THE UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE HISTOLOGY AND HISTOCHEMISTRY OF THE EMBRYONIC CHICK MANDIBLE FOLLOWING X-IRRADIATION A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY BY BARBARA ANN BAILEY Norman, Oklahoma 1970 HISTOLOGY AND HISTOCHEMISTRY OF THE EMBRYONIC CHICK MANDIBLE FOLLOWING X-IRRADIATION APPROVE DISSERTATION COMMITTEE ACKNOWLEDGEMENTS To Richard A. Goff, Professor of Zoology, for the suggestion of and direction of this problem; for the use of his research laboratory and equipment; for his trust, patience, time and encouragement; and for his invaluable assistance in the writing of this dissertation. To my Committee for their time spent reading and constructively criticising this dissertation. To Sister Barbara Hansen for her unending help and encouragement received during this study. To Mr. and Mrs. John R. Wallace and my Parents for their faith, trust and support during this study. To the many friends who have assisted me in various ways . To the Department of Zoology for a Graduate Teaching Assistantship and some of the materials used in this research. Ill TABLE OF CONTENTS Page LIST OF TABLES......................................... vi LIST OF FIGURES...................................... vii LIST OF PLATES......................................... ix Chapter I. INTRODUCTION................................. 1 Literature Review Aspects of the Problem II. MATERIALS AND METHODS X-irradiation Factors Gross Study Histology Histochemistry III. RESULTS...................................... 23 Radiation Effects— Gross Embryonic Deaths Surviving Embryos— General Effects Surviving Embryos— Anomaly Characteristics Histological Observations Normal Growth Patterns Normal Cell Death Patterns Effects of Radiation in the Sensitive Period— General in the Mandible Effects of Radiation in the Sensitive Period— Cell Death Patterns Effects of Radiation in the Sensitive Period— Growth Patterns, 1 - 2 Days Post Effects of Radiation in the Sensitive Period— Growth Patterns, 3 — ^ Days Post XV Page Histochemical Observations Cell Death Locations Normal Development Prior to the Sensitive Period, (Stages l8 to 26, 3-4 Days) Specific Structures in Control and Experimental Mandibles (Stages 27 to 35, 5-9 Days) IV. DISCUSSION................................... 100 Sensitive Period Anomalous Mandibular Development Normal Cell Death Normal Histogenesis Comparison with Congenital Anomalies General Comments V. SUMMARY AND CONCLUSIONS..................... II8 LITERATURE CITED..................................... 120 APPENDIX.............................................. 125 LIST OF TABLES Table Page 1. Acute and Delayed Deaths...................... 24 2. Gross Study— General Information............. 26 3. Maxilla and Mandibular Reduction............. 28 4. Maxilla/Mandible Length Ratios............... 30 5. Cartilage Measurements........................ 37 6. Histochemical Activity Prior to the Sensitive Period........................... 68 7. Histochemical Activity 1— Enzymatic......... 71 8. Histochemical Activity 11— Carbohydrate Tests........................................ 73 9. Histochemical Activity— Bone Development.... 82 10. Histochemical Activity— Supra-Angular Bone Development........................... 83 V I LIST OF FIGURES Figure Page 1. Typical location of a mandibular blister in a stage 35 (9 day) embryo.............. 2? 2. Reduction in mandibular length............... 29 3. Reduction in maxilla length.................. 29 4. Comparison of maxilla to mandibular lengths at time of sacrifice (623r)............... 31 5. Comparison of maxilla to mandibular lengths at time of sacrifice (700r)............... 31 6. Comparison of maxilla to mandibular lengths at time of sacrifice (800r and 750r)..... 31 7. Cartilage malformations occurring in embryos sacrificed after 9 or 10 days of incubation................ 33 8 . The contour of Meckel's cartilage in a 9-10 day embryo............................ 39 9. Proximal-distal cartilage ratios for mandibles x-irradiated at stages 24, 26+ and 27-............................... 40 10. The mandible at stage l8 (3 days)........... 4l 11. The plane in which serial sections of the histological study were cut............... 4l 12. Mandible regions are marked for orienta­ tion........................................ 4l 13. Mandible at sensitive period (stage 27, 5 days)..................................... 43 V l l Figure Page l4. "Spoon" shape of a stage 33 (8 day) man­ dible ...................................... . 4 3 15- Cartilage and "muscle" relationships in a stage 33 ( 8 day) mandible................ 43 l6. Bone formation in a stage 35 mandible 55 17- Mandible at stage 18 (3 days)................ 56 18. Stage 27 (5 days) embryonic mandible........ 56 19* Mandible at stage 30 (7 days)................ 58 20. Cell death pattern II......................... 6l 21. Cell death pattern III........................ 6l 22. Diagram of severe mandibular reduction, with a projected normal contour........... 66 23. Mandible at stage I8 (3 days)................. 69 24. Mandible at stage I8 ( 3 days)................. 69 25. The plane of serial sectioning for the histochemical study......................... 69 26. Diagrams of locations of mandibular primordia.................................... ~0 27. Stage 27 mandible ( 5 days).................... 78 28. Stage 31 mandible ( 7 days).................... 78 29. Stage 35 mandible ( 9 days).................... 78 30. Stage 27 mandible ( 5 days).................... 80 31. Stage 33 mandible ( 7 1/2 - 8 days)........... 80 32. Stage 35 mandible ( 9 days).................... 80 V I 1 1 LIST OF PLATES Plate Page I. Gross Study.................................. 35 II. Histological Study (A) Hematoxylin-Eosin... 45 III. Histological Study (B ) Hematoxylin-Eosin. . 47 IV. Histological Study (C) Hematoxylin-Eosin.. 49 V. Histological Study (D) Hematoxylin-Eosin.. 52 VI. Histological Study (E ) Hematoxylin-Eosin.. 64 VII. Acid and Alkaline Phosphatase Activity (A ) 86 VIII. Acid and Alkaline Phosphatase Activity (B) 88 IX. RNA, ATP and AMP Dephosphohydrolase Activity (A ).............................. 90 X. ATP and AMP Dephosphohydrolase Activity (B) 92 XI. RNA, ATP and AMP Dephosphohydrolase Activity (C)............................... 94 XII. Carbohydrate Activity.... 96 XIII. Carbohydrate Activity (B) 98 IX HISTOLOGY AND HISTOCHEMISTRY OF THE EMBRYONIC CHICK MANDIBLE FOLLOWING X-IRRADIATION CHAPTER I INTRODUCTION In the development of the chicken beak, the mandible in certain circumstances, may be differentially arrested or deformed. Such a unique localized anomaly appears to be an appropriate subject for the study of morphogenetic mechanisms. Previous workers described genetic mutants (Lamoreux, 1942; Marble, Harper & Hammers, 1944 and McGibbon, 1946) with retarded mandibular development. In addition numerous chemi­ cal teratogens cause reduced mandibles (Landauer, 1952, 1953î Steamer, Sanderson, Christian & Tyler, I96O and Kury & Craig, 1967). X-irradiation also causes anomalous mandibular development (Goff, unpublished data and Steamer, Sanderson, Christian & Tyler, I96O) in chicken embryos. Previous work dealing with anomalous mandibular development of chicken embryos consisted only of observations that malformations occurred. Developmental stages of chicken embryos are well defined and chicken embryos are easily main­ tained in relatively simple laboratory facilities. Thus, research was initiated to analyze anomalous mandibular development using techniques at the gross level and histological-histochemical procedures. Literature Review^ Congenital Mandibular Anomalies The lower beak consisted of a mere vestige in an anomaly described by Marble, Harper & Hammers (1944). McGibbon (1946) described mandibles seldom more than half the normal length. In extreme cases, the cartilage was bent up or down, almost at right angles to the normal plane. Both mutants were autosomal recessive alleles with the former being lethal in the homozygous condition and the latter semi-lethal. Chondrodystrophy resulting in an extreme shortening of the lower beak and overgrowth of the upper ("parrot beak") was caused by a homozygous autosomal recessive allele (Lamoreux, 1942). This abnormality was accompanied by shortening of the long bones of the chick. Chemically Caused Mandibular Anomalies Landauer (1952) induced abnormalities of the beak with boric acid. The most frequent form was a combination of shortening of the lower beak, cleft palate and bilateral facial coloboma. Each deformity varied in its extent and ^Unless otherwise stated all citations refer to the chicken embryo. 