1966; Smiley, 1970; Chaudhry and Shah, 1973; Shah and Chaudhry, 1974a, b; Greene, 1983; Shah, 1984). Thereafter, however, the basal epithelial cells show transient cytoplasmic alterations on day 13:18 of gestation (Fig. 47). The alterations seem to be reversible because subsequently on day 14:00 of gestation the epithelial cells appear healthy (Fig. 48). A similar latent response of the epithelial cells has not been noted in other ultrastructural studies on chemically induced cleft palate (Table II). One may perhaps suggest that the epithelial cells, having changed their path of differentiation, were discarding organelles which are normally involved in programmed elimination. During the present study no loss of epithelial cells was evident at any time. The lysosomally-mediated sublethal response in 6MP-induced cleft palate is different from the effect of other teratogens (Table II). The autolytic degeneration in the palatal epithelium of mice and rats was either reduced or prevented following treatment with B-aminoproprionitrile (Mato et al., 1975b), meclozine (Morgan, 1976), epidermal growth factor (Hassell, 1975; Hassell and Pratt, 1977), diazo-oxo-norleucine (Pratt and Greene, 1976; Morgan and Pratt, 1977), triamcinolone (Kurisu et al., 1981 phenylbutazone (Montenegro and Paz de la Vega, 1982), and Cortisol (Goldman et al., 1983). The response of the mesenchyme was not reported in these studies. In - 92 -hamster, following hydrocortisone and triamcinolone treatment (Shah and Travill, 1976b; Shah, 1980) a premature nonlysosomal necrosis of the epithelium was observed but the mesenchymal cells appeared unaffected. Shah et al. (1984a) observed a lysosomally-mediated sublethal response first in the epithelial, and then in the mesenchymal cells of developing hamster palate following 5-fluorouracil treatment. Ferguson (1978) reported cell death in the developing rat palatal mesenchyme following 5-fluoro-2-desoxyuridine treatment. It seems that the chemical nature of the drug, its species specific effect, and the metabolic state of the developing tissues at the time of treatment, among other factors, may determine the nature of the tissue response. In the present study, epithelial and mesenchymal cytoplasmic extensions into each others territory, and their contacts with one another were observed following sublethal injury. Cytoplasmic extensions of the epithelial cells and epithelial-mesenchymal contacts have also been observed in other studies on teratogen induced cleft palate (Shah, 1980; Shah et al., 1984a). It was suggested that the extension of epithelial cell processes into the mesenchyme increased the surface area of the cell to receive nutrients following injury (Shah, 1980). In the present study the cellular projections and contacts were seen late during cleft palate development, i.e., on day 12:18 of gestation. After day 11:00 of gestation the injured epithelial and mesenchymal cells appeared to have been repaired, and structurally resembled their normal counterparts. Furthermore, mesenchymal cytoplasmic extensions also extended into the epithelium (Fig. 47). Thus it is unlikely that the epithelial and mesenchymal cytoplasmic projections and contact between them, - 93 -as observed in the present study, develop for nutritional purposes. Epithelial-mesenchymal communications have been considered to play an important role during normal development in determining the differentiation and fate of the palatal epithelium (Pourtois, 1972; Tyler and Koch, 1977b; Tyler and Pratt, 1980; Shah et al., 1983; Shah, 1984). It is possible that, following 6MP treatment, since the injured epithelial and mesenchymal cells may have deviated from the normal path of differentiation, they prepared themselves for • a new role in the cleft palate by communicating with one another. The observations of light cells on day 13:18 of gestation (Fig. 47) may indeed be the response to an epithelial-mesenchymal interaction. Alterations in the configuration of the basal lamina have been observed in many pathological and experimental situations (Pearson and Spargo, 1961; Caulfield and Wilgram, 1962; Johnson and Fry, 1967; Frithiof, 1969; Woods and Smith, 1969, 1970; Vracko, 1972, 1979; Tarin, 1972; Yamanishi et al., 1972; Sugimoto et al., 1973; Takarada et al., 1974; Martinez-Hernandez et al., 1976; Jao et al., 1978; Frei, 1978; Ingber et al., 1981; White and Gohari, 1981; Otsubo and Kameyama, 1982; Yasuzumi et al., 1983). The alterations include irregularities in thickness, reduplication, detachment from the basal cells, fragmentation and complete disappearance. In the present study, the alterations in the basal lamina were seen long after injury to the epithelial and mesenchymal cells. The changes were characterized initially by breaks in the continuity of the basal lamina followed by its total loss. Similar observations on the basal lamina configuration have been made following treatment with several teratogens (Mato et al., 1975a, b; Shah and Travill, 1976b; Morgan, 1976; Ferguson, 1978; Shah, 1980, 1984; Shah et al., 1984a). - 94 -In pathological circumstances, the fragmentation and loss of basal lamina has been related either to its destruction by enzymes, or to a failure of epithelial cells to form it (Woods and Smith, 1969, 1970; Tarin, 1972; Yamanishi et al., 1972; Takarada et al., 1974; Martinez-Hernandez et al., 1976; Frei, 1978; White and Gohari, 1981). Although the present study lacks evidence to support either view, the latter seems more plausible. At the ultrastructural level there was no evidence that lysosomal enzymes escaped the epithelial and/or mesenchymal cells to affect the basal lamina. Fragments of cellular material, which appeared to be expelled from the mesenchymal cells on day 10:06 of gestation (Fig. 41) were membrane bound, and it is doubtful that enzymes would escape during their phagocytosis by macrophages. Furthermore, from a chronological viewpoint, the lysosome-mediated cellular injury subsided by day 11:00 of gestation, while early basal lamina changes were visible first on day 12:18 of gestation, i.e., after 42 hours. It is highly unlikely that enzymes would persist extracellularly over such a long duration. On the other hand, it is generally agreed that the basal lamina is an epithelial product (Kurtz and Feldman, 1962; Pearce et al., 1963; Briggman et al., 1971; Timpl et al., 1979; Stanley et al., 1982). Altered differentiation of the epithelial cells due to sublethal injury, as implicated in the earlier paragraphs, could affect the synthesis of molecules required for basal lamina formation and maintenance, thus causing the defect. Merker et al. (1975) indicated that, following 6MP treatment, the mesenchymal cell damage in the developing rat embryonic limb bud occurred after 5 hours. They further indicated that the epithelial cells were not - 95 -affected. Scott et al. (1980), on the other hand, noted that the effect of 6MP treatment on the rat embryo limb bud was observed in the mesenchymal cells, and then in the epithelial cells, after 24 hours. Adhami and Noack (1975), and Adhami (1979) also noted a duration of 24 hours for 6MP to affect the rat embryonic brain tissues. Observations of the present study in hamster show that the initial adverse effect of 6MP is seen after 24 hours in the mesenchymal cells, and later on in the epithelial cells. The difference in the interval for cellular response observed by Merker et al. (1975) and others may possibly be attributed to the vehicle used to inject the drug. Merker et al. (1975) used 0.9% NaCl and 2.5% propylene glycol to administer 6MP, whereas Adhami and Noack (1975), Adhami (1979), Scott et al. (1980), and in the present study water was used as a vehicle for 6MP injection. Furthermore Scott et al. (1980) injected 6MP on day 11:00 whereas Merker et al. (1975) injected on day 12:00 of gestation to induce limb malformations. It is possible that these differences in vehicle may affect the duration of placental transfer, and that of metabolism of 6MP in the maternal, placental and fetal tissues, and hence in the eventual timings of the tissue response. It is also possible that cells at the initial stages of differentiation are highly susceptible to 6MP induced injury (Merker et al. 1975). The proposition is further strengthened by the observations that 6MP affected the primordial stage of the development of limb (Merker et al., 1975; Scott et al., 1980), and palate (Present study) to induce the malformations. As indicated in the INTRODUCTION, several biochemical pathways have been suggested by which 6MP may exert its action. The mechanism, however, by which 6MP affects differentiating cells and tissues in a developing embryo is - 96 -not known. It is suggested that 6MP goes through the placenta and is anabolized by the fetus into active antimetabolite (Neubert et al., 1977, 1980). The activated antimetabolite then may affect the cellular functions by one of the following mechanisms: (1) directly affecting RNA synthesis, especially that of mRNA and consequently affecting protein synthesis (Roy-Burman, 1970; Neubert et al., 1970; Mewes et al., 1971; Kawahata et al., 1980), or (2) interference with DNA synthesis either via directly inhibiting enzymes such as adenylosuccinate synthetase, or feedback inhibition of aminotransferase (Neubert et al., 1977), thus affecting purine metabolism (Fig. 2) (El ion and Hitchings, 1965; El ion, 1967; Roy-Burman, 1970; Malamud et al., 1972; Paterson and Tidd, 1975; or (3) by direct incorporation into DNA (Martin et al., 1972; Tidd and Paterson, 1974; Zimmerman et al., 1974; Breter and Zahn, 1979; Ding and Benet, 1979; Lennard and Maddocks, 1983). Merker et al. (1975) proposed that the