3 independent occurrence. Reduced mandibles resulted mainly when treatment occurred between the 84th and 120th hours o( incubation. Addition of riboflavin and nicotinamide reduced the teratogenic effects, with riboflavin having the greatest effect. It was postulated that the effects of boric acid may be complex formation with riboflavin linked enzymes. Injection of 3 mg of pilocarpine (Landauer, 1953) into 95 hr embryos resulted in "parrot beak" and upper beak reduction. Treatment with 6-12 mg caused mainly short lower beaks, suggesting a sensitive period for the mandible. Nicotinamide provided partial reduction in the severity of the anomaly. Such reduction indicated that boric acid and pilocarpine may alter the same metabolic pathways. Azaserine (0—diazoacetyl—L—serine) injected into the yolk sac of 4 day embryos caused shortened lower beaks, "parrot beaks" and facial coloboma with cleft palate (Dagg & Karnofsky, 1955)« Severely shortened mandibles occurred with the least and most severe upper beak defects. Nico­ tinamide and riboflavin addition did not reduce anomaly formation. It was suggested that azaserine affects a dif­ ferent or later site in the same pathway affected by boric acid and pilocarpine. "Parrot beaking" with shortened lower jaws was a common effect of eserine sulphate injection between the 2nd and 6th day of incubation (Agarwal, 1956). Maximum sensi­ tivity resulted when the injection occurred on the 4th day. k Edema was often associated with abnormal bending of the lower jaw cartilage and was suggested as a cause of the curvature. Limb skeleton abnormalities also occurred, suggesting a metabolic relationship of facial and limb parts, at least during the sensitive period. It was also suggested that the formation of cartilaginous primordia was altered. Mitomycin C injection into the yolk sac caused reduced lower jaws when treatment occurred during the 3rd and 4th days of incubation (Kury & Craig, 1967). It is generally considered that mitomycin C inhibits DNA synthesis in intact cells with little initial effect on RNA or protein synthesis. Radiation Caused Mandibular Anomalies Most studies using ionizing radiation deal with major abnormalities and their correlation with the stage of irradiation (Clapp, 1964; Goff, 1959; Karnofsky, Patterson & Ridgeway, 1950; and Steamer, Sanderson, Christian & Tyler, i960). Irradiation between the 2nd and 5th days of incuba­ tion resulted in localized accumulations of fluid (Steamer, Sanderson, Christian & Tyler, I96O). Production of fluid filled sacs beneath the mandible, which was often reduced, was a frequent result. A common response of chicken embryos x-irradiated at 5 days of incubation was hemorrhage in the mandible (Goff, 1959). It was also noted (Goff, unpublished data) that mandibular reduction resulted. Cell Death Cell death in certain locations is a normal event in embryonic development (Bellairs, I96I; Jacobson & Fell, 1941; Glucksmann, 1951; Saunders & Gasseling, 1962 and Whitten, I968). Mention was made of the occurrence of necrosis in the mid-line of mandibular arches during the period of union of bilateral anlagen (Jacobson & Fell, 194l). Necrotic cells were also noted in pre—osteogenic centers. Two developmental analysés combining the study of cell death patterns with histochemistry showed that cell death can be responsible for anomaly production (Hinchliffe & Ede, 1967 and Milaire, I962). In the study of limb development in the polydactylous talpid^ mutant of the fowl (Hinchliffe & Ede, I967), normally occurring cell death, accompanied by the presence of acid phosphatase, was absent from the superficial mesenchyme of the limbs. It was postulated that absence of cell death was responsible for the abnormal sculpturing of the limbs and excessive elongation of the apical ectodermal ridge. Failure of the occurrence of normal necrosis was considered the causative factor in anomaly production. Oligosyndactylism, a dominant congenital malformation in the mouse, occurs because of fusion between the 2nd and 3rd digits. Gruneberg (I96I) suggested that a reduction in mesoderm in the preaxial foot plate was responsible. 6 Excessive necrosis, with acid phosphatase, was in the pre­ axial mesoderm of 10 1/2 day limbs (Milaire, 1962) and continued to be present in the limbs of much older embryos. Milaire concluded that massive regional degenerations resulted in reduction of mesoderm during early foot forma­ tion. Histochemistry There is little histochemistry of even normal embry­ onic mandibles. The histochemistry of the adventitious cartilage of membrane bones of chick embryos was studied (Hall, 1968). Observations involved the primary cartilage of the mandible only in its relationship to adventitious cartilage. Additional histochemical work with the embryonic beak was primarily concerned with the process of Keratiniza- tion (Kingsbury, Allen & Rotherain, 1953 and Yasui & Hayashi, 1968). Aspects of the Problem Some gross similarities appear in mandibular anoma­ lies as a result of treatment by teratogens. Effects do not indicate identical responses, but illustrate that the mandible is most sensitive during the 4th and 5th days of incubation and that the cartilage is often abnormally curved. Radiation, as well as other teratogens, causes unlike effects when applied at different embryonic stages. X-irradiation was selected as the teratogen for this study 7 because its period of application occurs only during radia­ tion intervals, with possible secondary effects ensuing. The precise time when chemical teratogens reach their destination and cause effects cannot be accurately deter­ mined. Timing is critical in elucidating factors of development. Killing of highly proliferative undifferentiated cells has been established as an early response to radiation. Removal of dead cells, followed by proliferation of remaining cells occurs after this initial damage (Hicks, 1952; Hicks, 195^; and Skalko, I965). Analysis of cell death localiza­ tions before and after x— irradiation enabled determination of primordia most sensitive to radiation, therefore, aiding in resolving causes and evaluation of anomalous formation. The contribution of Jacobson & Fell (1941) on the developmental potencies of the primitive mesenchyme of the mandible provide an important background for this problem. They determined the location of the cartilage, muscle and membrane bone primordia of the embryonic chick mandible by culturing in vitro sections of 3 through 7 day mandibles. They found: (l) Myogenic cells do not originate in the mandibular arches, but migrate laterally from head mesen­ chyme between the 40th and 60th hr of development and are determined at 3 days. (2) Presumptive cartilage cells are determined at 4 days and originate within the mandibular arches in the proximal mesenchyme and migrate distally as 8 the muscle primordia move to the mid-line of the mandible. (3) There are two sites of membrane bone primordia, both determined at 4 days; the supra—angular, angular, and opercular bone primordia originate in the proximal mesen­ chyme and migrate distally; the dentary bone primordium originates in the distal-lateral mesenchyme near its site of initial formation. Basic mandibular primordia are thus determined before the ^th day, i.e., the suggested sensitive period resulting from x-irradiation. Biochemical events such as the chondrification of Meckel's cartilage begin near the end of the 5th day. Histo­ chemical methods were selected to investigate alterations in biochemical events. By visualizing precise localizations of chemical constituents in developing tissues and detecting some of their enzymatic properties, it was possible to identify some dynamic aspects of normal and abnormal morpho­ genesis. This developmental analysis reports the x-irradiation sensitive period of the mandible. Abnormal cartilage con­ tours were determined and correlated with the range of the sensitive period and the various doses applied. Normal and x-irradiation induced necrotic patterns were recorded. Alterations in developing connective tissue, cartilage, bone and musculature were related to the various forms of the anomaly. CHAPTER II MATERIALS AND METHODS White Leghorn eggs for gross and histological studies and some of the histochemical control studies were obtained from the Capitol Hill Hatchery, Oklahoma City, Oklahoma. Eggs for histochemical experiments were obtained from Parkin's Hatchery in Shawnee, Oklahoma and were from a White Leghorn X California Gray stock and from pure White Leghorns. No differences in the x-irradiation effect were detected in the two strains. Storage periods and incubation conditions are summarized on Table A (Appendix). A forced-draft incubator was used for all experiments. Daily records of temperature and humidity were kept. Eggs were turned three times daily. X-Irradiation Factors Some infertility and inviability of eggs prior to utilization occurred (Table B, Appendix). Eggs were generally candled the day of irradiation to eliminate infertile or inviable eggs. Occasionally eggs were candled the day before x-irradiation. During radiation intervals, the eggs were placed in circular boxes with masonite bottoms and cardboard sides. 9 10 The sides were anchored to the bottom by plumbers tape. The boxes were 22.86 cm in diameter and 6.98 cm in height. Foam rubber lined the bottom of the boxes and was molded around a core of plastic foam. Eggs were cushioned with blunt ends toward the periphery of the box. Each box held twelve or thirteen eggs. Eggs were held in position by a Incite cover that rested lightly on their exposed surfaces. A hole in the center of the cover fit over a wooden peg mounted in the center of the box. The cover was held in place by a clamp that grasped the peg. The foam rubber lining made allowance for any size variations so that all eggs were equidistant from the target. Boxes were preheated prior to placing eggs in them. For x-irradiation the boxes were placed on a turn­ table within an incubator. The incubator had bottom and sides of masonite and top of 8 mm thick Incite. As the eggs were irradiated, they were rotated at l6 rpm. Incubation temperature was maintained, but humidity was not controlled. The incubator was placed on a table and centered beneath the head of the x-ray machine. X-irradiation was by a Westinghouse Medium Therapy Unit, with a Machlett CT-2 50 tube and with inherent filtra­ tion of 2 mm of Al. Maximum rating was 250 KV and 15 MA. The machine was operated with added filtration of 1/2 mm Cu and 1 mm Al. Some irradiations were 200 KV and others 250 KV, with no differences detected in the response of embryos. 11 In order to maintain a constant exposure dose per min, the head to target distance was 52. 