rapid necrosis, which occurs in the limb mesenchyme within 5 hours after 6MP treatment cannot be solely due to reduced nucleic acid synthesis, because other rapidly proliferating tissues such as the epithelium and endothelium were not affected. They suggested that the rapid effect of 6MP may be due to inhibition of a short lived substance such as mRNA or cAMP which play a role in carrying information for cytodifferentiation. A lack of availability of such a substance, Merker et al. (1975) indicated, would not produce information necessary for differentiation and lead to reduced protein synthesis and eventual necrosis. It is unlikely, therefore, that the initial cellular lesions observed in the present study, and those observed earlier by Adhami and Noack (1975), Adhami (1979) and Scott et al. (1980) could be due to reduced protein synthesis, - 97 -because the duration between drug administration and theinitial cellular effect was long (24 hours). Furthermore, there was no electron microscopic evidence of alterations in the protein synthetic machinery or in the polyribosomal content of the epithelial and mesenchymal cells of developing palatal primordia. Hence one may deduce that 1n the present study perhaps 6MP did not directly affect mRNA and protein synthesis in the palatal cells, as implicated by Merker et al. (1975) for differentiating limb mesenchymal cells. Taylor et al. (1962), Webster et al. (1973), Scott (1977) and Adhami (1979) suggested that if 6MP or its metabolites rapidly inhibited DNA synthesis the first morphologically demonstrable lesion could be expected within a few hours after drug administration. In the delayed cytotoxic response following 6MP administration, however, Tidd et al. (1972) and Horakova et al. (1974) suggested that the cells must go through the DNA synthesis-phase at least once. Also in their study on the effect of 6MP on HeLa cells vn vitro Horakova et al. (1974) observed that following DNA synthesis and mitosis, HeLa cells showed reduced DNA and RNA content per cell eventually leading to cell damage or necrosis. In the present study, the observation that after 6MP treatment the palatal primordia indeed developed, suggests that perhaps at least some of its mesenchymal and epithelial cells may have had the opportunity to undergo DNA synthesis and mitosis before manifesting injury caused by the drug or its metabolite. The delayed cytotoxicity thus may reflect incorporation of 6MP into DNA as proposed by Tidd et al. (1972) and Tidd and Paterson (1974). The mechanism by which lysosomes develop in the mesenchymal and epithelial cells following 6MP administration, and how it affects - 98 -cytodifferentiation is not clear. Menkes et al. (1970), Scott (1977), Scott et al. (1980) and others have suggested that teratogen-induced lysosomal alterations may alter some pathways involved in cellular differentiation, which consequently can delete or injure one or more cellular functions essential for histo- and morphodifferentiation. Information on metabolic pathways involved in the early differentiation of palatal tissue is, however, virtually non-existent. It is perhaps reasonable to suggest that 6MP induced lysosome-mediated sublethal injury could perturb the intracellular homeostasis, or vice versa, and subsequently alter the programmed differentiation of palatal mesenchyme and epithelium by affecting DNA synthesis, either by affecting purine metabolism, or by direct incorporation into DNA (Fig. 2) (El ion and Hitchings, 1965; El ion 1967; Tidd and Paterson, 1974). Eventually the assaulted cells of the palatal primordia, following their repair, may adapt themselves for a different role in the cleft palate. Foregoing RESULTS and DISCUSSION indicate that normal palatal development in hamster consists of an orderly sequence of events involving growth, differentiation, and death at both cellular and tissue levels. 6MP treatment injured first the mesenchymal and then the epithelial cells of the developing palatal primordia, which in turn appear to irreversibly alter the differentiation of cells and tissues to cause the cleft palate. The regulatory mechanisms of cell and tissue differentiation during normal palatal development, however, are only partially understood. Also, the biochemical mechanism(s) of the action of 6MP on developing palatal tissues and cells are still unclear. Future investigations may be directed toward understanding these mechanisms before the pathogenesis of cleft palate following 6MP treatment can be completely defined. - 99 -SUMMARY AND CONCLUSIONS 1. Formation of the secondary palate in hamster occurs in distinct but sequential stages. 2. The age, weight, and crown-rump length of the fetus are reliable indicators of stages of normal palatal development in hamster. 3. The initial formation of the palatal shelves is independent of tongue development. 4. The timely appearance of lysosomes play a crucial role in the autolytic elimination of medial edge epithelia during palatal closure. 5. Macrophages are involved in removal of epithelial cellular debris. 6. A single intraperitoneal injection of 6-mercaptopurine on day 9:00 of gestation affects the vertical development of palatal shelves, and thus produces cleft palate. 7. An association exists between the reduction in weight and crown-rump length of the fetus and the cleft palate. 8. At cellular and subcellular levels, four main differences were observed which distinguished palatal development between the 6-mercaptopurine treated and control fetuses. In order of appearance in the treated palates, these were (a) alterations in the nuclear membrane, (b) premature appearance of lysosomes first in the mesenchymal, and then in the epithelial cells, (c) subsequent changes in the epithelial cells, and (d) alterations in the basal lamina. - 100 -9. administration of 6-mercaptopurine induces sublethal injury in the mesenchymal and epithelial cells during vertical development of the palatal shelves. The injury altered the path of cytodifferentiation. 10. The alterations in the basal lamina were secondary in response to the epithelial and mesenchymal injury following 6-mercaptopurine treatment. 11. Inhibition of DNA synthesis following 6-mercaptopurine administration may have disrupted the process of differentiation in both the epithelial and mesenchymal cells and thus caused, cleft palate. - 101 -REFERENCES Abramovici, A., Rachmuth-Forschmidt, P., Liban, E. and Sandbank, U. 1980. Experimental limb dysmorphogenesis as a model of chemical injury response in undifferentiated embryonic tissues: A light and electron microscopical study. J. Path. 131:289-308. Adams, C.E., Hay, M.F. and Lutwak-Mann, C. 1961. The action of various agents upon the rabbit embryo. J. Embryol. exp. Morphol. 9:468-491. Adhami, H. 1979. Influence of 6-mercaptopurine on the prenatal development of the rat cortex. Z. mikrosk.anat. Forsch., Leipzig 93:21-32. Adhami, H. and Noack, W. 1975. Histological effects of 6-mercaptopurine on the fetal rat central nervous system: A light microscopic study. Teratology 11:297-312. Anderson, H and Matthiessen, M. 1967. Histochemistry of the early development of the human face and nasal cavity with special reference to the movement and fusion of the palatine processes. Acta. Anat. 68:473-508. Andrew, F.D. and Zimmerman, E.F. 1971. Glucocorticoid induction of cleft palate in mice; no correlation with inhibition of mucopolysaccharide synthesis. Teratology 4:31-38. Angelici, D.R. 1968. Reopening of fused palatal shelves. Cleft Palate J. 5:205-210. Angelici, D. and Pourtois, M. 1968. The role of acid phosphatase in the fusion of the secondary palate. J. Embryol. exp. Morphol. 20:15-23. Arstila, A.U., Hirsimaki, P. and Trump, B.F. 1974. Studies on the subcellular pathophysiology of sublethal chronic cell injury. Beitr. Pathol. 152:211-242. Asisi, A. and Merker, H.J. 1975. A specific malformation of the extremities in rats following administration of 6-mercaptopurine on day 12 of gestation (Ger.). Arzneimittel-Forsch./Drug Res. 25:1924-1926. Asling, C.W. and Von Wagenen, G. 1967. A note on development of the secondary palate in rhesus monkey (Macaca mulatta). Arch, oral Biol. 12:909-910. Asling, C.W., Nelson, M.M., Dougherty, H.D., Wright, H.V., Evans, H.M. 1960. The development of cleft palate resulting from maternal pteroylglutamic (folic) acid deficiency during the latter half of gestation in rats. Surg. Gynec. Obst. 111:19-28. Babiarz, B.S., Allenspach, A.L. and Zimmerman, E.F. 1975. Ultrastructural evidence of contractile systems in mouse palates prior to rotation. Dev. Biol. 47:32-44. - 102 -Baeckeland, E, Heinen, E. and Renard, A.M. 1982. Mobility of Con A receptors at the surface of palatal shelves before closure of the secondary palate. In: lectins-Biology, Biochemistry, Clincial Biochemistry, Vol. II, Ed. T.C. Bog-Hansen, Walter de Gruyter and Co., Berlin, New York, pp. 295-304 Barka, T. and Anderson, P.J. 1962. Histochemical methods for acid phosphatase using hexazonium pararosanilin as a coupler. J. Histoe hem. Cyto. Chem. 10:741-753. Barry, A. 1961. Development of the branchial region of human embryos with special reference to the fate of epithelia. In: Congenital Anomalies of the Face and Associated Structures. Ed. S. Pruzansky, CC. Thomas, Springfield, pp. 46-62. Baud, C.A. 1963. Nuclear membrane and permeability. In: Intracellular Membrane Structure. Ed. S. Seno and E.V. Cowdry. Okayama Press, Okayama. Baxter, H. and Fraser, F.C. 1950. The production of congenital defects in the offspring of female mice treated with cortisone. McGill Med. J. 19:245-249. Bekhor, I., Mirell, C. and Anne, L. 1978. Induction of cleft palate by triamcinolone acetonide: Re-examination of the problem. Cleft Palate J. 15:220-232. Berkson, J. 1957. Tables for use in estimating the normal distribution function by normit analysis. Biometrika 44:411-435. Bieber, S., Nigrelli, R.F. and Hitchings, G.H. 1952. Effects of purine and pyrimidine analogues on development of Rana pipiens. Proc. Soc. Exp. Biol. Med. 79:430-432. Bilski-Pasquier, G., Charon, P. and Bousser, J. 1962. Leucose et grossesse. Nouv. Revue fr. Hemat. 