39 cm at 250 KV and 35-̂ ^̂ cm at 200 KV. Exposure rates ranged from y8r to 86r/min. Dose was determined with a Victoreen ionization chamber. The chamber was placed in a box identical to those in which the eggs were rotated, except that a special plastic foam platform supported the chamber in a position correspond­ ing to the surface of an egg. Three 1 min readings were taken with the chamber, prior to egg irradiation, and averaged. Readings were corrected for barometric pressure and the characteristics of the ionization chamber. At the end of each experiment, three 1 min readings were repeated. The variation in the before and after readings was never greater than 2 or 3 r . The initial value was taken as the dose rate. The incubator was located within 3 yards of the x-ray room. The eggs were unincubated during the short period they were carried to the chamber and back. Controls were treated just as the experimental embryos, with the exception that the X —ray machine was not in operation while eggs were rotated. In the gross and the histochemical studies, the number of control eggs was 1/4 to 1/3 the number of expéri­ mentais used. Equal numbers of expérimentais and controls were used in the histological study. X-irradiation condi­ tions and the stages for x-irradiation and sacrifice are summarized in Tables A and B (Appendix). Following 12 x-irradiation, eggs were returned to the incubator and not removed from the boxes or turned for a period of at least 12 hr after x-irradiation. Gross Study Twenty to twenty— four hr following x-irradiation all eggs were candled to determine acute deaths, i.e., those embryos dying within 24 hr after x-irradiation. These embryos were examined to determine any similarities in their response to x-irradiation. Dead embryos were fixed in neutral buffered 10% formalin (Lillie, 1948). Those embryos undergoing delayed deaths, i.e., those surviving 3 or 4 days after x-irradiation, but dying before the 9th or 10th day of incubation, were also fixed and stored for closer examination. Embryos were consi­ dered alive if blood was circulating in the major vitelline vessels. General observations were made at the time of sacrifice. Evidence of hemorrhage, beak deformation and other abnormalities were noted. Control embryos were staged according to Hamburger & Hamilton (1951). Criteria for staging included: beak and third toe length; presence of protuberance on the posterior side of digit 2 of the wing; presence of labial grooves; number of rows of feather germs dorsal to the eyelid and on the breast; number of scleral papillae; and the position of the nictitating membrane. A stage was assigned for each of these developmental features and an average stage was deter­ mined for each embryo. Since the experimental embryos 13 exhibited a more varied response, they could only be approxi­ mately staged. Measurements of the upper beak length were made from the interior corner of the nostril to the beak tip. The third toe was measured from its tip to the middle of its metatarsal joint. In addition to the standard measurements for staging, each embryo was weighed and the length of the lower jaw was measured. Each formalin fixed embryo was blotted with paper towels prior to weighing. The lower jaw was measured from the corner of the mouth to the tip of the mandible. A number of embryos in each experiment were set aside for cartilage staining. The head was removed and then stained in Van Wijhts solution for cartilage (Gray, 1952). Embryos were then dehydrated with ethylene glycol monoethyl ether (Cellosolve) and cleared in dimethoxyethyl phthalate (Nelsen, 19^5)» All cartilage measurements were made using a calibrated ocular micrometer at a magnifica­ tion of lOx to 20x. Meckel's cartilage was measured from the distal tip to the articular process. When the cartilage was bent or doubled back on itself, each segment was measured and then summed. Histology Five-day, or approximately stage 27, embryos were irradiated at doses of 625r and ?50r in experiments VIII and IX respectively. Control and experimental embryos were l4 sacrificed at the following intervals after x-irradiation: 0, 2, 4, 8, 12, 24, 48, 72, and 96 hr. These embryos were fixed in Bouin's solution for 24 hr and then transferred to 70% ethanol for storage. Each embryo was staged and examined for any gross abnormalities. Mandible reductions could be detected in embryos as early as stage 32. Embryos were then prepared for histological examina­ tion. Generally at least two controls and four experimental embryos were selected from each period of sacrifice. Samples from each group were selected so that all types of mandible malformations could be analyzed more closely. Such a selec­ tion of mandibular abnormalities could only be done with any degree of assurance when embryos were stage 32 and older. Mandibular blisters could be detected as early as stage 28, so several embryos exhibiting this response were included in each group. Each embryonic head was removed and trimmed to remove all but a moderate amount of tissue above and below the beak. Heads were then dehydrated and embedded in paraffin.^ One series of heads was mounted so that long sections of the mandible could be made, while a second series was mounted for cutting sagittal sections. Ten micron sections were cut and mounted on slides with albumin in glycerin. Sections were then stained in Harris' hematoxylin and an aqueous solution of eosin and then dehydrated and cleared. Cellosolve ^Peel-A—Way, Lipshaw Manufacturing Company. 15 was used for dehydration and toluene for clearing purposes. This series of embryos was studied for locations, shapes and relationships of the early cartilage, muscle and bone primordia. Sites of cellular deaths were also noted. Criteria for describing necrosis were those of Glucksmann (1951). Histochemistry This study consisted of four experiments (X through XIII), in which groups of embryos at the critical period (stage 27) were x-irradiated and sacrificed serially as in the histological experiments. Embryos were frozen as fresh tissues and not fixed directly. Each embryo was staged, trimmed as for histological preparation, but then placed in a paper ring filled with OCT. 2 The embryos rested upon an object disc on the stage of a CO^ Quick Freeze Chamber 3 used to freeze the tissue rapidly with a minimum of distor­ tion. The embryo, frozen in a cube of OCT was then either sectioned or removed for storage or re—mounted on an object disc if reorientation was required. It was not always possible to process frozen embryos immediately. When this was the case, frozen embryos were wrapped individually in aluminum foil and stored in a deep freeze at — 20°C. 2Tissue-Tek-Mounting Compound, Ames Company, Elkhart, Indiana. 3American Optical Company. 16 In addition to these four experiments, 8l normal embryos were used to perfect the histochemical reactions prior to the x-irradiation experiments. These embryos pro­ vided controls additional to those used in the actual experi­ ments. Embryos of stages l8 through 26 were included in these reactions in order to determine enzymatic localiza­ tions up to the sensitive period. Mandibles were sectioned longitudinally with an American Optical Cryostat, model #84$. Mandibles were sectioned at: stage l8 through 28 - 15 microns; stage 29 through 33 - 20 microns and stages jk, 35 - 25 microns. Cutting temperatures were between -10 and -20°C. As each section was cut it was picked up by lightly touching a warm cover slip to its surface. Cover slips were pre—warmed by placement on a grooved aluminum plate that rested on a warming plate. The aluminum plate averaged 37°C. As sections were cut they were placed on the warming plate. In most cases all sections of one mandible remained on the warming plate until the entire mandible was cut. Mandibles in experiment X were analyzed for non specific acid and alkaline phosphatase. Those in experiment XI were analyzed for activity of AMP phosphohydrolase, ATP phosphohydrolase, ribonucleic acid and deoxyribonucleic acid. Acid mucopolysaccharides were visualized in experiments XII and Xlll and the presence of chondroitin sulfate and kera— tosulfate was checked. 17 The method of Mulnard (1955) was used for the alka­ line phosphatase reactions. Molarity of the substrate, cal­ cium B glycerophosphate (disodium salt, pentahydrate, 0.1% max. L a isomer, Sigma Co.) was 0.019. Extremes of pH were from 8.9 to 9.13 at room temperature. Incubation time varied with age of the embryos. A 2 to 3 hr interval was most suited for embryos at stages l8 through 29, while a 1 to 2 hr interval was optimum for embryos at stages 30 through 35. Incubation temperature was 37°C. Control sections were treated under the same conditions with the exception that substrate was not added to the medium. All sections, post­ fixed in 10% neutral formalin + 1% CaCl^ (formal calcium), were dehydrated in cellosolve and cleared in toluene before mounting in permount. Gomori's (1941) method for acid phosphatase was utilized. Sodium glycerophosphate, the substrate, (disodium salt, 75% isomer. Sigma Co.) was used at a molarity of 0.014. Optimum incubation time intervals were always shorter than those of alkaline phosphatase. With younger embryos the intervals varied from 1 to 2 hr while the oldest embryos required only 30 to 45 min. The substrate was not added to the control medium. pH values ranged from 4.96 to 5.06 at room temperature. Incubation temperature was 37°C. Sec­ tions, post—fixed in formal calcium, were mounted on slides with water soluble glycerin jelly. 18 Adenosine 5' monphosphoric acid (AMP) (Type II, isolated from yeast, 99-100%, Sigma Co.) and adenosine 5' triphosphoric acid (ATP) (from equine muscle. Sigma Co.) were used at molarities of 0.001 and 0.008 respectively in the nucleotide tests. The procedure of Wachstein & Meisel (1957) was used for the localization of these two phospha­ tases. Values for the pH of the incubation medium for both phosphatases were 7.00-7.1 at room temperature. Incubation time intervals ranged from 45 min to 2 hr for ATP and from 1 hr to 3 hr for AMP, with the younger embryos receiving the longer interval. AMP and ATP were not added to the incubation medium of the control sections. The incubation temperature was 37°C in all cases. Sections of both nucleo­ tide reactions were mounted on slides with water soluble glycerin jelly. All embryos of the phosphatase reactions were post fixed in formal-calcium prior to dehydration and or mounting. DNA and RNA localization was obtained by using the Methyl-Green Pyronin method of Kurnick (1955). Kurnick recommends the use of pyronin g; however after testing the staining clearness of pyronin y versus pyronin g, pyronin y (Allied Chemicals) appeared to be the better stain and was used exclusively in experiment XI (Methyl green, Hartman- Leddon Co.). All sections were prefixed in formal— calcium for 5 min and then rinsed for 5 min in Chick Ringers'. Con­ trol sections were incubated for JO min at 37°C in a solution 19 of ribonuclease (Type I-A from Bovine pancreas, Sigma Co.) in glass distilled water (1.0 mg/10 ml). Alternate sections were incubated in glass distilled water. The pH of the solutions ranged from 5.4 to 5.7 at room temperature. No buffer was used in the incubation medium since these sol­ vents may extract proteins and RNA (Opie & Lavin, 1946), thus interfering with the evaluation of the digestion. After the incubation period, sections were rinsed several times in distilled water and then immersed in the staining mixture for 30 min. Sections were rinsed again in distilled water, immersed in two changes of n-butyl alcohol (5 min each), followed by immersion in two changes of tolu­ ene (5 min each). The dehydrated sections were then mounted on slides with permount. Chromatin appeared blue and cyto­ plasmic RNA pinkish red. The periodic acid-Schiff (PAS) reaction (Mowry, 1963) was used for the localization of complex carbohydrates. Presence of glycogen was attempted by using diastase (Type V A, from Malt, Sigma Co.) in phosphate buffer (pH - 6) on alternate sections (Barka & Anderson, 1963). Sections were always prefixed in neutral buffered formalin (5 min) and then rinsed in Chick Ringers' (5 min). Alternate sections were then placed in phosphate buffer and phosphate buffer plus diastase and were incubated for a 30 min period at 37°C. Sections were then rinsed in distilled water. In order to remove any aldehyde groups added by the fixative. 20 an aldehyde blockade was carried out next by placing the sections in a solution of 2,4-dinitrophenylhydrazine (East­ man Organic Chemical) for 10 min. This was followed by a distilled water rinse (5-10 min), and then immersion in a 5% solution of periodic acid for 5 min. Sections were then placed in Schiffs' reagent for 30 min followed by 3 rinses (2 min each) in a sodium bisulfite solution. Sections were then washed and mounted on slides with glycerin jelly. Several attempts were made to perfect the utiliza­ tion of diastase in order to localize glycogen. The results are in question and therefore, reference was made only to PAS positive structures. Chondroitin sulfate and keratosulphate presence was checked by using 0.1% alcian blue (alcian blue 8GN — Mathe- son, Coleman and Bell) in acetate buffer (pH - 5*6) contain­ ing .4 M MgCl^ and .9 M MgCl^ respectively (Scott, Dorling & Quintarelli, 1964). This procedure was carried out at room temperature (24°C). Hyaluronidase (.1 mg/ ml) (Type I, Bovine Testis, Sigma Co.) in O.IM phosphate buffer (pH - 6) was used to verify the presence of chondroitin sulfate (Barka & Anderson, 1963). All sections were pre-fixed in neutral buffered formalin and rinsed in Chick Ringers' prior to any staining or incubation. Alternate sections were placed in buffer alone. The incubation time (37°C) was 30 to 60 min depending upon the age of the embryo tested. Sections were then rinsed 21 several times in distilled water and then placed in a 0.1% solution of toluidine blue for 10 to 30 men. Toluidine blue O powder (Coleman and Bell Co.) had been previously treated to improve its staining quality (Robinson & Bacsich, 1958). Sections were then rinsed for 2 min in a solution containing equal quantities of 1% potassium ferrocyanide and 3% ammonium molybdate. This latter rinse, before dehydration, has been shown to improve the retention of the metachromatic reaction (Bensley, 193^). The rinse was followed by dehydration in two changes of n butyl alcohol (5 min each) and then clearing in toluene (two 5 min intervals). Sections were mounted on slides with permount. Only alcohol stable metachromasia was accepted. The presence of sulfated and carboxylated polyanions were determined by the procedure of Lev & Spicer (1964). A 1/0 solution of alcian blue in .IN HCl (pH - l.l4) was used for the presence of sulfated anions and a 1% solution of alcian blue in 3% acetic acid (pH - 2.47) was used for the presence of carboxylated anions. Alternate sections were used in the two tests. Staining was carried out at room temperature (24°C) for 30 min. After staining, those sec­ tions at a pH of l.l4 were blotted dry with filter paper, because an Immediate rinse with distilled water will raise the pH of the tissues enough so that carboxylated groups will also be stained (Lev & Spicer, 1964). Following blot­ ting the sections were dehydrated in 2 changes of n—butyl 2 2 alcohol and cleared in 2 changes of toluene (5 min each). Sections at pH of 2.47 were rinsed immediately in distilled water and dehydrated and cleared. Cover slips were mounted on slides with permount. Slides were examined with an American Optical Micro­ star Microscope and a Photomax (Olympus, Tokyo, Japan). Photomicrographs were taken with the Photomax. Kodak Plus X film (35 mm) was used in photographing the histological series, while Kodak High Contrast Copy film (35 mm) was used to photograph the histochemical reactions. A stage micrometer was photographed so that exact magnifications could be determined. CHAPTER III RESULTS Radiation Effects--Gross Embryonic Deaths Embryos suffering acute death following irradiation (Table l) were generally characterized by severe hemorrhages, particularly in the brain and limbs. Blood of the vitel­ line system often collected in the sinus terminalis. Blood collection in the sinus terminalis was a common occurrence in early embryonic death. Embryos undergoing delayed death (Table l) were generally eviscerated and had severe body hemorrhages. Hemorrhages were common throughout the body, with the mesencephalon, cornea, back and thighs being heavily effected areas. The yolk stalk connection near the body was generally constricted with stagnant blood on both sides of the constriction. There was also tendency for blood to collect in the sinus terminalis. Very pronounced mandibular reduction was generally apparent in these embryos, along with digital and limb reduction. 23 TABLE 1 ACUTE AND DELAYED DEATHS Exp. Dose Ave . Stage No. No. No. No. No. No. (r) X-Rayed Acute Delayed Sur­ Wi th X-Rayed Deaths Deaths viving Blisters la 800 26 + 36 21 3 12 1 Ib 800 25 33 Ic 25 1 7 1 800 24 33 8 1 24 0 Ila 700 29- 43 22 7 l4Ilb 750 28 42 5 10 7 lie 750 27 4l 27 3 17 6 18 8 to Ilia 550 27- 46 0 5 4l 0Illb 625 27- 46 0 4 42 IIIc 13750 27 24 23 1 0 0 I Va 700 27- 44 29 5 10 2 IVb 665 27- 46 21 20 IVc 625 5 727- 45 4 2 39 10 Va 625 29 + 36 9 6 21 6 VI 625 24 24 0 3 21 0 Vila 650 24 11 1 0 0 Vllb 700 24 11 2 9 0 9 0 25 Surviving Embryos— General Effects Surviving embryos (Table 2) sacrificed 4 or 5 days following X - irradiation usually had weaker extraembryonic circulations than control embryos. Embryos surviving doses between 750r and 800r usually had severe brain hemorrhages, as well as hemor­ rhages on the thighs, wings, cornea and mandibular regions. Hemorrhages occurred in embryos receiving lower doses, but with decreasing frequency, with the mesencephalon remaining the most susceptible location. Embryoni c blisters. A number of x-irradiated embryos had either clear blisters or hematomas beneath their mandibles (Table 1, Fig. l). There was no correla­ tion between severity of a lower jaw anomaly and presence of blisters. Blisters were often present on embryos that had slight, if any, lower jaw reduction. Of 13 surviving embryos with blisters beneath the mandible (experiment Illb) 7 were below mandibles with slight reductions in length and 6 were below severely reduced mandibles. No blisters were on embryos receiving 550r. Embryos x-irradiated at 4 days of incubation did not develop blisters. All degrees of mandibular reduction were observed in embryos without blisters. TABLE 2 GROSS STUDY--GENERAL INFORMATION Exp . Ave . No. X-Irradiated Controls Stage Staged Dose No. Ave. No. Ave . No. Ave . No. Ave. No. X-Rayed Prior (r) Stage of Weight Stage of Weight t o Sacri- Survivors Exp. f iced (g) Sacri- Survivors f iced (g) la 26 11 800 36 34- 12(33.3 )* 0.96 6 35 6(100.0)* 1.56 Ib 25 10 Boo 33 34 7(21.2 ) 1.10 B 35 7(87.5 ) 1.72 Ic 2k 11 Boo 33 34 24(72.7 ) O.B9 10 35 B(Bo.o) 1.52 Ila 29- II 700 43 34 + 14(12.7) 1.23 12 12(100.0) 1.83lib 28 12 750 42 35 10(23.8 ) 1.42 12 3 356 +- 12(100.0) 1. 42 lie 27 11 750 4i 34 + 18(43.9 ) 1.26 12 35 + 9 (75.0 ) 1.26 Ilia 27- 8 550 46 27- 41(8911) 1.52 12 12(100.0) 2.23 to Illb 8 625 46 35 + 35 +35 + 42(91:3) 1.58 12 36 10(83.3 ) 2.69 as IIIc 27 8 750 24 -- 0(00.0) - 24 35-38 23(95.8 ) - IVa 27- 6 700 44 35 10(22.7 ) 1.46 12 36 10(83.3 ) 2.45 IVb IVc 27- 6 665 46 35 + 20(45.6) 1.64 12 36 + 12(100.0) 2.72 27- 6 625 45 35 + 39(86.6 ) 1.77 12 36 12(100.0) 2.61 Va 29 + 12 625 36 12 35 + 21(58.3 ) 1.80 12 36 io(83.3 ) 2.40 Vb 29 + 235 36 10(83.3 ) 2.24 2. 40 VI 2k 13 625 24 35 + 21(87.5 ) 1.62 11 36 11(100.0) 2.64 Vila 2k 8 650 11 36 9(81.8 ) 10 36 + 10(100.0) Vllb 2k 8 700 11 36 9(81.8 ) — — ) = % of embryos survi Ving. 27 Figure 1. Typical location of a mandibular blister (B) in a stage 35 embryo (9 days). Sensitive per iod. The greatest mandibular reduction occurred at 5 days of incubation (approximately stage 27) and this was consistent for all roentgen doses utilized (Table 3, Figs. 2 and 3). Maxilla reduction was much less at corresponding doses, but there was a trend toward greater maxilla reduction at 6 days of incubation (stage 29 ) . Comparison of maxilla to mandibular lengths as ratios (Table 4) showed that there was very little varia­ tion in control values (.43 to .48) while experimental ratios varied considerably (.39 to 1.24). The greatest mandibular reduction occurred when embryos were x-irradi­ ated at 5 days of incubation (Figs. 4, 5, and 6). Control and experimental data (Table 4) were significantly differ­ ent (P< 0.10). TABLE 3 MANDIBLE AND MAXILLA REDUCTION Ave. Mandibular Le nffth Maxilla Length Dose Stage No. No. Control X-Rayed % Control X-Rayed % X-Rayed Controls X-Rayed ( mm. ) (mm. ) Reduc- (mm. ) (mm.) Reduc- tion tion 800 24 8 24 3.45 1.80 47.8 1.63 1.23 24. 