2:289-311. Blattner, R.J., Williamson, A.P., Simonsen, L. and Robertson, G.G. 1960. Teratogenesis with cancer chemotherapeutic agents. Pediatrics 56:285-293. Boggs, D.R., Wintrobe, M.M. and Cartwright, G.E. 1962. The acute leukemias. Medicine (Baltimore) 41:163-225. Bollert, J.A. and Hendrickx, A.G. 1971. Morphogenesis of the palate in the baboon (Papio cynocephalus). Teratology 4:343-354. Bonaiti, C., Briard, M.L., Feingold, J., Pavy, B., Psaumes, J., Migne-Tufferaud, G. and Kaplan, J. 1982. An epidemiological and genetic study of facial clefting in France. I. Epidemiology and frequency in relatives. J. Med. Genetics 19:8-15. - 103 -Bower, C. and Stanley, F.J. 1983. Western Australian . congenital malformations register. Med. J. Aust. 2:189-191. Bragonier, J.R. and Carver, M.J. 1968. The influence of 6-mercaptopurine on rat placenta and fetus. Biochem. Pharmocol. 17:1689-1697. Breter, H.J. and Zahn, R.K. 1979. Quantitation of intracellular metabolites of [35S]-6-mercaptopurine in L51784 cells grown in time-course incubates. Cancer Res. 39:3744-3748. Briggman, R., Doldorf, F.G. and Wheeler, CF. 1971. Formation and origin of basal lamina and anchoring fibrils in adult human skin. J. Cell. Biol. 51:384-395. Brinkley, L. 1980. In vitro studies on palatal elevation. In: Current Research Trends in Prenatal Craniofacial Development. Ed. R.M. Pratt and R.L. Christiansen. Elsevier-North Holland Press, New York, pp. 201-220. Brinkley, L. 1984. Role of extracellular matrix in palatal development. Curr. Topics Dev. Biol. 19:17-36. Brinkley, L.L. and Vickerman, M.M. 1982. The effects of chlorcyclizine-induced alterations of glycosaminoglycans on mouse palatal shelf elevation in vivo and,in vitro. J. Embryol. Exp. Morphol. 69:193-213. Brusati, R. 1969. Ultrastrucutral study of the processes of formation and involution of the epithelial sheet of the secondary palate in the rat. J. Submicr. Cytol. 1:215-234. Burdi, A.R. and Faist, K. 1967. Morphogenesis of the palate in normal human embryos with special emphasis on the mechanisms involved. Am. J. Anat. 120:149-160. Burdi, A.R. and Silvey, R.G. 1969. Sexual differences in closure of the human palatal shelves. Cleft palate J. 6:1-7. Buresh, J.J. and Urban, T.J. 1964. The teratogenic effect of the steroid nucleus in the rat. J. Dent. Res. 43:548-554. Calabresi, P. and Parks, R.E. Jr. 1975. Chemotherapy of Neoplastic Diseases. In: The Pharmacological Basis of Therapeutics. Ed. L.S. Goodman, and A. Gilman, 5th edition, MacMillan, New York, pp. 1254-1307. Callas, G. and Walker, B.E. 1963. Palate morphogenesis.in mouse embryos after x-irradiation. Anat. Rec. 145:61-71. Caul field, J.B. and Wilgram, G.F. 1962. An electron microscopic study of blister formation in erythema multiforme. J. Invest. Dermatol. 39:307-316. Chapman, C.J. 1983. Ethnic differences in the incidence of cleft lip and/or cleft palate in Auckland. 1960-1976. New Zealand Med. J. 96:327-329. - 104 -Chaudhry, A.P. and Shah, R.M. 1973. Pathogenesis in hamster II. Ultrastructural observations on the closure of palate. J. Morph. 139:329-350. ' Chaudhry, A.P. and Shah, R.M. 1979. Light and electron microscopic observations on the closure of the secondary palate with the primary palate and the nasal septum. Acta Anat. 103:384-394. Chaudhry, A.P., Schwartz, D., Schwartz, S. and Schmutz Jr., J.A. 1967. Some aspects of experimental induction of cleft palates. J. Oral Ther. 4:98-103. Chung, C.S., Rao, D.C. and Ching, G.H.S. 1980. Population and family studies of cleft lip and palate. Prog. Clin. Biol. Res. 46:325-352. Cleaton-Jones, P. 1976. A scanning electron microscope study of the developing rat secondary palate. South African J. Med. Sci. 41:3-9. Coleman, R.D. 1965. Development of the rat palate. Anat. Rec. 151:107-117. Constantinides, P. 1984. Ultrastructural pathobiology. Elsevier, Amsterdam. Cooper, R. 1972. Alterations during immobilization and regeneration of skeletal muscle in cats. J., Bone Joint Surg. 54:919-953. Crawford, A.M., Kerr, J.F.R. and Currie, A.R. 1972. The relationship of acute mesodermal cell death to the teratogenic effects of 7-ohm-12-MBA in the foetal rat. Br. J. Cancer 26:498-503. Czeizel, A. 1980. Studies of cleft lip and cleft palate in East European populations. Prog. Clin. Biol. Res. 46:249-296. Dagg, CP. 1966. Teratogenesis. In: Biology of the Laboratory Mouse. Ed. E.L. Green. McGraw-Hill, New York, pp. 309-328. Daniel, M.R., Dingle, J.T., -Glauert, A.M. and Lucy, J.A. 1966. The action of excess vitamin A alcohol on the fine structure of rat dermal fibroblasts. J. Cell Biol. 30:465-475. De Angelis, V. and Nalbandian, J. 1968. Ultrastructure of mouse and rat palatal processes prior to and during secondary palate formation. Arch, oral Biol. 13:601-608. DeDuve, C. 1963. The lysosome. Sci. Am. 208:64-72. Del Balso, A.M. and Kauffman, F.C. 1975. The effect of e-aminopropionitrile on acid hydrolases and selected metabolites in oro-facial structure of foetal rats. Archs. oral Biol. 20:251-255. - 105 -De Paola, D., Drummond, J., Lorente, C, Zarbo, R. and Miller, S.A. 1975. Glycoprotein biosyntehsis at the time of palate fusion by rabbit palate and maxilla cultured in vitro. J. Dent. Res. 54:1049-1055. De Sesso, J.M. 1981. Comparative ultrastructural alterations in rabbit limb-buds after a teratogenic dose of either hydroxyurea or methotrexate. Teratology 23: 197-215. Diamond, I., Anderson, M.M. and McCreadie, S. 1960. Transplacental transmission of busulfan (Myleran) in a mother with leukemia. Pediatrics 25:85-90. Didcock, K., Jackson, D and Robson, J.M. 1956. The- action of some nucleotoxic substances on pregnancy. Brit. J. Pharmacol. 11:437-441. Diewert, V.M. 1974. A cephalometric study of orofacial structures during secondary palate closure in the rat. Arch. Oral Biol. 19:303-315. Diewert, V.M. 1979. Correlation between mandibular retrognathia and induction of cleft palate with 6-aminonicotinamide in the rat. Teratology, 19:213-228. Diewert, V.M. and Pratt, R.M. 1979. Selective inhibition of mandibular growth and induction of cleft palate by diazo-oxo-norleucine (DON) in the rat. Teratology 20:37-52. Ding, T.L. and Benet, L.Z. 1979. Determination of 6-mercaptopurine and azathioprine in plasma by high performance liquid chromatography. J. chromatogr. 163:281-288. Donelli, M.G., Colombo, T., Forgione, A. and Garattini, S. 1972. Distribution of 6-mercaptopurine in tumour-bearing animals. Pharmacology 8:311-320. Dostal, M. and Jelinek, R. 1970. The morphogenesis of cleft palate induced by exogenous factors. II. Induction of cleft by cortisone in random bred mice. Acta Chirurgiae Plasticae 12:206-208. Dostal, M. and Jelinek, R. 1971. Induction of cleft palate in rats with intra-amniotic corticoids. Nature 230:464. Dostal, M. and Jelinek, R. 1972. Morphogenesis of cleft palate induced by exogenous factors V. Quantitative study of the process of palatal closure of different strains of mice. Folia Morph. 20:362-374. Dryden, R. 1977. Before Birth. Heinemann Educational Books, London, pp. 53-54. Dursy, E. 1869. Zur entwicklungsgeschichte des kopfes des menshen und der hoheren wirbeltiere. Verlag der H. Lauppschen Buchhandlung, Tubigen. - 106 -Elion, G.B. 1967. Biochemistry and pharmacology of purine analogues. Fed. Proc. Fed. Am. Soc. Exp. Biol. 26:898-903. Elion, G.B. and Hitchings, G.H. 1965. Metabolic basis for the action of analogs of purines and pyrimidines. Adv. chemotherap. 2:91-177. Elion, G.B., Burgi, E. and Hitchings, G.H. 1952. Studies on condensed pyrimidine systems. IX. The synthesis of some 6-substituted purines. J. Am. Chem. Soc. 74:411-414. Elion, G.B., Callahan, S., Rundles, R.W. and Hitchings, G.H. 1963. Relationship between metabolic rates and antitumor activities of thiopurines. Cancer Res. 23:1207-1217. Elizorava, N.A. and Stupnitskaya, V.M. 1962. Leukemia in Pregnancy (Russ.) Klin. Med. (Mosk.) 40:104-106. Elwood, J.M. and Rogers, J.R. 1975. The incidence of congenital abnormalities in British Columbia, Alberta, Manitoba and New Brunswick, 1966-1969. Can. J. Public Health 66:471-476. Erickson, J.L.E. 1969. Mechanism of cellular autophagy. In: Lysosomes in Biology and Pathology. Ed. J.T. Dingle and H.B. Fell. North-Holland Publishing Co., Amsterdam pp. 345-394. Fabro, S. (1983) Reproductive toxicology: State of the art, 1982. Prog. Clin. Biol. Res. 117:391-393. Fairweather, D.V.I. 1982. Screening in pregnancy for congenital abnormality. Brit. J. Hosp. Med. 27:601-607. Fara, M. 1971. Congenital defects in the hard palate. Plast. Reconstr. Surg. 48:44-47. Farbman, A.I. 1968. Electron microscope study of palatal fusion in mouse embryos. Dev. Biol. 18:93-116. Farbman, A.I. 1969. The epithelium-connective tissue interface during closure of the secondary palate in rodent embryos. J. Dent. Res. 48:617-624. Ferguson, M.W.J. 1977. The mechanism of palatal shelf elevation and the pathogenesis of cleft palate. Virchows Arch. A. path. Anat. Histol. 375:97-113. Ferguson, M.W.J. 1978. Palatal shelf elevation in the Wistar rat fetus. J. Anat. 125:555-577. Ferguson, M.W.J. 1981. The structure and development of the palate in Alligator mississippiensis. Archs. oral Biol. 26:427-443. - 107 -Flint, O.P. 1980. Cell behaviour and cleft palate in the mutant mouse, amputated. J. Embryol. exp. Morph. 58:131-142. Franke, W.W. 1974. Structure, biochemistry and functions of the nuclear envelope. Int. Rev. Cytol. Suppl. 