5 800 25 6 7 4.29 1.88 56.2 1.88 1.37 27.1 800 26 + 6 12 3.79 1.48 61.0 1.68 1.1 34.5 750 27 9 17 4. 52 1.67 63.1 2.16 1 . 58 26.9 to00 750 28 12 10 4.67 2.39 48.6 2.23 1.46 34.6 700 24 6 9 7.47 4.11 45.0 3.19 2.91 8.8 700 27- 10 10 5.17 1. 76 66.0 2.23 1. 76 21.1 700 29- 12 14 4. 30 1.98 56.3 1.94 1. 30 33.0 625 24 9 21 5.22 4.02 23.0 2.37 2.09 11.8 625 27- 20 67 5.13 3.12 39.2 2.27 2.07 8.9 625 29 + 10 21 5.06 4.21 16.8 2.4l 1.63 32.4 29 60 OZ —— • 785000rr P 40 a3 u - 2S?f Ç0 20 5 I o 4 5 6 INCUBATION AGE IN DAYS Zo 40 T 3u PZ O 20 ■ X I INCUBATION AGE IN DAYS Figure 2. Reduction in mandibular length. Figure 3- Reduction in maxilla length. TABLE 4 MAXILLA/MANDIBLE LENGTH RATIOS Dose Age No. Days Stage Embryos (Clis.d. )* (Ells.d. )*) p 800 4.0 24 24 O.47IO.O4 O.69IO.I5 0.0002 Boo 4.5 25 7 0 .4410.04 O.81I0.22 0.0020 Boo 5.0 26 + 12 0.44lo.o4 0.77I0.26 0.0010 750 5.0 27 18 0 .4810.05 I.I7I0.63 0.0005 750 5.5 2B 10 0.4710.03 0 .6410.18 0.0100 700 4.0 24 9 O.43IO.I3 0.7110.25 0.0100 700 5.0 27- 10 O.43IO.52 1.2410.72 0.0100 700 6.0 29- l4 0.4510.04 0.7210.14 0.0001 625 4.0 24 21 O.47I0.07 0.5210.08 0.0900 625 5.0 27- 81 0 .4410.02 0.7210.29 0.0001 625 6.0 29 + 21 0 .4810.03 0.3910.05 0.0001 aControl index - standard deviation. ’Experimental index t standard deviation. 31 "EXPERIMENTAIS £0 . 8 T 5 ’CONTROLS g TABLE 5 CARTILAGE MEASUREMENTS Control Slight to Moderate Reduction^ Stage Length Moderate^ Tota 1 Proxima1 Distal Ratio ( S.D. )(̂ No. Severe Dose ReductionX-Rayed (Mean) Reduct i on (Mean) (Mean) (Mean) P/D With mm (mean) mm mm mm Minor (Mean) mm Bends mm 800 2k 11.4(5)^ 7.48(2)* 7.2(6 )* 4.0 3.2 1.3 0.42 — — — — 800 26 + 11.5(6 ) - 6.5(8 ) 4.2 2.3 2.1 1.09 1 5.17(2 )* 750 27 14.8(9) -- 10.1(8) 7.4 2.7 2.9 1.23 7 5.66(9 ) 700 2k 11.8(6) 8.8(1) 7.8(5 ) 3.3 4.5 0.7 0.20 -- -- 700 27- 18.7(6 ) -- 10.8(4) 7.8 3.0 3.0 0.96 4 6.38(2 ) 665 27- 18.7(6 ) 12.1(1 ) 14.2(2) 10.5 3.7 3.0 0.30 2 6 .38(1 ) 625 27- 17.2(16) 13.8(20) 11.3(27) 8.4 2.9 3.2 1.99 15 6.60(4) 625 29 -- 6.4(3) -- - — — — — -- — — -- 550 27- 11.8(6) 8.8(9 ) -- -- — — — — — — -- — — (̂ ) = number of embryos. ^Reduction without cartilage curvature. 'Reduction with cartilage curvature. ^Standard deviation. 'Without cartilage bends. 38 marked cartilage bending (Plate I, Ic) and moderate reduc­ tion with -slight curvature (Plate I, Id) are illustrated. Cornification occurred in both mandibles. A major cartilage bend (Fig. 7 ) was considered the first bend that occurred moving from the proximal (articulation) end of the cartilage to the distal end. Minor bends occurred within the distal portion of a cartilage rod with a major bend. Embryo If (Plate I) demonstrates a typical major bend and embryo Ig (Plate I) very severe folding of cartilage in a minor bend. Minor bends were considered part of the distal end of the carti­ lage when proximal/distal ratios were determined (Table 5 ). Measurements 9 and 10 day embryos (Table 5 ) indicated a significant trend for the location of the major cartilage bond to be 2/3 the length of the cartilage from the prox­ imal end, in embryos x-irradiated at 5 days of incubation. Analysis of variance showed high correlation (P > 0.1 of a larger F). Minor bends occurred within the distal end. Nine and 10 day control embryos (cartilage stained) were examined for cartilage stress points. The midpoint of the distal curvature occurred within the distal 1/3 of the cartilage (Fig. 8) and most major bends occurred along this distal curve. Major cartilage bends tended to occur more toward the proximal end of the cartilage in embryos x-irradiated at k days of incubation. Cartilage bends for 4 1/2 day 39 Figure 8 . The contour of Meckel's cartilage in a 9-10 day embryo. A— articulation point, D--distal, P--proximal. embryos occurred between the average bend location in 4 and 5 day embryos. Maxilla/mandibular ratios verify this (Fig. 9). Extreme reducti on— no curvature. In the severest mandibular reduction only a stub of the jaw was present (Plate I, le). Meckel's cartilage consisted of short rods not meeting in the mid-line, but protruding as two separ­ ate prongs. Average cartilage stub lengths were less than average lengths of the proximal portion of the cartilage in embryos with major bends (Table 5). Histological Observations Normal Growth Patterns Stage iB (3 days). The mandible was shaped like an archer's bow (Fig. 10) with distinct symphysis in the 40 4.0 3.0 «/I STAGE 2 7- 2.0 STAGE 26+ STAGE 24 1.0- STAGE 24 0.0 650 750 800 DOSE (ROENTGENS) Figure 9. Proximal-distal cartilage ratios for mandibles x-irradiated at stages 24, 26+., and 27-. 4l 10. 1 1 . PO 12 . Figure 10. The mandible at stage l8 (3 days). T-trigemi- nal "muscle," N-nerve. Figure 11. The plane in which serial sections of the histological study were cut. M-mandible (stage 29). Figure 12. Mandibular regions are marked for orientation (stage 32). A-anterior, D-distal, P-proximal, Po-posterior. 42 mid-line (see Figures 11 and 12 for orientation). The greatest dimension was in a medial-lateral direction. Cell condensations of presumptive muscle were present on either side of the mid-line with nerve fibers of the mandibular rami (of trigeminus nerve) located laterally (Fig. 10). Loose mesenchyme filled the remain­ der of the mandible. There was no evidence of Meckel's cartilage. Stage 2 6 , 27 (5 days)— Sensitive Period. Meckel's cartilage primordia formed as two rod-like mesenchymal condensations in the lateral mesenchyme of the mandible (first visceral arches). Nerve fibers were found near lateral condensation surfaces. The quadrates, which form the surfaces for articulation of the lower jaw were pre­ sent as mesenchymal condensations and were the most anterior structures in the mandible. It was impossible to distinguish a clear separation between the primordia of Meckel's car­ tilages and the quadrates. Two sets of presumptive muscle condensations were visible. One set was located medial to the cartilage pri­ mordia (Fig. 13)) while the second set was found on either side of the mid-line posterior to Meckel's cartilage. Muscle primordia of the mandible will be referred to as trigeminal "musculature." The muscle condensation in the mid-line (hyoid "musculature") was located on the anterior-distal surface of the cartilage primordium of the 43 13. 14. 15. Figure 13. Mandible at sensitive period (stage 27, 5 days). C--Meckel's cartilage primordium, D--distal-lateral mesenchyme, DC--dense cell condensation, H — hyoid "musculature," P— proximal mesenchyme, T--trigeminal "musculature," N— nerve, HC— hyoid precartilage. Figure l4, "Spoon"shape of stage 33 (8 day) mandible. C--Meckel's cartilage. F i gur e 13 Cartilage and "muscle" relationship in a stage 33 (8 day) mandible. C--Meckel’s car­ tilage, PT--pretendon, T--longitudinal tri­ geminal "musculature," TT— transverse trigemi­ nal "musculature." 44 hyoid skeleton (Fig. 13; Plate II, 2c and 2d). In addition to cartilage and muscle blastema, a dense cellular condensation was present in the lateral mesenchyme distal to the point of juncture with the max­ illa. Stage 28, 29 (5 1/2 - 6 days). The quadrate was chondrified by the end of the 6th day as indicated by an increase in cytoplasmic basophilia. Meckel's cartilage was chondrifying in a proximal-distal direction with the distal half still unchondrified by the end of the 6th day. Trigeminal muscle primordia filled most of the area between Meckel's cartilages, which curved medially. Dis­ tal cartilage ends were beginning to curve, elongating parallel to one another (Plate III, 3a and 3b). Develop­ ing "muscle" was now observed around proximal ends of Meckel's cartilage and will be referred to as "proximal musculature." Stage 30 - 31 (6 1/2 - 7 days). Distal tips of Meckel's cartilage were nearly parallel. "Muscle" strands (trigeminal) paralleled Meckel's cartilage to the distal tips (Plate IV, 4d). Posterior trigeminal "muscle" devel­ oped transversely between the more proximal ends of Meckel's cartilage. Hyoid "musculature" had elongated distally. Meckel's cartilage was completely chondrified, with the exception of the distal tip (perichondral cap) by the end of the 7th day. 45 PLATF II Histological Study (A)— Hematoxylin — Eosin 2a Normal embryo--four days of incubation. The mandibular arch is shown. The mid-line region is the site of con­ siderable necrosis (arrow). (x 49) 2b Detail of the mid-line of the mandibular arch of 2a. Several large macrophages with phagocytosed cells are evident (arrows). (x 37l) 2c Control embryo--stage 27. This illustrates the mandi­ bular development at approximately the sensitive period. Necrotic cells may be found along the nerve laterally and in the mid-line, particularly near the distal tip of the cartilage condensation. C--cartilage primordium, N--nerve fibers, T--trigeminal "musculature." (x 49) 2d Same as 2c. The proximal ends of the precartilage are visible. C--Meckel's cartilage primordium, H--hyoid "musculature." (x 49) 2e Control embryo--stage 27+--sacrificed 30 minutes after experimental embryos were x-irradiated. A very small blister is present at the symphysis of the arch (arrow). ( X 49 ) 2f Experimental embryo— estimated stage 27— sacrificed 2 hours after x-irradiation (dose 750r). A blister is present at the symphysis of the arch. Hemorrhages are evident (arrows). (x 49) 4 6 P L A T E I I 2 a 2 c 2 d m 2 e 2 f 47 PLATE III Histological Observations (B)— Hematoxylin— Eosin 3a Control embryo--stage 29— sacrificed 24 hours after experimental embryos were x-irradiated. Proximal ends of the cartilage are shown. Muscle primordium is grow­ ing distally from the developing hyoid apparatus. A strand of developing trigeminal "muscle" can be barely observed running parallel to Meckel's cartilage. B-- blood vessel, C— Meckel's cartilage, H--hyoid "muscula­ ture," N--nerve, T--trigemina1 "musculature." (x 49) 3b Same as 3a. Section is located posterior to that in 3a. Transverse trigeminal "musculature" extends between the proximal ends of Meckel's cartilage. C— Meckel's cartilage, T--trigeminal "musculature." (x 49) 3c Control embryo--stage 27--sacrificed 8 hours after exper­ imental embryos were x-irradiated. Detail of a portion of mandibular nerve running lateral to Meckel's car­ tilage condensation is shown. A number of necrotic cells (arrows) are located along the borders of the nerve fibers. N--nerve. (x 371) 3d Experimental embryo — estimated stage 29 — sacrificed 24 hours after x-irradiation. There is evidence of the early cartilage. The trigeminal "musculature" (arrow) along with the perichondral cap is heavily necrotic. C--Meckel's cartilage, T--trigeminal "musculature." ( X 49 ) 3e Same as 3d. This is a greater magnification of the mid­ region of the section. Necrotic cells can be disting­ uished in the mesenchyme and developing muscle (arrow). C--cartilage bend, T--trigeminal "musculature." (x I85) 3f Same as 3d. This section was taken posterior to the cartilage condensations. The tip of a clear blister is visible. S--blister. (x 49) 48 P L A T E I I I 3 a 3 b 3 c 3 d 3 f 49 PLATE IV Histological Observations (C)— Hematoxylin--Eosin 4a Experimental embryo— stage 28+--sacrificed 24 hours after x-irradiation. Numerous macrophages are located in the lateral mesenchyme. Some necrosis is evident in the muscle primordia medial to the cartilage and in the mid-line. Muscle primordia have more necrotic figures than the surrounding less dense mesenchyme. C--Meckel's cartilage, H--Hyoid "muscle," T— trigeminal "muscle." Nerve--arrow, LM--lateral mesenchyme. (x 49) 4b Same as 4a. Detail of the right cartilage illustrating the abundant necrotic figures in the lateral mesenchyme. C--Meckel's cartilage, LM--lateral mesenchyme, N— nerve. (x I85) 4c Same as 4a. Detail of the mid-region of the mandibular arch. Necrotic cells are particularly present in the muscle primordia. C— Meckel's cartilage, T— trigeminal "musculature." (x I85) 4d Control embryo— stage 31--sacrificed 2 days after experimental embryos were x-irradiated. The distal extent of the cartilage growth is shown. The tri­ geminal "musculature" extends almost to the distal tip of the cartilage. C--Meckel's cartilage, T--trige- minal muscle. (x 49) 4e Experimental embryo— estimated stage 30— sacrificed 2 days after x-irradiation. Meckel's cartilage has not extended distally as in controls (4d). Mandible has not elongated properly in the proximal-distal dimen­ sion. C--Meckel's cartilage, T--trigeminal "muscula­ ture." ( X 49) 4f Experimental embryo — estimated stage 31 — sacrificed 2 days after x-irradiation. Mandible is severely reduced. Little evidence of "muscle" exists and cartilage con­ sists of short stubs. Necrosis is very intense through­ out the distal mesenchyme. C--Meckel's cartilage, D-- distal mesenchyme. (x 49) 50 P L A T E IV # 1 # » 4 b 4 a 4 c 4 d a*' a 4 e 4 f 51 Stage 32 - 33 (7 1/2 - 8 days) . The mandible was "spoon" shaped (Fig. l4) and Meckel's cartilage was S shaped (Fig. I3 ). Mesenchyme was dense in the peri­ chondral cap. Narrow bands of trigeminal "muscle" paral­ leled the cartilage, ending short of the distal tips. (Plate V, 3d). A pre-tendon condensation passed between the cartilage tips (Plate V, 5e) and extended proximally to the transverse trigeminal "muscle." Proximal "muscu­ lature" (around Meckel's cartilage articulation) was well developed (Plate V, 5a). Primordia of the supra-angular and angular bones appeared first as dense cell condensations in mesenchyme lateral to proximal ends of Meckel's cartilage. Ossifi­ cation began by the 7th and 8th days respectively in proximal portions of each bone (Plate V, 5b and 5c). Cell condensations for the dentary and opercular bones were present by the 8th day in mesenchyme distal-lateral and distal-medial respectively to Meckel's cartilage. The articulation of Meckel's cartilage and the quadrato- jugal bone of the upper jaw, with the quadrate was well established (Plate V, 5a). Presumptive skeletal structures of the hyoid appar­ atus were distinct. Hyoid "musculature" along with developing paraglossum extended into the developing jaw (Plate V, 5f). Stage 34 - 35 (9 days). Relative proportions of 52 PLATE V Histological Study (D)— Hematoxylin--Eosin 5a Control embryo— stage 33--sacrificed 3 days after experimental embryos had been x-irradiated. This sec­ tion was taken through the quadrate-Mecke1 ’s cartilage articulation. The proximal "muscles" are well defined. QJ--quadrato-jugal bone, Q--quadrate, C--Meckel's cartilage, P--proximal "muscle." (x 49) 5b Same as 5a* C— Meckel's cartilage, S— supra-angular bone, QJ--quadrato-jugal bone. (x 49) 5c Same as 5a- C— Meckel's cartilage, S— supra-angular bone A--angular bone. (x 49) 5d Same as 5a. The distal extent of the trigeminal muscle is shown between the Meckel's cartilages. C--Meckel's cartilage, T-trigeminal "musculature." (x 49) 5e Same as 5a. Pretendon condensation passes between the distal cartilage tips. C— Meckel's cartilage, PT-pretendon. (x 49) 5f Same as 5a. Tongue development is shown. H--hyoid "musculature," P — developing paraglossum. (x 49) 53 PLATE 5 b 5 c 5 d 5 e 5 f 54 Meckel's cartilage and muscle had changed and the tri­ geminal "muscle" paralleling the cartilage was a compara­ tively narrow band. Transverse trigeminal "muscle" was a relatively smaller structure. Mandibular nerve rami continued to parallel the lateral surface of Meckel's cartilage. Opercular and dentary bones appeared as thin plates on the medial and lateral surfaces of Meckel's cartilage respectively. Initial ossification began by approximately 8 1/2 - 9 days in the opercular and dentary bones respectively. Angular and supra-angular bones had well developed trabeculae. Figure l6 is a composite drawing of a series of photomicrographs of the mandible showing respective locations of the 4 membrane bones at stage 35+ (9 days). Cell condensations indicated the initial appear­ ance of the mentomandibular-gonial bone (endochonral) at the distal ends of Meckel's cartilage. The tongue was now shaped like a wedge between Meckel's cartilages. Hyoid "musculature" elongated dis­ tally as the paraglossum developed. Normal Cell Death Patterns Prior to the sensitive period. Necrotic cells, singly or in clumps within macrophages were found in the mid-line of 3 day mandibles (Fig. 1?). Isolated necrotic cells were found in lateral regions. 55 Figure l6. Bone formation in a stage 35 man­ dible . Supra-angular bone Angular bone Dentary bone Opercular bone Meckel's cartilage Mentomandibular-gonial bone 56 0 <3. 17. 18. Figure 1?. Mandible at stage l8 (3 days). N--nerve, T--trigeminal "muscle," Ty--thyroglossal duct, .--necrotic cells, o--macrophages. Figure l8. Stage 27 (5 days) embryonic mandible. C-- Meckel's cartilage, DC--dense cell condensa­ tion, H--hyoid "muscle," HC--hyoid precartilage, N--nerve, T— trigeminal "muscle," .— necrotic cells. 57 By the 4th day (stages 22 - 24), necrotic cells were nearly all in the mid-mesenchyme of the mandible (Plate II, 2a and 2b). Necrotic cells were present in ectoderm where mandibular arches join the neck of the embryo and in ectoderm of pharyngeal pouches. Near the end of the 4th day, necrotic cells were found along the ramus mandibularis. The sensitive period. Cell death localizations are shown (Fig. l8). Small clumps of necrotic cells were found on both sides of the most proximal extent of the precartilage and along the entire length of the nerve (Plate III, 3c). Some necrosis was still found in the mid-line, particularly at the distal tip of Meckel's cartilage. Small clear blisters were observed in the mid-line of the symphysis of the arches in several con­ trol mandibles (Plate II, 2e). Later development. Necrotic cells were found lateral to and around the proximal end of Meckel's car­ tilage through stage 30 (6 1/2 - 7 days) (Fig. 19). During this period, scattered cell deaths wereo bserved near the distal tip of the cartilage and in presumptive hyoid and trigeminal "muscle." Necrotic cells were also observed around the spinal nerves and around the nerves of the presumptive tongue (hyoid) musculature. 58 19. Figure 19• Mandible at stage 30 (7 days). C--Meckel's cartilage, N--nerve, .--Necrotic cells, T-- trigeminal "muscle." 59 Effects of Radiation in the Sensitive Period — General in Mandible Cell death--30 minutes--2 hr. post. There was little evidence of cell damage during the interval immedi­ ately following x-irradiation up to approximately 30 min. Most striking observation was complete absence of mitotic figures throughout the mandible. Necrosis directly attrib­ uted to x-irradiation was evident 1 to 2 hr later. Cell death— ^ to 48 hr. post. Necrotic cells were being phagocytosed by macrophages by 4 hr. after x-irradi- ation. Patterns of cell death (to be described) remained the same in this period. Macrophages became very abundant from 12 to 24 hr. post. From 24 to 48 hr. post-x-irradia- tion, the contents of macrophages were generally disinte­ grating cells (blue-purple dots within macrophage cyto­ plasm). Nearly all dead cells were cleared out by 48 hr. Hemorrhages. Embryos receiving 750 r had severe t mandibular hemorrhages (Plate II, 2f). Such extreme tis­ sue alterations could not be studied for radiation sensi­ tivity. The bulk of the histological study was therefore on embryos receiving 625r. Occasionally a mandible receiv­ ing 625r had a number of small hemorrhages or dilated ves­ sels, particularly in the lateral mesenchyme. One mandible (625r) had a severe hemorrhage in the mid-mesenchyme with a small blister, however, blisters were often present with­ out evidence of hemorrhage. Mitotic activity. A few mitotic figures were 6o observed in mandibles by 4 hr. after x-irradiation. Mitotic activity was not conspicuously different than normal by 12 to 24 hr. post x-irradiation in mesenchyme and cartilage primordia. Mitotic activity was observed throughout the entire length of Meckel's cartilage. Effects of Radiation in the Sensitive Period -- Cell Death Patterns A large number of mandibles without excessive hemorrhages exhibited patterns of necrosis which varied depending upon severity of damage. Pattern No distinct pattern could be determined for a number of mandibles. Necrotic cells were fairly uniformly scattered throughout the mandible. Radiation damage was not severe. Pattern II. The lateral mesenchyme was heavily necrotic, with the dense cell condensation of this mesen­ chyme being the most severely affected. Distal mesenchyme exhibited a lighter degree of necrosis than any of the lateral mesenchyme (Fig. 20; Plate IV, 4a, 4b, and 4c). Pretendon, hyoid and trigeminal "musculature" contained abundant necrotic cells. "Musculature" and pretendon was always more severely affected than the loose mesenchyme present within the mid-mandible. Meckel's cartilage was not severely affected. Scattered necrotic cells were found throughout the entire length, with no area exhibiting greater necrotic intensity. 