4:72-236. Franke, W. 1974. Nuclear envelopes: Structure and biochemistry of the nuclear envelope. Phil. Trans. Roy. Soc. (London) Ser B 268:67-93. Franke, W.W. and Scheer, U. 1974. The Cell Nucleus. In: Structures and functions of the nuclear envelope. Ed. H. Busch, Academic Press, New York and London, pp. 220-328. Franke, W.W., Scheer, U., Krohne, G. and Jarasch, E.D. 1981. The nuclear envelope and the architecture of the nuclear periphery. J. Cell Biol. 91:393-503. Fraser, F.C. and Fainstat, T.D. 1951. Production of congenital defects in the offspring of pregnant mice treated with cortisone. Pediatrics 8:527-533. Fraser, J.E.S. 1947. A manual of embryology: The development of human body. Williams and Wilkins, Baltimore, pp. 283-285. Frei, J.V. 1978. Objective measurement of basement membrane abnormalities in human neoplasm of colorectum and of breast. Histopathology 2:107-115. Frenkel, E.P. and Meyers, M.C. 1960. Acute leukaemia and pregnancy. Ann. Int. Med. 53:656-671. Frithiof, L. 1969. Ultrastructure of the basement membrane in normal and hyperplastic human oral epithelium compared with that in preinvasive and invasive carcinoma. Acta Path. Microbiol. Scand. (Suppl.) 200:3-63. Fulton, J.T. 1957. Closure of the human palate in embryo. Am. J. obst. Gynec. 74:179-182. Gedigk, P. and Totovic, V. 1983. Lysosomes and Lipopigments. In: Cellular Pathobiology of Human Disease. Ed. B.F. Trump, A. Laufer, and R.T. Jones, Gustav Fischer, New York. Giroud, A. and Martinet, M. 1956. Malformations de la face et hypervitaminose A. Rev. Stomatol. 57:454-463. Glauert, A.M., Daniel, M.R., Lucy, J.A., Dingle, J.T. 1963. Studies on the mode of action of excess vitamin A. VII. Changes in the fine structure of erythrocytes during haemolysis. J. Cell Biol. 17:111-121. Goldman, A., Shapiro, B. and Katsumata, M. 1978. Human fetal palatal corticoid receptors and teratogens for cleft palate. Nature (London) 272:464-466. - 108 -Goldman, A.S., Baker, M.A., Piddington, R. and Herold, R. 1983. Inhibition of programmed cell death in mouse embryonic palate Jjn vitro by Cortisol and phenytoin: Receptor involvement and requirement of protein synthesis. Proc. Soc. Exp. Biol. Med. 174:239-243. Goldman, A., Katsumata, M., Yaffe, S. and Gasser, D. 1977. Palatal cytosol cortlsol-binding protein associated with cleft palate susceptibility and H-2 genotype. Nature (London), 265:643-645. Goldstein, L. 1974. Movement of molecules between nucleus and cytoplasm. In: The cell nucleus Vol. 1. Ed. H. Busch, Academic Press, New York pp. 388-440. Goss, A.N. 1975. Human palatal development in vitro. Cleft Palate J. 12:210-221. Greene, R.M. 1983. Hormonal involvement in palatal differentiation. In: Handbook of Experimental Pharmacology Vol. 65. Teratogenesis and Reproductive Toxicology. Ed. E.M. Johnson and D.M. Kochhar, Springer-Verlag, Berlin, pp. 75-92. Greene, R.M. and Kochhar, D.M. 1973. Palatal closure in the mouse as demonstrated in frozen sections. Am. J. Anat. 137:477-482. Greene, R.M. and Kochhar, "D.M. 1974. Surface coat on the epithelium of developing palatine shelves in the mouse as revealed by electron microscopy. J. Embryol. Exp. Morph. 31:683-692. Greene, R.M. and Pratt, R.M. 1976. Developmental aspects of secondary palate formation. J. Embryol. Exp. Morph. 36:225-245. Greene, R.M. and Pratt, R.M. 1977. Inhibition by diazo-oxo-norleucine (DON) of rat palatal glycoprotein synthesis and epithelial cell adhesion in vitro. Exp. Cell Res. 105:27-37. Greene, R.M. and Pratt, R.M. 1978. Inhibition of epithelial cell death in the secondary palate in vi tro by alteration of lysosomal function. J. Histochem. Cytochem. 26TT109-1114. Greene, R.M. and Pratt, R.M. 1979. Correlation between cyclic AMP levels and cytochemical localization of adenylate cyclase during development of the secondary palate. J. Histochem. Cytochem. 27:924-931. Greene, R.M., Lloyd, M.R. and Nicolaou, K.C. 1981. Agonist-specific desensitization of prostaglandin stimulated cyclic AMP accumulation in palatal mesenchymal cells. J. Craniofacial Genet. Dev. Biol. 1:261-272. Greene, R.M., MacAndrew, V.I and Lloyd, M.R. 1982. Stimulation of palatal glycosaminoglycan synthesis by cyclic AMP. Bioc. Bioph. Res. Comm. 107:232-238. - 109 -Greene, R.M., Shanfeld, J.L., Davidovitch, Z. and Pratt, R.M. 1980. Immunohistochemical localization of cyclic AMP in the developing rodent secondary palate. J. Embryol. Exp. Morphol. 60:271-281. Greene, R.M., Goldman, A.S., Lloyd, M., Baker, M., Brown, K.S., Shanfeld, J.L. and Davidovitch, Z. 1981. Glucocorticoid inhibition of cyclic AMP in the developing secondary palate. J. Craniofacial Genet. Dev. Biol. 1:31-44. Gringauz, A. 1978. Drugs: How they act and why. CV. Mosby Co., St. Louis. Grubb, R.B. and Montiegel, E.L. 1975. The teratogenic effects of 6-mercaptopurine on chick embryo in ovo. Teratology 11:179-186. Gulamhusein, A.P. and England, M.A. 1982. The developing ferret palate - A scanning electron microscope study: 1. Primary palate and secondary palatal shelves. J. Craniofacial Genet. Dev. Biol. 2:107-123. Hale, F. 1933. Pigs born without eyeballs. J. Hered. 24:105-106. Hale, F. 1935. The relation of vitamin A to anophthalmos in pigs. Am. J. Ophth. 18:1087-1093. Hale, F. 1937. The relation of maternal vitamin A deficiency to microphthalmia in pigs. Texas J. Med. 33:228-232. Hamilton, L. and El ion, G.B. 1954. The fate of 6-mercaptopurine in man. Ann. N.Y. Acad. Sci. 60:304-314. Hassel, J.R. 1975. The development of rat palatal shelves in vitro. An ultrastructural analysis of the inhibition of epithelial ceD death and palate fusion by the epidermal growth factor. Dev. Biol. 45, 90-102. Hassel, J.R. and Pratt, R.M. 1977. Elevated levels of cAMP alters the effect of epidermal growth factor in vitro on programmed cell death in the secondary palatal epithelium. Exp.Tell Res. 106:55-62. Hayward, A.F. 1969. Ultrastructural changes in the epithelium during fusion of the palatal processes in rats. Archs. oral Biol. 14:661-678. Hayward, J.R. and Avery, J. 1957. A variation in cleft palate. J. Oral Surg. 15:320-326. Heinen, E., Baeckeland, E. and Renard, A.M. 1982. The role of the cell coat in palatal shelves adhesion. The action of neuraminidase or Limulus  polyphemus lectin on shelves cultivated in vitro. In: Lectins-Biology, Biochemistry, Clinical Biochemistry, Vol. TT, Ed. T.C. Bog-Hansen, Walter de Gruyter and Co., Berlin, New York, pp. 285-294. - 110 -Hemminki, K., Axelson, 0., Niemi, M.L. and Ahlborg, G. 1983. Assessment of methods and results of reproductive occupational epidemiology: Spontaneous abortions and malformations in the offspring of working women. Prog. Clin. Biol. Res. 117:293-307. Herken, R., Merker, H.J. and Krowke, R. 1978. Investigation of the effect of hydroxyurea on the cell cycle and the development of necrosis in the embryonic CNS of mice. Teratology 18:103-118. Hill, J.M. cited by Yahia, C, Hyman, G.A. and Phillips, L.L. 1958. Acute leukemia and pregnancy. Obstet. Gynec. Surv. 13:1-21. Hill, J.M. and Loeb, E. cited by Sokal and Lessmann 1960. Effects of cancer chemotherapeutic agents on the human fetus. JAMA 172:1765-1771. Hinrichsen, C.F.L. and Stevens, G.S. 1974. Epithelial morphology during closure of the secondary palate in the rat. Archs. oral Biol. 19:969-980. Hirsch, K. and Hurley, L.S. 1978. Relationship of dietary zinc to 6-mercaptopurine teratogenesis and DNA metabolism in the rat. Teratology 17:303-314. Holmstedt, J.O.V. and Bagwell, J.N. 1977. Morphogenesis of the secondary palate in the mongolian gerbil (Meriones unguiculatus). Acta Anat. 97:443-449. Hoist, P. and Mills, B. 1975. Tissue phosphatase changes following triamcinolone associated with cleft palate in rats. Teratology 11:57-64. Hoover, B.A. and Schumacher 1966. Acute leukaemia in pregnancy. Am. J. Obst. Gynec. 96:316-320. Horakova, K., Navarova, J. and Paterson, A.R.P. 1974. The delayed cytotoxic effect of 6-mercaptopurine. Biochim. Biophys. Acta 366:330-340. Hudson, C. and Shapiro, B.L. 1973. A radioautographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death. Arch, oral Biol. 18:77-81. Humphrey, T. 1969. The relation between human fetal mouth opening reflexes and closure of the palate. Am. J. Anat. 125:317-344. IARC 1981. IARC monographs on the evaluation of carcinogenic risk of chemicals to humans Vol. 26. Some Antineoplastic and Immunosuppressive Agents, Lyon, pp. 249-266. Im, M.J. and Mulliken, J.B. 1983. Microanalysis of epithelial and mesenchymal acid hydrolase activities in the developing palate. J. Craniofacial Genet. Dev. Biol. 3:281-288. - Ill -Ingber, D.E., Madri, J.A. and Jamieson, J.D. 19.81. Role of basal lamina in neoplastic disorganization of tissue architecture. Proc. Natl. Acad. Sci. USA 78:3901-3905. Innes, P.B. 1978. The ultrastructure of the mesenchymal element of the palatal shelves of the fetal mouse. J. Embryol. exp. morph. 43:185-194. Innes, P.B. 1981. The ultrastructure of murine secondary palatal ectomesenchyme during shelf reorientation. J. Craniofacial Genet. Dev. Biol. 1: 359-371. Iregbulem, L.M. 1982. The incidence of cleft lip and palate in Nigeria. Cleft Palate J. 19:201-205. Jacobs, R.M. 1964a. Histochemical study of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec. 149:691-697. Jacobs, R.M. 1964b. 35S-liquid-scinti11ation count analysis of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec. 150:271-278. Jacobs, R.M. 1966. Effects of cortisone acetate upon hydration of embryonic palate in two inbred strains of mice. Anat. Rec. 156:1-4. Jacobson, B. 1982. Effect' of teratogens on connective tissue development. Molec. Aspects. Med. 5:401-459. Jacobson, B. and Shah, R.M. 1981. The glycosaminoglycan compositon of fetal palate from normal and hydrocortisone treated hamster. Teratology 23:42A-43A. Jao, W., Vazquez, L.T., Keh, P.C. and Gould, V.E. 1978. Myoepithelial differentiation and basal lamina deposition in fibroadenoma and adenosis of the breast. J. Pathol. 126:107-112. Jelinek, R. and Dostal, M. 1973. The role of mitotic activity in the formation of the secondary palate. Acta. Chir. Plast 15:216-222. Johnson, F.R., and Fry, L. 1967. Ultrastructural observations on lichen planus. Arch. Dermatol. 95:596-607. Jurand, A. 1966. Early changes in limb buds of chick embryos after thalidomide treatment. J. Embryol. exp. Morph. 16:289-300. Jurand, A. 1968. The effect of hydrocortisone acetate on the development of mouse embryos. J. Embryol. exp. Morph. 20:355-366. Kalter, H. and Warkany, J. 1959. Experimental production of congenital malformations in mammals by metabolic procedures. Physiol. Rev. 39:69-115. - 112 -Karnofsky, D.A. 1960. Influence of antimetabilites inhibiting nucleic acid metabolism on embryonic development. Trans. Assoc. Amer. physicians 73:334-347. Kasper, C.B. 1974. The Cell Nucleus. In: Chemical and biochemical properties of the nuclear envelope. Ed. H Busch, Academic Press, New York and London, pp. 349-382. Kawahata, R.T., Holmberg, C.A., Osburn, B.I., Chuang, L.F. and Chuang, R.Y. 1980. Effect of 6-mercaptopurine ribonucleotides on DNA-dependant RNA polymerase activity. Mol. Pharmacology 18:503-506. Keibel, K. and Mall, F.P. 1912. Manual of Human Embryology. Lippincott, Philadelphia, pp. 340-343. Kerr, J.F.R. 1971. Shrinkage necrosis. A distinct mode of cellular death. J. Pathol. 105:13-20. Kitamura, H. 1966. Epithelial remnants and pearls in the secondary palate in the human abortus: A contribution to the study of the mechanism of cleft palate formation. Cleft Palate J. 3:240-256. Koch, W. and Smiley, G.R. 1981. In vivo and J_n vi tro studies of the development of the avian secondary pala~te. ATchs. oral Biol. 26:181-189. Kochhar, D.M. and Johnson, E.M. 1965. Morphological and autoradiographic studies of cleft palate induced in rat embryos by maternal hypervitaminosis A. J. Embryol. Exp. Morphol. 14:223-238. Kochhar, D.M., Penner, J.D. and McDay, J.A. 1978. Limb Development in mouse embryos II. Reduction defects, cytotoxicity and inhibition of DNA synthesis produced by cytosine arabinoside. Teratology 18:71-92. Koguchi, H. 1980. Population data on cleft lip and cleft palate in the Japanese. Prog. Clin. Biol. Res. 46:297-323. Koziol, C.A. and Steffek, A. 1969. Acid phosphatase activity in palates of developing normal and chlorcyclizine treated rodents. Arch, oral Biol. 14:317-321. Krawczyk, W. and Gi11 on, D. 1976. Immunofluorescent detection of actin in non-muscle cells of the developing mouse palatal shelf. Archs. oral Biol. 21:503-508. Kurisu, K., Saskaki, S., Shimazaki, K., Ohsaki, Y., and Wada, K. 1981. Light and electron microscopic studies on the effect of triamcinolone acetonide on the medical edge epithelia of palatal shelves of mouse fetuses in vivo. J. Craniofacial Genet. Dev. Biol. 1:273-284. Kurtz, S.M. and Feldman, J.D. 1962. Experimental studies on the formation of the glomerular basement membrane. J. Ul trastruct. Res. 6:19-27. - 113 -Kury, G., Chaude, S. and Murphy, M.L. 1968. Teratogenic effects of some purine analogues on fetal rats. Arch. Pathol. 86:395-402. Kusanagi, T. 1983. Occurance of cleft palate, palatal slit, and fetal death in mice treated with a glucocorticoid: An embryo transfer experiment. Teratology 27:395-400. Langman, J. and Cardell, E.L. 1978. Ultrastructural observations on FUdR-induced cell death and subsequent elimination of cell debris. Teratology 17:229-270. Larsson, K.S. 1962a. Closure of the secondary palate and its relation to sulfo-mucopolysaccharides. Acta. Odontol. Scand. 20, Suppl. 31:1-35. Larsson, K.S. 1962b. Studies on the closure of the secondary palate IV. Autoradiographic and histochemical studies of mouse embryos from cortisone-treated mothers. Acta. Morph. Neer-Scand. 4:369-386. Larsson, K.S. and Bostrom, H. 1965. Teratogenic action of salicylates related to the inhibition of mucopolysaccharide synthesis. Acta. Paediat. Scand. 54:43-48. Lee, R.A., Johnson, C.E. and Hanlon, D.G. 1962. Leukemia during pregnancy. Amer. J. Obstet. Gynec. 84:455-458. Lennard and Maddocks, J.L. 1983. Assay of 6-thioguanine nucleotide, a major metabolite of azathioprine, 6-mercaptopurine and 6-thioguanine in human red blood cells. J. Pharm. Pharmacol. 35:15-18. Lenz, W.V. 1961. Die thaiidomid-embryopathie. Dtsch med. Wschr. 86:2555. Levin, S.M. 1983. Problems and pitfalls in conducting epidemiological research in the area of reproductive toxicology. Prog. Clin. Biol. Res. 117:349-364. Lorente and Miller, S.A. 1978. The effect of hypervitaminosis A on rat palatal development. Teratology 18:277-284. Lorente, C.A., DePaola, D.P., Drummond, J.F. and Miller, S.A. ; 1974. Lysosomal enzyme activity associated with palatal development in the rabbit. J. Dent. Res. 53:65(Abstract 43). Lowry, R.B. and Trimble, B.K. 1977. Incidence rates for cleft lip and palate in British Columbia 1952-71 for North American Indian, Japanese, Chinese and total populations: Secular trends over twenty years. Teratology 16:277-284. Loyd, H.0. 1961. Acute leukemia complicated by pregnancy. J.A.M.A. 178:1140-1143. Luke, D. 1976. Development of the secondary palate in man. Acta Anat. 94:596-608. - 114 -Lynch, J.B., Lewis, S.R. and Blocker, T.G. 1966. Cleft palate not explained by embryology. Plast. Reconstr. Surg. 38:552-554. Malamud, D., Gonzales, CM., Chin, H. and Malt, R.A. 1972. Inhibition of cell proliferation by azathiopurine. Cancer Rec. 32:1226-1229. Mangiameli, S. 1961. Leucosi e gravidanza. Minerva ginec. (Torino) 13:785-792. Martin, W.R., Crichton, I.K., Yang, R.C and Evans, A.E. 1972. The metabolism of thioinosinic acid by 6-mercaptopurine sensitive and resistant leucemic leucocytes. Proc. Soc. Exp. Biol. Med. 140:423-428. Martinez-Hernandez, A., Fink, L.M. and Pierce, G.B. 1976. Removal of basement membrane in the involuting breast. Lab. Invest. 34:455-461. Mato, M. and Uchiyama, Y. 1972. Specific changes in the epithelium covering the palatine shelves in the teratogen treated embryos. Gunma Rep. Med. Sc. 3:377-380. Mato, M. and Uchiyama, Y. 1975. Ul trastructures of glosso-palatal fusion after treatment of meclozine-hydrochloride. Virchows Arch. A Path. Anat. and Hist. 369:7-17. Mato, M., Aikawa, E. and Katahira, M. 1966. Appearance of various types of lysosomes in the epithelium covering lateral palatine shelves during a secondary palate formation. Gunma J. Med Sc. 15:46-56. Mato, M., Aikawa, E. and Katahira, M. 1967a. Alteration of fine structure of the epithelium on the lateral palatine shelf during the secondary palate formation. Gunma, J. Med. Sc. 16:79-99. i'iato, M., Aikawa, E. and Katahira, M. 1967b. Studies on cell-reaction of the nasal epithelium during the fusion of palatine shelves. Anat. Anz. 121:504-517. Mato, M., Uchiyama, Y. and Aikawa, E. 1975a. Studies on normal and heterotypic fusion during morphogenesis. 10th Int. Cong. Anat., Tokyo, 396. Mato, M., Uchiyama, Y., Aikawa, E. and Smiley, G.R. 1975b. Ultrastructural changes in rat palatal epithelium after 3-am1noproprionitrile. Teratology 11:153-168. McBride, W.G. 1961. Thalidomide and congenital abnormalities. Lancet. 2:1358. McConnell, J.B. and Bhoola, R. 1973. A neonatal complication of maternal leukaemia treated with 6-mercaptopurine. Postgrad, med. J. 49:211-213. Meller, S.M. and Barton, L.H. 1978. Extracellular coat in developing human palatal processes: Electron microscopy and ruthenium red binding. Anat. Rec. 190:223-232. - 115 -Meller, S.M., DePaola, D.P., Barton, L.H. and Mandella, R.D. 1980. Secondary palatal development in the New Zealand white rabbit : A scanning electron microscopic study. Anat. Rec. 198:229-244. Melnick, M., Shields, E.D. and Bixler, D. 1980. Studies of cleft lip and cleft palate in the population of Denmark. Prog. Clin. Biol. Res. 46:225-248. Menkes, B., Sandor, S. and Hies, A. 1970. Cell death in teratogenesis. Adv. Teratol. 4:170-215. Merchant-Larios, H. and Coello, J. 1978. The effect of busulfan on rat primordial germ cells at the ultrastructural level. Cell Differentiation 8:145-155. Mercier-Parot, L. and Tuchmann-Duplessis, H. 1967. Production of limb malformations by 6-mercaptopurine in three species: rabbit, rat and mouse (Fr.). C.R. Soc. Biol. Paris 161:762-768. Merker, H.J., Pospisil, M. and Mewes, P. 1975. Cytotoxic effects of 6-mercaptopurine on the limb-bud blasternal cells of rat embryos. Teratology 11:199-218. Merskey, C. and Rigal, W. 1956. Pregnancy in acute leukemia treated with 6-mercaptopurine. Lancet 2:1268-1269. Mewes, P., Siebert, G. and Neubert, D. 1971. Effect of 6-mercaptopurine on RNA metabolism of rat embryos. Teratology 4:495(abstract). Mitts, T.F., Garrett, W.S. and Hurwitz, D.J. 1981. Cleft of the hard palate with soft palate integrity. Cleft Palate J. 18:204-206. Mollenbauer, H. 1964. Plastic embedding mixture for use electronmicroscopy. Stain. Technol. 39:111-114. Montenegro, M.A. and Paz de la Vega, Y. 1982. Light and electron microscopic study on the effect of phenylbutazone on developing mouse palatal epithelium in vitro. Archs. oral Biol. 27:771-775. Morgan, P.R. 1969. Recent studies on the fusion of the secondary palate. London Hosp. Gaz. 72:6-19. Morgan, P.R. 1976. The fate of the expected fusion zone in rat fetuses with experimentally-induced cleft palate - an ultrastructural study. Dev. Biol. 51:225-240. Morgan, P.R. and Pratt, R.M. 1977. Ultrastructure of the expected fusion zone in rat fetuses with diazo-oxo-norleucine (DON)-induced cleft palate. Teratology 15:281-289. Morriss, G.M. 1973. The ultrastructural effects of excess maternal vitamin A on the primitive streak stage rat embryos. J. Embryol. Exp. Morphol. 