61 HC JO 0 LM 2 0 0 M 21. Figure 20. Cell death pattern II. (Estimated stage 27-28, 5 1/2 days). C--Meckel's cartilage, H--hyoid "muscle," HC--hyoid precartilage, LM— lateral mesenchyme, N— nerve, T — trigeminal "muscle," .--necrotic cells, o--macrophages. F i gur e 21, Cell death pattern III. (Estimated stage 29- 30, 6 1/2 days). C— Meckel's cartilage, DM-- distal mesenchyme, N--nerve, .--necrotic cells, o--macrophages. 62 The perichondral cap was more affected than the surround­ ing loose mesenchyme. Necrotic cells appeared to be fairly evenly distributed in the proximal mesenchyme. Pattern III. Mandibles had massive necrosis in distal and mid-mesenchyme. Muscle primordia and lateral mesenchyme contained abundant necrotic cells (Fig. 21; Plate IV, 4f). Radiation damage was severe. Effects of Radiation in the Sensitive Period — Growth Patterns— 1-2 Days Post Pattern 2» Overall size of mandible was reduced, but conformation was normal. Meckel's cartilage and mus­ cle primordia had normal growth patterns. Pattern II. Meckel's cartilage did not appear to be growing as rapidly as in controls of the same incu­ bation period (Plate IV, 4e ) . Mandibular contour was blunted in its long dimension. Trigeminal muscle pri­ mordia remained more compact and formed a less acute angle (between cartilage rods) than in controls. Some embryos exhibited evidence of bend formation at distal ends of Meckel's cartilage (Plate III, 3d, 3e, and 3f)• The mandibular nerve paralleled the cartilage laterally as in controls. Pattern III. Mandibular development was severely stunted (Plate IV, 4f). Meckel’s cartilage consisted of two short stubs. Most of the distal mesenchyme and tri­ geminal "musculature" were missing. Extensive necrosis 63 was still present in the distal mesenchyme of several mandibles. Effects of Radiation in the Sensitive Period— Growth Patterns--3-^ Days Post Pattern Growth Patterns were similar to those of controls. Pattern II. Proximal "musculature" appeared to be developing normally, although muscle bundles were smaller than in controls. Cartilage bends were evident (Plate VI, 6a, 6b, 6c, 6d, and 6e). Trigeminal "musculature" gener­ ally terminated at the initiation, or apex, of a bend. "Muscle" fibers apparently stopped growing distally abruptly in several mandibles. Developing fibers were compacted at this point (Plate VI, 6a). Transversely devel­ oping trigeminal "muscle" was more compact than in controls of the same incubation age. Mandibular contours were blunted in their long dimensions. Bent Meckel's cartilages appeared to just fill this contour. Angular and supra-angular bones were fairly well developed proximally. Opercular and dentary bones were barely present in mandibles sacrificed 4 days post x irradiation. Bones curved, molding with abnormally curved cartilage. Dentary (and mentomandibular-gonial) bone extended around distal cartilage tips in several mandibles (Plate VI, 6b). Developing bones appeared short and thick. The tongue was blunted in its long dimension; its 64 PLATE VI Histological Study ( E )--Hematoxylin--Eosin 6a Experimental embryo--estimated stage 32--sacrificed 3 days after x-irradiation. A bend is present in the left cartilage (note the two pieces). The transverse fibers of the trigeminal "musculature" are present proximally. The trigeminal "musculature" that normally parallels the cartilage ends at the initiation of the bend. The muscle primordia does not appear to have been able to elongate properly (developing fibers are clumped). C--Meckel's cartilage, T--trigeminal "musculature." (x 49) 6b Experimental embryo— estimated stage 35--sacrificed 4 days after x-irradiation. A severe bend exists in the left cartilage. A band of trigeminal "muscle" ends at the point of the bend. The developing dentary and mento­ mandibular-gonial bone appears united in the mid-line. C— Meckel's cartilage, D--dentary bone, M--mentomandibular- gonial bone, T--trigeminal "musculature." (x 49) 6c Experimental embryo--estimated stage 31--sacrificed 3 days after x-irradiation. This is a section anterior to the apex of a cartilage bend. Trigeminal "muscle" extends to the initiation of this bend. C--Meckel's cartilage, T--trigeminal "muscle." (x 49) 6d Same as 6c. This section was taken to illustrate the apex of the left cartilage bend. Trigeminal "muscle" extends to this point. C--Meckel's cartilage, T— tri­ geminal "muscle." (X 49) 6e Same as 6c. The right cartilage bend is shown. The pretendon condensation is visible between the cartilages. C— Meckel's cartilage, PT--pretendon. (x I85) 6 5 PLATE VI 6 b 6 a 6 d 6 e 66 distal and lacked the wedge shaped contour of controls and was bluntly rounded. HyoLd "musculature" was con­ siderably reduced. Pattern III. No stub mandibles were sectioned at this incubation age. It was apparent after two days of incubation (Fig. 22) that perichondral caps and the bulk of trigeminal "musculature" were destroyed along with the distal mesenchyme. / \ \ N N. Figure 22. Diagram of severe mandibular reduction, with a projected normal contour. C— Meckel's cartilage, D--distaI mesenchyme. Histochemical Obs ervat ions Cell Death Locations Necrotic cells were sites of intense acid phos­ phatase activity. Normal and x-irradiat ion induced cell death patterns were identified, and were consistent with histological observations. In addition to acid phosphase activity in necrotic cells, the mesenchyme surrounding 67 areas of cell death (resulting from x-irradiation) con­ tained considerable phosphatase activity. Normal Development Prior to the Sensitive Period (Stages 18 to 26, 3-4 Days) Semi-quantitative evaluations of the amounts of reaction products in major primordia (Table 6 ) showed that the ectoderm, nerve and muscle were the main sites of enzymatic activity (Figs. 23, 24) (see Fig. 25 for orientation of sections). Acid (Plate VII, 7a) and alka­ line phosphatase and ATP dephosphohydrolase (Plate IX, 9a) exhibited moderate to strong activity in these loca­ tions. AMP dephosphohydrolase activity was negative throughout the mandible until stage 26 (Table 6 ) when moderate activity occurred in the ectoderm and proximal lateral mesenchyme. By stage 26, there was moderate RNA activity in cartilage and muscle primordia (Table 6 ). In addition RNA and PAS+ were moderate in the ectoderm. Specific Structures in Control and Experimental Mandibles (Stages 27 to 351 5-9 Days) Mandibular sites analyzed in detail are shown in Fig. 26. Cartilage, muscle and bone followed the same growth patterns described for the histology of control and experimental embryos. Ectoderm. The dephosphorylating enzymes had uni­ form activity within the ectoderm, although there was some variation in the amount of activity (Table 7 ). Alcian TABLE 6 HISTOCHEMICAL ACTIVITY PRIOR TO THE SENSITIVE PERIOD Acid P. Aik. P. ATP De • AMP De. RNA PAS + Location Stages Stages Stages Stages Stage Stage 18 22 26 18 22 26 18 22 26 18 22 26 26 26 CARTILAGE PRIMORDIA M M W + 4- + N N N + MUSCLE PRIMORDIA + + + + + + + + •f + + + + + + + N N N + - NERVE + + + + + + + + - + + -h N N N N - ECTODERM + + + + + 4- + + + + + f N N + - - PROXIMAL- LATERAL N N 00- - f - - 4- + - - MESENCHYME DISTAL- LATERAL — — + - - - - - 4- N N N - - MESENCHYME KEY: N--Negative reaction — Trace or background activity + Moderate activity ++ Strong activity +++ Intense activity ++++ Very intense activity 69 23. 24 . 25. Figure 23 Mandible at stage l8 (3 days). Activity of alka­ line phosphatase and ATP dephosphohydrolase is strong in nerve and muscle primordia on either side of the mid-line and in the ectoderm. Figure 24, Mandible at stage l8 (3 days). Activity of acid phosphatase is strong in nerve and muscle pri­ mordia on either side of the mid-line and in the ectoderm. Activity is strong in necrotic cells in the mid-line. Figure 25, The plane of serial sectioning for the histo- chemical study. M--mandible. 70 PM ML DL 26. Figure 26. Diagram of location of mandibular primordia. C— Meckel's cartilage, DL--distal-lateral mesenchyme, H— hyoid "musculature," HC--hyoid precartilage, ML--medial-lateral mesenchyme, N--nerve, P--proximal mesenchyme, PC--perichon- dral cap, PM--proximal "musculature," T--tri- geminal "musculature." TABLE 7 HISTOCHEMICAL ACTIVITY I--ENZYMATIC ATP AMP Sta es Acid Alkaline Dephospho- Dephospho- Phosphatase Phosphatase hydrolase hydrolase RNA 27 29 31 33 35 2? 29 31 33 35 27 29 31 33 35 27 29 31 33 35 2? 