30:219-242. - 116 -Mott, W.J., Toto, P.D. and Holgers, D.C. 1969. Labelling index and cellular density in palative shelves of cleft-palate mice. J. Dent. Res. 48:263-265. Murphy, M.L. 1960. Teratogenic effects of tumour-inhibiting chemicals in the fetal rat. In: Ciba Foundation Symposium on Congenital Malformations. Ed. Wolstenholme, G.E., Little, Brown and Company, Boston, pp. 78-107. Murphy, M.L. 1962. Teratogenic effects in rats of growth inhibiting chemicals, including studies on thalidomide. Child. Hosp. Dist. Columbia Proc. 18:307-322. Nanda, R. 1970. The role of sulfated mucopolysaccharides in cleft palate production. Teratology 3:237-244. Nanda, R. 1971. Tritiated thymidine labelling of the palatal processes of rat embryos with cleft palate induced by hypervitaminosis A. Archs. oral Biol. 16:435-444. Nanda, R. and Romeo, D. 1975. Differential cell proliferation of embryonic rat palatal processes as determined by incorporation of tritiated thymidine. Cleft Palate J. 12:436-443. Neu, L.T. Jr. 1962. Leukemia complicating pregnancy. Missouri Med. 59:220-221. Neubert, D., Barrach, H.J. and Merker, H.J. 1980. Drug-induced damage to the embryo or fetus. In: Drug-Induced Pathology. Ed. E. Grundmann, Springer-Verlag, Berlin, Heidelberg, New York, pp. 241-331. Neubert, D., Lessmollmann, U., Hinz, N., Dillmann, I. and Fuchs, G. 1977. Interference of 6-mercaptopurine riboside, 6-methylmercaptopurine riboside, and azathioprine with the morphogenetic differentiation of mouse extremities in vivo and in organ culture. Naunyn-Schmiedeberg's Arch. Pharmacol. 298:93-105. Neubert, D., Merker, H.J., Kohler, E., Krowe, R. and Barrach, H.J. 1970. Biochemical aspects of teratology. In: Advances in the Biosciences. Ed. G. Raspe, Pergamon Press, London, pp. 575-622. Nicholson, H.O. 1968a. Cytotoxic drugs in pregnancy. J. Obst. Gynacol. Br. Common. 75:307-312. Nicholson, H.O. 1968b. Leukemia and pregnancy. A report of five cases and discussion of management. J. Obstet. Gynaec. Brit. Comm. 75:517-520. Novikoff, A. 1963. Lysosomes in physiology and pathology of cells: Contribution of staining methods. In: Lysosomes. Ed. A.V.S. De Reuk and M.P. Cameron, Churchill, London, pp. 36-77. O'Leary, J.A. and Bepko Jr., F.J. 1963. Obstetrical clinics: Acute leukemia and pregnancy. Georgetown Medical Bulletin 16:162-164. - 117 -Oltner, J. and Carcassonne, Y. 1962. Leucenrie et grossesse. Marseille Med. 99:31-36. Olson, F.C. and Massaro, E.J. 1977. Effects of methyl mercury on murine fetal amino acid uptake, protein synthesis and palate closure. Teratology 16:187-194. Otsubo, Y. and Kameyama, Y. 1982. Ultrastructural changes of epithelium-connective tissue junction in experimental lingual tumors. J. oral Pathol. 11: 159-173. Parekh, J.G., Hanson, T.A. and Smith, R.S. 1959. Acute leukaemia and pregnancy. J.J.J. Hosp. Grant, med. Coll. 4:49. Paterson, A.R.P. and Tidd, D.M. 1975. 6-thiopurines. In: Handbook of Experimental Pharmacology, Vol. 38, Ed. 0. Eichler, A. Farah, H. Herken and A.D. Welch, Springer, Berlin, pp. 384-403. Patten, B.M. 1971. Embryology of the palate and maxillofacial region. In: Cleft Lip and Cleft Palate, Eds. W.C. Grabb and S.W. Rosenstein and K.R. Bzoch, Little, Brown Co., Boston, pp. 21-53. Paul, M.A. and Piazza, F. 1979. Cost of treating birth defects in state crippled children's services, 1975. Pub. Health Rep. 94:420-424. Pearce, G.B., Midgley, A.R. and Sri Ram, J. 1963. The histogenesis of basement membranes. J. Exp. Med. 117:339-347. Pearson, R.W. and Spargo, B. 1961. Electron microscope studies of dermal-epidermal separation in human skin. J. Invest. Dermatol. 36:213-225. Persaud, T.V.N. 1979. Advances in the study of birth defects Vol. 3. Ed. T.V.N. Persaud. MTP Press Ltd., Lancaster. Peters, P.W.J., Dormans, J.A.M.A. and Geelen, J.A.G. 1979. Light microscopic and ultrastructural observations in advanced stages of induced exencephaly and spina bifida. Teratology 19:183-196. Pick, J.B. and Evans, C.A. 1981. Growth inhibitition and occurrence of cleft palates due to hypervitaminosis A. Experientia 37:1189-1191. Plagemann, P.G.W., Marz, R., Wohlhueter, R.M., Graff, J.C. and Zylka, J.M. 1981. Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation. Biochimica et Biophysica Acta 49-62. Pourtois, M. 1966. Onset of the acquired potentiality for fusion in the palatal shelves of rats. J. Embryol. exp. Morph. 16:171-182. - 118 -Pourtois, M. 1972. Morphogenesis of the primary and secondary palate. In: Developmental Aspects of Oral Biology. Ed. H.C. Slavkin and L.A. Bavetta, Academic Press, London, pp. 81-108. Pratt, R.M. 1983. Mechanisms of chemically-induced cleft palate. Trends Pharmacol. Sci. 4:160-162. Pratt, R.M. and Greene, R.M. 1976. Inhibition of palatal epithelial cell death by altered protein synthesis. Dev. Biol. 54:135-145. Pratt, R.M. and Hassel, J.R. 1975. Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf. Dev. Biol. 45:192-198. Pratt Jr., R.M. and King, C.T.G. 1972. Inhibition of collagen cross-linking associated with 6-aminopropionitrile-induced cleft palate in the rat. Dev. Biol. 27:322-328. Pratt, R.M. and Martin, G.R. 1975. Epithelial cell death and cyclic AMP increase during palatal development. Proc. Nat. Acad. Sci. USA 72:874-877. Pratt Jr., R.M., Goggins, J.F., Wilk, A.L. and King, C.T.G. 1973. Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation and fusion. Dev. Biol. 32:230-237. Pruzanski, S. 1961. Congenital anomalies of the face and associated structures. C.C. Thomas, Springfield, Illinois, pp. 3-11. Puget, A., Cros, S., Oreglia, J. and Tollon, Y. 1975. Study on the embryonal sensitivity of the Afghan pika (Ochotona rufescens rufescens) to two teratogenic agents, azathioprine and 6-mercaptopurine (Fr.). Zbl. Veterinarmed A 22:38-56. Raichs, A. 1962. Gestacion y leucemia agunda: Description de cuatro observations. Sangre 7:194-212. Ravenna, P. and Stein, P.J. 1963. Acute monocytic leukemia in pregnancy. Amer. J. Obstet. Gynec. 85:545-548. Ree, K., Rugstad, H.E. and Bakka, A. 1982. Ultrastructural changes in the nucleus of a human epithelial cell line exposed to cytotoxic agents. Acta path, microbiol. immunol. scand. Sect A 90:427-435. Reimers, T.J. and Sluss, P.M. 1978. 6-mercaptopurine treatment of pregnant mice: Effects on second and third generation. Science 201:65-67. Reimers, T.J., Sluss, P.M., Goodwin, J. and Seidel Jr., G.E. 1980. Bigenerational effects of 6-mercaptopurine on reproduction in mice. Biol. Reprod. 22:367-375. Reynolds. E.S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-213. - 119 -Rezende, J. de, Coslovsky, S. and Agin'ar, P.B. de. 1965. Leukemia e gravidez. Rev. Ginec. obstet. (Rio de J} 117:46-50. Rigby, P.G., Hanson, T.A. and Smith, R.S. 1964. Passage of leukaemic cells across the placenta. New. Eng. J. Med. 271:124-127. Ross, L. and Walker, B.E. 1967. Movement of palatine shelves in untreated and teratogen treated mouse embryos. Am. J. Anat. 121:509-521. Rothberg, H., Conrad, M.E. and Cowley, R.G. 1959. Acute granulocytic leukemia in pregnancy: Report of four cases, with apparent acceleration by prednisone in one. Amer. J. Med. Sci. 237:194-204. Roy-Burman, P. 1970. Recent results in cancer research. In: Analogues of Nucleic Acid Components vol. 25. Ed. R. Rentchnick, Springer-Verlag, New York., pp. 59-62. Sabatini, D.D., Bench, K. and Barnett, R.S. 1963. Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzyme activity by aldehyde fixation. J. Cell Biol. 17:19-58. Sadler, T.W. and Cardell, R.R. 1977. Ultrastructural alterations in neuroepithelial cells of mouse embryos exposed to cytotoxic doses of hydroxyurea. Anat. Rec. 188:103-124. Salomon, D.S. and Pratt, R.M. 1979. Involvement of glucocorticoids in the development of the secondary palate: A review. Differentiation 13:141-154. Sandberg, A.A. cited by Sokal and Lessmann. 1960. Effects of cancer chemotherapeutic agents on the human fetus. J.A.M.A. 172:1765-1771. Sauer, G.J.R. and Evans, C.A. 1980. Hypervitaminosis A and matrix alterations in maxillary explants from 16-day rat embryos. Teratology 21:123-130. Saxen, L. and Rapola, J. 1969. Methods in teratology. In: Congenital Defects. Holt, Rinehart and Winston Inc., New York, p. 7-34. Schardein, J.L. 1976. Drugs as teratogens. CRC Press, Cleveland, Ohio. Schumacher, H.R. 1957. The use of 6-mercaptopurine 1n treatment of acute leukemia in late pregnancy. Am. J. Obst. Gynec. 74:1361-1362. Schupbach, P.M. 1983. Experimental induction of an incomplete hard palate cleft in the rat. Oral. Surg. 55:2-9. Schupbach, P.M. and Schroeder, H.E. 1983. Cell release from the palatal shelves and the fusion line. J. Biol. Bucc. 11:227-241. Schupbach, P.M., Chamberlain, J.G. and Schroeder, H.E. 1983. Development of the secondary palate in the rat: A scanning electron microscopic study. J. Craniofac. Gen. Dev. Biol. 3:159-177. - 120 -Schweichel, J.U. and Merker, H.J. 1973. The morphology of various types of cell death in prenatal tissues. Teratology 7:253-266. Scott Jr., W.J. 1977. Cell death and reduced proliferative rate. In: Handbook of Teratology, Vol. 2. Eds. J.G. Wilson and F.C. Fraser, Plenum Press, New York. Scott, W.J., Ritter, E.J. and Wilson, J.G. 1980. Ectodermal and mesodermal cell death patterns in 6-mercaptopurine riboside-induced digital deformities. Teratology 21:271-279. Shah, R.M. 1977a. Effects of prenatal administration of hadacidin, a cancer chemotherapeutic agent, on the development of hamster fetuses. J. Embryol. Exp. Morph. 39:203-220. Shah, R.M. 1977b. Palatomandibular and maxillo-mandibular fusion, partial aglossia and cleft palate in a human embryo. Report of a case. Teratology 15:261-272. Shah, R.M. 1979a. A cellular mechanism for the palatal shelf reorientation from a vertical to a horizontal plane in hamster: light and electron microscopic study. J. Embryol. Exp. Morph. 53:1-13. Shah, R.M. 1979b. The distribution of desmosomes and ruthenium red-bound cell surface carbohydrates during palatal closure in the hamster. Invest. Cell Pathol. 2:319-331. Shah, R.M. 1979c. Current concepts on the mechanisms of normal and abnormal secondary palate formation. In: Advances in the Study of Birth Defects, Vol. 1, Teratogenic Mechanisms. Ed. T.V.N. Persaud, MTP Press Ltd., Lancaster, pp. 69-84. Shah, R.M. 1979d. Usefulness of golden Syrian hamster in experimental teratology with particular reference to the induction of orofacial malformations. In: Advances in the study of birth defects, Vol. 2, Teratological testing. Ed. T.V.N. Persaud, MTP Press, Lancaster, pp. 25-39. Shah, R.M. 1979e. Cleft palate development in hamster embryos following triamcinolone treatment. J. Anat. 129:531-539. Shah, R.M. 1980. Ultrastructural observations on the development of triamdnolone-induced cleft palate in hamsters. Invest. Cell pathol. 3:281-284. Shah, R.M. 1984. Morphological, cellular, and biochemical aspects of differentiation of normal and teratogen-treated palate in hamster and chick embryos. Current Topics in Dev. Biol. 19:103-135. Shah, R.M. and Burdett, D.N. 1979. Developmental abnormalities induced by 6-mercaptopurine in the hamster. Can. J. Physiol. Pharmacol. 57:53-58. - 121 -Shah, R.M. and Burton, A.F. 1980. Metabolism and nuclear-cytosol binding of 14C-glucocorticoids during cleft palate development. J. Dent. Res. 59(Special Issue A): 304. Shah, R.M. and Chaudhry, A.P. 1974a. Light microscopic and histochemical observations on the development of palate in golden hamsters. J. Anat. 117:1-15. Shah, R.M. and Chaudhry, A.P. 1974b. Ultrastructural observations on closure of the soft palate in hamsters. Teratol. 10:17-30. Shah, R.M. and Crawford, B.J. 1980. Development of the secondary palate in chick embryo: A light and electron microscopic and histochemical study. Invest. Cell Pathol. 3:319-328. Shah, R.M. and Kilistoff, A.J. 1976. Cleft palate induction in hamster by various glucocorticoid hormones and their synthetic analogues. J. Embryol. Exp. Morphol. 36:101-108. Shah, R.M. and MacKay, R.A. 1978. Teratological evaluation of 5-fluorouracil and 5-bromo-deoxyuridine on hamster fetuses. J. Embryol. Exp. Morphol. 43:47-54. Shah, R.M. and Travill, A. .1974. Cellular organization during early palate formation. Teratology 9:A-36. Shah, R.M. and Travill, A.A. 1976a. Morphogenesis of the secondary palate in normal and hydrocortisone-treated hamsters. Teratology 13:71-84. Shah, R.M. and Travill, A.A. 1976b. Light and electron microscopic observations on hydrocortisone induced cleft palate in hamsters. Am. J. Anat. 145:149-166. Shah, R.M. and Wong, D.T.W. 1980. Morphological study of cleft palate development in 5-fluorouracil-treated hamster fetuses. J. Embryol. Exp. Morph. 57:119-128. Shah, R.M., Crawford, B. and Suen, R. 1983. Tissue interaction and cytodifferentiation during palatogenesis in chick and hamster. J. Dent. Res. 62:236(Abstract). Shah, R.M., Donaldson, D. and Burdett, D. 1979. Teratogenic effects of diazepam in the hamster. Can. J. Physiol. Pharmacol. 57:556-561. Shah, R.M., Wong, D.T.W. and Suen, R.S.K. 1984a. Ultrastructural and cytochemical observations on 5-fluorouracil induced cleft-palate development in hamster. Am. J. Anat. 1/0:567-580. Shah, R.M., Cheng, K.M., Suen, R. and Wong, A. 1984b. An ultrastructural and histochemical study of the development of secondary palate in Japanese quail, Coturnix coturnix japonica. J. Craniofacial. Genet. Dev. Biol, (in press). - 122 -Shah, R.M., Crawford, B.J., Greene, R.M., Suen, R.S., Burdett, D.N., King, K.O. and Wong, D.T.W. 1985. In vitro development of the hamster and chick secondary palate. J. Craniofac. Genet. Develop. Biol. (In Press). Shields, E.D., Bixler, D. and Fogh-Andersen, P. 1981. Cleft palate: A genetic and epidemiologic investigation. Clinical Genetics 20:13-24. Sinykin, M.B. and Kaplan, H. 1962. Leukemia in pregnancy. A case report. Amer. J. Obstet. Gynec. 83:220-224. Smetana, K. and Hermansky, F. 1966. On the ultrastructure of leukaemic monocytes. Folia heamatol. 86:35-46. Smiley, G.R. 1970. Fine structure of mouse embryonic palatal epithelium prior to and after midline fusion. Arch, oral Biol. 15:287-296. Smiley, G.R. 1972. A possible genesis for cleft palate formation. Plast. Reconstr. Surg. 50:390-394. Smiley, G.R. and Koch, W.E. 1971. Fine structure of mouse secondary palate development in vitro. J. Dent. Res. 50:16/1-1677. Smiley, G.R. and Koch, W.E. 19/5. A comparison of secondary palate development with different in vitro techniques. Anat. Rec. 181:711-724. Smithells, R.W. 1966. Drugs and human malformations. In: Advances in Teratology Vol. 1, pp. 251-278. Sokal, R.R. and Rohlf, F.J. 1969. Biometry. The principles and practice of statistics in biological research. Wilt. Freeman and company, San Francisco. Sonawane, B.R. and Goldman, A.S. 1981. Susceptibility of mice to phenytoin-induced cleft palate correlated with inhibition of fetal palatal RNA and protein synthesis. Proc. Soc. Exp. Biol. Med. 168:175-179. Souchon, R. 1975. Surface coat of the palatal shelf epithelium during palatogenesis in mouse embryos. Anat. Embryol. 147:133-142. Stanley, J.R., Woodley, D.T., Katz, S.I. and Martin, G.R. 1982. Structure and function of basement membrane. J. Invest. Dermatol. (Suppl.) 79:69-72. Steffek, A.J., Verrucio, A.C. and King, C.T.G. 1968. The histology of palatal closure in the rhesus monkey (Macaca mulatta). Teratology 1:425-430. Stempak, J.G. and Ward, R.T. 1964. An improved staining method for electron microscopy. J. Cell Biol. 22:697-701. Stewart, J.O. 1964. Leukemia in pregnancy. A case report of acute lymphatic leukemia. J. Nat. Med. Ass. INY) bb:8/-89. - 123 -Sugimoto, A., Rose, G.G., Takarada, H. and Cattoni, M. 19/3. Ultrastructural changes of basal lamina and anchoring fibrils of gingiva in vitro. J. Periodont. Res. /:12/-142. Swift, H. and Hruban, Z. 1964. Focal degeneration as a biological process. Fedn. Proc. Fedn. Am. Spcs. Exp. Biol. 23:1026-1037. Takarada, H. , Cattoni, M., Sugimoto, A. and Rose, G.G. 1974. Ultrastructural studies of human gingiva. III. Changes of the basal lamina in chronic periodontitis. J. Periodontol. 45:288-302. Tarin, D. 1972. Morphological studies on the mechanism of carcinogenesis. In: Tissue Interactions in Carcinogenesis. Ed. D. Tarin, Academic Press, New York, pp. 227-290. Tassin, M.T. and Weill, R. 1980. Scanning electron microscopic study of the medio palatal epithelium: Simultaneous modifications characterizing fusion and degenerescence processes. Wilhelm Roux's Archives 188:13-21. Taylor G.R. 1978. Craniofacial growth during closure of the secondary palate in the hamster. J. Anat. 125:361-3/0. Taylor, J.H., Hart, W.F. and Tung, J. 1962. Effects of fluorodeoxyuridine on DNA replication, chromosome breakage and reunion. Proc. Nat. Acad. Sci. 48:190-198. Theodosis, D.T. and Fraser, F.C. 1978. Early changes in the mouse neuroepithelium preceeding exencephaly induced by hypervitaminosis A. Teratology 18:219-232. Thiersch, J.B. 1954. The effect of 6-mercaptopurine on the rat fetus and on reproduction of the rat. Ann. N.Y. Acad. Sci. 60:220-227. Thiersch, J.B. 1956. The control of reproduction in rats with the aid of antimetabolites and early experiences with antimetabolites as abortifacient agents in man. Acta, endocr. (Kbh) Suppl. 28:37-45. Tidd, D.M. and Dedhar, S. 1978. Specific and sensitive combined high-performance liquid chromatographic-flow fluorometric assay for intracellular b-thioguanine nucleotide metabolites of 6-mercaptopurine and 6-thioguanine. J. Chromatogr. 145:237-246. Tidd, D.M. and Paterson, A.R.P. 1974. A biochemical mechanism for the delayed cytotoxic reaction of 6-mercaptopurine. Cancer Res. 34:738-746. Tidd, D.M., Kim, S.C, Horakova, K., Moriwaki, A. and Paterson, A.R.P. 1972. A delayed cytotoxic reaction for b-mercaptopurine. Cancer Res. 32:317-322. Timpl, R., Rohde, H., Gehron, R.P., Rennard, S.I., Foidart, J.M. and Martin, G.R. 1979. Laminin-A glycoprotein from basement membranes. J. Biol. Chem. 254:9933-9937. - 124 -Trump, B.F., Berezesky, I.K., Phelps, P.C. and Jones, R.T. 1983. An overview of the role of membranes in human disease. In: Cellular Pathobiology of Human Disease. Ed. B.F. Trump, A. Laufer, R.T. Jones. Gustav Fischer, New York. Tterlikkis, L. , Ortega, E., Soloman, R. and Day, J.L. 1977. Pharmacokinetics of mercaptopurine. J. Pharm. Sci. 66:1454-1457. Tuchmann-Duplessis, H. 1983. The teratogenic risk. Prog. Clin. Biol. Res. 117:245-258. Tuchmann-Duplessis, H. and Haegel, P. 1974. Illustrated human embryology: Volume 2 Organogenesis. Masson and Co. Paris, pp. 16-19. Tuchman-Duplessis, H. and Mercier-Parot, L. 1958. On the teratogenic action of several antimitotic substances in the rat (Fr.). C.R. Hebd. Acad. Sci. 247:152-154. Tuchmann-Duplessis, H. and Mercier-Parot, L. 1966. Production of limb malformations in the rabbit by administration of azathioprine and 6-mercaptopurine (Fr.). C.R. Soc. Biol. Paris 160:501-507. Tuchmann-Duplessis, H. and Mercier-Parot, L. 1968. Experimental production of limb malformations (Fr.). Union med. Can. 97:283-288. Tyan, M.L. 1982. Differences in the reported frequencies of cleft lip plus cleft lip and palate in asians born in Hawaii and the continental United States. Proc. Soc. Exp. Biol. Med. 171:41-45. Tyler, M.S. and Koch, W.E. 1975. In vitro development of palatal tissues from embryonic mice. I. DifferentiaTTon of the secondary palate from 12-day mouse embryos. Anat. Rec. 182:297-303. Tyler, M.S. and Koch, W.E. 1977a. In vi tro development of palatal tissues from embryonic mice II. Tissue isolation and recombination studies. J. Embryol. Exp. Morph. 38:19-36. Tyler, M.S. and Koch, W.E. 1977b. In vitro development of palatal tissues from embryonic mice III. Interactions between palatal epithelium and heterotypic oral mesenchyme. J. Embryol. Exp. Morph. 38:37-48. Tyler, M.S. and Pratt, R.M. 1980. Effect of epidermal growth factor on secondary palatal epithelium in vitro: tissue isolation and recombination studies. J. Embryol. Exp. Morph-. 58:93-106. Vargas, V., Nasjleti, C. and Azcurra, J. 1972. Cytodifferentiation of the mouse secondary palate in vi tro: morphological, biochemical, and histochemical aspects. J. Elibryol. Exp. Morphol. 27:413-430. Veau, V. 1931. Division palatine. Masson and Cie., Paris. Verrusio, A.C. 1970. A mechanism for closure of the secondary palate. Teratology 3:17-20. - 125 -Vischniakov, Y.S. 1968. The specificity of the damaging effect produced by 6-mercaptopurine at different stages of embryogenesis in rats (Russ). Farmakol. Toksikol. 31:480-481. Vishniakov, Y.S. 1969. Teratogenous effect of 6-mercaptopurine on albino rat embryos (Russ.). Arkh. Anat. 57:37-41. Yracko, R. 1972. Significance of basal lamina for regeneration of injured lung. Virchows Arch. (Pathol. Anat.) 355:264-274. Vracko, R. 1979. Basal lamina scaffold. Its role in maintenance of tissue structure and in pathogenesis of basal lamina thickening. In: Biochemistry and Pathology of Basement Membrane. Ed. A.M. Roberts, R. Bonifaca and L. Roberts, S. Karger, New York, pp. 78-90. Walker, B.E. 1961. The association of mucopolysaccharides with morphogenesis of the palate and other structures in mouse embryos. J. Embryol. Exp. Morph. 9:22-31. Walker, B.E. 1967. Induction of cleft palate in rabbit by several glucocorticoids. Proc. Soc. exp. Biol. Med. 125:1281-1284. Walker, B.E. 1969. Correlation of embryonic movement with palatal closure in mice. Teratology 2:191-198. Walker, B.E. 1971. Palate morphogenesis in the rabbit. Archs. Oral Biol. 16:275-286. Walker, B.E. and Crain, B. 1960. Effects of hypervitaminosis A on palate development in two strains of mice. Am. J. Anat. 107:49-58. Walker, B.E. and Fraser, F.C. 1956. Closure of the secondary palate 1n three strains of mice. J. Embryol. Exp. Morph. 4:176-189. Walker, B.E. and Fraser, F.C. 1957. The embryology of cortisone induced cleft palate. J. Embryol. Exp. Morphol. 5:201-209. Walker, B.E. and Ross, L.M. 1972. Observations of palatine shelves in living rabbit embryos. Teratology 5:97-102. Warkany J. and Nelson, R.C. 1941. Skeletal abnormalities in offspring of rats reared on deficient diets. Anat. Rec. 79:83-100. Warkany, J. and Schraffenberger, E. 1943. Congenital malformations induced in rats by maternal nutritional deficiency. V. Effects of a purified diet lacking riboflavin. Proc. Soc. Exp. Biol. Med. 54:92. Warkany, J., Roth, C.B. and Wilson, J.G. 1948. Multiple congenital malformations: A consideration of etiologic factors. Pediatrics 1:462-471. Waterman, R. and Meller, S.M. 1974. Alterations in the epithelial surface of human palatal shelves prior to and during fusion: A scanning electron microscopic study. Anat. Rec. 180:111-136. - 126 -Waterman, R., Ross, L. and Meller, S.M. 1973. Alterations in the epithelial surface of A/Jax mouse palatal shelves prior to and during fusion: A scanning electron microscopic study. Anat. Rec. 176:361-375. Webster, W., Shimada, M. and Langman, J. 19/3. Effect of 6-fluorodeoxyuridine, colcemid, and bromodeoxyuridine on developing neocortex of the mouse. Am. J. Anat. 135:67-86. Wee, E.L., Babiarz, B.S., Zimmerman, S. and Zimmerman, E.F. 1979. Palate morphogenesis IV. Effects of serotonin and its antagonists on rotation in embryo culture. J. Embryol. Exp. Morph. 53:75-9U. Whaley, W.G., Dauwalder, M. and Kephart, J.E. 1971. Assembly, continuity, and exchanges in certain cytoplasmic membrane systems. In: Origin and continuity of cell organelles. Ed. J. Reinert and H. Ursprung. Springer-Verlag, New York. White, F.H. and Gohari, K. 1981. A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis. J. Pathol. 135:277-294. Wilk, A.L., King, C.T.G. and Pratt, R.M. 1978. Chlorocyclizine induction of cleft palate in the rat: Degradation of palatal glycosaminoglycans. Teratology 18:199-210. Windholz, M. 1976. The merck index, 9th ed., Ed. M. Windholz, Merck and Co., Rahway, N.J., p. 763. Wood, P.J. and Kraus, B.S. 1962. Prenatal development of the human palate. Some histological observations. Arch, oral Biol. 7:137-150. Woods, D.A. and Smith, C.J. 1969. Ultrastructure of the dermal-epidermal junction in experimentally induced tumors and human oral lesions. J. Invest. Dermatol. 52:259-263. Woods, D.A. and Smith, C.J. 1970. Ultrastructure and development of epithelial cell pseudopodia in chemically induced premalignant lesions of the hamster cheek pouch. Exp. Mol. Pathol. 12:160-174. Wragg, L., Diewert, V. and Klein, M. 1972. Spatial relations in the oral cavity and the mechanisms of secondary palate closure in the rat. Arch, oral Biol. 17:683-690. Wyllie, A.H. 1981. Cell death: A new classification separating apoptosis from necrosis. In: Cell death in biology and pathology. Ed. I.D. Bowen and R.A. Lockshin. Chapman and Hall, London, New York. Yamanishi, Y., Dabbous, M.K. and Hashimoto, K. 1972. Effect of col 1agenolytic activity in basal cell epithelioma of the skin on reconstituted collagen and physical properties and kinetics of the crude enzyme. Cancer Res. 32:2551-2555. - 127 -Yasuzumi, G., Aoyama, N. and Yabumoto, N. 1983. Ultrastructural changes of basal lamina and protoplasmic astrocytes in craniostenosis with epilepsy. J. Submicrosc. Cytol. 15:583-592. Zar, J.H. 1974. Biostatistical analysis. Prentice-Hall Inc. Englewood Cliffs, N.J. Zeiler, K.D., Weinstein, S. and Gibson, R.D. 1964. A study of the morphology and the time of closure of the palate in the albino rat. Arch, oral Biol. 9:545-554. Zelena, J., Smetana, K. and Jirmanova, I. 1978. Abnormalities of the nuclear envelope in porcine muscle affected with congenital myofibrillar hypoplasia. Virchows. Arch. B. Cell Path. 28:157-165. Zimmerman, E.F. 1979. Palate morphogenesis: Role of contractile proteins and neurotransmitters. In: Advances in the study of birth defects. Vol. 3., Ed. T.V.N. Persaud, MTP Press Limited, Lancaster, pp. 143-159. Zimmerman, E.F., Andrew, F. and Kalter, H. 1970. Glucocorticoid inhibition of RNA synthesis responsible for cleft palate in mice: A model. Proc. Nat. Acad. Sc. (USA) 67:7/9-785. Zimmerman, E.F., Wee, E.L., Clark, R.H. and Venkatasubramanian, K. 1980. Neurotransmitters and teratogen involvement in cell mediated palatal elevation. In: Current Research Trends in Craniofacial Developments. Ed. R.M. Pratt, and R.L. Christiansen, Elsevier North Holland, Amsterdam, pp. 187-202. Zimmermann, T.P., Chu, L.C., Bugge, C.J.L., Nelson, D.J., Lyon, G.M. and Elion, G.B. 1974. Identification of 6-methylmercaptopurine ribonucleoside 5'-diphosphate and 5'-triphosphate as metabolites of 6-mercaptopurine in man. Cancer Res. 34:221-224. Zunin, C. and Borrone, L. 1955. The teratogenic effect of 6-mercaptopurine (Ital.). Minerva Pediatr. 7:66-71. - 128 -APPENDIX 1 Preparation of solutions for Acid Phosphatase Cytochemistry (Barka and Anderson, 1962). Formol-Calcium: Fresh formol-calcium was prepared by dissolving one gram anhydrous calcium chloride (Allied Chemical Canada Ltd., catalog* 1502, Lot# X070) in 60 ml of distilled water. To this solution, 10 ml of 40% formaldehyde was added, and adjusted to pH 7.1. The volume was then brought to 100 ml with distilled water. Pararosanll1n: With gentle warming, one gram of pararosanilin hydrochloride (J.T. Baker Chemical Co., Phil 1ipsburg, N.J., catalog # 2903, Lot # 508512) was dissolved in 20 ml of distilled water and 5 ml concentrated hydrochloric acid. After the solution cooled, it was filtered and stored at room temperature for future use. 4% Sodium Nitrite: A fresh solution of sodium nitrite (Fisher Scientific Co., Fair Lawn, N.J., catalog # S-347, Lot # 781768) was prepared by the addition of 4 g sodium nitrite to 100 ml of water. Michael Is Veronal Acetate Buffer: 5.85 g anhydrous sodium acetate (Fisher Scientific Co., Fair Lawn, N.J., catalog # S-210, Lot# 774397) was added to 14.714 g Barbital Sodium (BDH Chemicals Canada Ltd., Vancouver). They were then dissolved in 500 ml of distilled water. - 129 -Naphthol AS-TR Solution: 100 mg naphthol AS-TR phosphate (Nutritional Biochemlcals Co., Cleveland, Ohio, catalog # 4308) was dissolved in 10 ml N, N-dimethyl formamide (Fisher Scientific Co., Fair Lawn, N.J., catalog* D-131, Lot # 786597) and then quickly chilled in the freezer to prevent the formation of a precipitate. The solution was freshly prepared for each incubation. Methyl Green Stain: 1 g of methyl green (Fisher Scientific Co., Fair Lawn, N.J., catalog # M-295, Lot # 773099) was dissolved in 100 ml of veronal acetate buffer, and adjusted to pH 4.0. Incubation Solution: The incubation solution was prepared in two parts. In the first part, 30 ml of Michael is veronal acetate buffer was mixed with 72 ml of distilled water and 6 ml of the substrate naphthol AS-TR solution. In the second part 4.8 ml of pararosanilin solution was diazotized by mixing with 4.8 ml of 4% sodium nitrite solution. The two parts were mixed and adjusted to pH 5.0 with 0.5 N NaOH. The solution was then filtered and used as the incubation medium. . 

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 7 0
China 4 0
Sudan 2 0
France 1 0
Japan 1 0
City Views Downloads
Unknown 6 0
Beijing 4 0
Ashburn 2 0
Mountain View 2 0
Tokyo 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096007/manifest

Comment

Related Items