29 31 33 35 C A R T I L A G E + + + + + + + -+-+ + — — N + + + — — N — — — N + + + + + + + + P E R I C H O N - + D R I U M — + + + + + + — + 4- — — — + 4-4- 4- +4- N — + — — — + 4" 4- 4- P E R I C H O N D R I - A L C A P - 4-4- 4-4- 4-4- 4- 4- 4- 4 4- N - 4-4-4- 4- - N N N N N - 4-4-4-4-4- B O N E — — 4- 4- 4 -4- 4- S U P R A — — — -4 -4 4- -4- -4- — — 4 - 4 - 4 4 1 — -4- 4-44-4-4- — -4 4- 4- -4 4- 4- 4- N — 4 -4-44- A N G U L A R M E D I A L M E S E N C H Y M E N N - - - - - + + . -4 4 - + 4- N N N - - - - - - - P R O X I M A L - + L A T E R A L -4 -4 -4-4 -4 -4 -4-4 -4-4 -4-4 4 N 4- 4-4- -44- -44- -44- -4 -44- -4 -4 - - - - - M E S E N C H Y M E N E R V E -4 -4 4 - 4- - 4 -4 -4 4 - - N 4 -4 4 - - 4 -4 N - - - N N N N N N E C T O D E R M + + -4 + + + 4- + 4- -4 -4 -4 -4 4 -44- P R O X I M A L 4 4 4 4 4 4 4 M U S C L E 4 4 4 4 4 4 4 4 4 — 4 — N N 4 4 4 4 4 4 4 4 4 4 N N N — — 4 4 4 4 4 4 4 4 H Y O I D 4 4 4 4 4 M U S C L E 4 4 4 4 4 4 4 4 _ 4 - N N 4 4 4 4 4 4 4 4 N N N - - 4 4 4 4 -1 - 4 4 T R I G E M I N A L 4 M U S C L E — 4 4 4 4 4 4 4 — 4 — — N 4 4 4 4 4 4 4 N N N — — — — 4 4 — 4 4 K e y : N N e g a t i v e A c t i v i t y S t r o n g a c t i v i t y - T r a c e o r g e n e r a l b a c k g r o u n d a c t i v i t y 4 4 4 I n t e n s e a c t i v i t y 4 M o d e r a t e a c t i v i t y 4 4 4 4 V e r y i n t e n s e a c t i v i t y 72 blue at pH l.l4 appeared specifically in the basement membrane in control and experimental mandibles (Plate XIII, 13f and 13g). Ectoderm was fairly uniformly stained in all other carbohydrate tests (Table 8 ). There was no dif­ ference in the enzymatic activities or carbohydrate stain­ ing of control and experimental mandibles. Muscle development. The proximal and hyoid "mus­ culature" developed more rapidly than the trigeminal "musculature," as indicated by the intensity of the reac­ tion (Tables 7 and 8 ). A. Alkaline phosphatase activity appeared during the early development of muscle and diminished (Table 7)« By stage 31 (Plate VIII, 8b) there was little activity in muscle primordia. B. Acid phosphatase activity increased (in mus­ cle fibers) (Plate VIII, 8a) as muscle development pro­ ceeded. C. ATP dephosphohydrolase activity (Plate IX, 9c) increased considerably as fibers began to form. Activity increased progressively from strong to very intense in the proximal musculature (Table 7) (Plates IX, 9f and XI, 11c). D. AMP dephosphohydrolase activity occurred only in trace amounts during the 7th and 8th days of development (Table 7). E. RNA increased as fiber development advanced TABLE 8 HISTOCHEMICAL ACTIVITY II--CARBOHYDRATE TESTS Alcian Blue^ Alcian Blue^ Toluidine Blue PASx Position Stages Sta ges Metachromasiao Lagea Stage s 27 29 32 35 27 29 32 35 27 29 32 35 27 29 32 35 CARTILAGE 4- + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + PERICHONDRIUM - + + + - + + + + + + + + - + + 4- + + - BONE-SUPRA- ANGULAR N N + N N - + + N N N N N N + + NERVE N N N N N N N N N N N N - + + + ECTODERM + + + + + + + + + + + - — — - + + + •f BASEMENT MEMBRANE ■vj PROXIMAL MUSCLE + + + + + + + N -(- + + HYOID MUSCLE + + + N + TRIGEMINAL MUSCLE + + N N PERICHONDRAL CAP MESENCHYME IN GENERAL ^Alcian Blue- -pH 1.14 KEY: N negative activity Trace or general background ^Alcian Blue --pH 2.4? activity + Moderate activity + + Strong activity + + + VI+ + + + enrtye nsien teancstei viatcyt ivity 74 (Table 7). F. Carbonhydrate staining intensity increased progressively (Table 8). Connective tissue within the muscle stained positively with alcian blue at pH l.l4 and at pH 2.24, while the muscle fibers were PAS+. Muscle fibers also demonstrated some toluidine blue metachromisia. Muscle development in experimental embryos fol­ lowed the same histochemical pattern as in control mandi­ bles, even though muscle bundles were smaller and develop­ ing fibers sometimes irregularly aligned. Cartilage development. A. Alkaline phosphatase (Plate VII, 7b and 7e) was found both cytoplasmically and extracellularly within cartilage and perichondrium. Activ­ ity was strong prior to chondrification and moderate during the period of chondrification (Table 7)« Perichondral activity remained for a longer time than phosphatase activity in the cartilage matrix. B. Acid phosphatase was confined to the cyto­ plasm of the small cartilage cells (Plate VII, 7c and 7d). Activity became stronger (Table 7) and was found both cytoplasmically and extracellularly. C. ATP dephosphohydrolase was also confined to the cytoplasm of cartilage cells (Plate IX, 9e and 9f). As development proceeded, activity became strong intra- cellularly and intercellularly in the perichondrium (Table 7>. 75 D. AMP dephosphydrolase activity appeared in trace to moderate quantities in the perichondrium and matrix of the cartilage in stage 30 to 33 embryos (7 and 8 days) (Plates IX, 9g and X, 10b). By stage 35 activity was negative in the cartilage and trace activity was present in the perichondrium (Plate XI, lie). E. RNA was located cytoplasmically in cartilage cells and in the perichondrium during the entire interval of development observed (Plates IX, qd and XI, 11a, 11b and Ilf) . F. Intensity of the carbohydrate tests demonstrated that chondrification occurred first in the proximal end of Meckel's cartilage and moved distally. Carbohydrates were uniformly distributed by the end of the 7th day of incubation (stages 29-30) and were located cytoplasmically and extracellularly. Alcian blue at pH l.l4 did not stain nuclei (Plate XII, 12d and 12f), while alcian blue at pH 2.47 stained nuclei (Plate XII, 12c and 12e). Alcian blue (Plate XIII, 13a and 13b) and PAS+ (Plate XIII, 13e) staining intensities increased as growth proceeded (Table 8). Presence of chondroitin sulfate in the cartilage was indicated by toluidine blue metachromasia (Table 8). Hyaluronidase removed this metachromasia (Plates XII, 12a and 12b; XIII, 13c and 13d). Tests for keratosulfate were negative; it was not detected in even the oldest control 76 mandibles tested (stage 35» 9 days). No differences were observed in the histochemistry of the cartilage in experimental mandibles. Photomicro­ graphs of acid phosphatase activity (Plate VII, 7f) and ATP and AMP dephosphohydrolase activity (Plate X, 10a and 10b) illustrate normal cartilage development in irradi­ ated mandibles. Perichondral cap. A. Alkaline phosphatase activ­ ity (Plate VIII, 8b) was moderate in the perichondral cap, while the surrounding mesenchyme had moderate to strong activity. B. Acid phosphatase activity (Plate VII, 7c and VIII, 8a) was much stronger in the cap than the adjacent mesenchyme. C. ATP dephosphohydrolase activity (Plate IX, 9e) was moderate to strong and was also stronger in the cap than in the adjacent mesenchyme. D. AMP dephosphohydrolase activity was negative at all times. E. RNA activity was greater in the cytoplasm of mesenchymal cap cells during the 6th and 7th days of devel­ opment than in those of the lateral perichondrium (Table 7)« F. All carbohydrate stains exhibited only trace quantities in the cap (Table 8). Similar patterns of activity occurred in the peri­ chondral caps of experimental embryos. 77 Mesenchymal development. A. Strong alkaline phosphatase activity appeared in the lateral and medial mesenchyme prior to the actual appearance of the membrane bones. As soon as the supra-angular bone (stage 30, 7 days) appeared, activity declined in the surrounding mesenchyme. Activity followed a similar pattern with the formation of the angular, dentary and opercular bones. By stage 35 (9 days) alkaline phosphatase activity was mainly confined to developing membrane bones, i.e., see Figures 27 through 29 for distribution of alkaline phos­ phatase activity. B. Acid phosphatase was strong to moderate in the proximal-lateral and distal-lateral mesenchyme, but was present as general background activity in the medial mesenchyme (Table l ) . C. ATP dephosphohydrolase activity was moderate in the distal-lateral and medial mesenchyme. Strong activity was present in the proximal mesenchyme, where the greatest developmental activity occurred (Table 7). Proximal muscle, quadrate and proximal Meckel's cartilage (earliest chondrification) and the angular and supra- angular bone (earliest bone development) occurred in this region. D. Two days after the first AMP dephosphohydrolase activity occurred (stages 26-27, 5 days) in the proximal mesenchyme (Table 7) (Plate IX, 9b), the supra-angular ALKALINE PHOSPHATASE ACTIVITY (Shaded Areas Denote Activity) s- o- 27. •n] CO DM 28. 29. F i gur e 2 7 - Stage 27 mandible (5 days). C--Meckel's cartilage primordia, E— ectoderm. Figure 28. Stage 31 mandible (7 days). C--Meckel's cartilage, S--supra-angular bone. Figure 29. Stage 35 mandible.(9 days). A-- angular bone, D--dentary bone, DM-- mentomandibular-gonial bone, 0--opercular bone, S--supra-angular bone. 79 bone appeared with strong activity (Plate X, lOd). Prox­ imal mesenchymal activity declined at this time. Activait y extended the entire length of the mandible in the lateral mesenchyme of stage 32-33 (8 days) mandibles before the appearance of the dentary and mentomandibular-gonial bones; see Figures 30 through 32 for the distribution of AMP dephosphohydrolase activity. Strong activity in the distal-lateral mesenchyme preceded dentary-mentomandibular- gonial bone formation. E. RNA was distributed fairly uniformly through­ out the mesenchyme, with activity somewhat greater in the proximal mesenchyme. In experimental mandibles acid phosphatase activity was greater than in controls, in the lateral and distal mesenchyme. Active site locations depended upon the pattern of cell death found in the mandible, and correlated with histological observations of cell deaths. Acid phosphatase was very intense in mandibles with severe distal and distal lateral necrosis (Plate VII, 7f). In experimental embryos, alkaline phosphatase and AMP dephosphohydrolase activities in the distal mesenchyme conformed to blunted mandibular contours. Enzymatic intensi­ ties did not appear different than in control mandibles. No alterations were apparent in the ATP dephosphohydrolase and RNA activities or in carbohydrate staining intensities. Bone development. The developmental pattern was AMP DEPHOSPHOHYDROLASE ACTIVITY (Shaded Areas Denote Activity) A _ ^ 3 0 . DL DM 0o0 31. 32 . Figure 30- Stage 27 mandible (5 days). C--Meckel's cartilage primordia, E-- ectoderm, P--proximal mesenchyme. Figure 31. Stage 33 mandible (7 1/2 - 8 days). C--Meckel's cartilage, DL— distal- lateral mesenchyme, S— supra-angular bone. Figure 32. Stage 35 mandible (9 days). A--angular bone, D--dentary bone, DM-- dentary-mentomandibular-gonia1 bone, 0--opercular bone, S--supra- angular bone. 81 the same for all 4 membrane bones. Order of bone develop­ ment (Table 9) was the supra-angular (Table 10), angular, opercular and dentary. A. Strong alkaline phosphatase activity occurred in the mesenchyme prior to the appearance of all bones (Plate VIII, 8b). All cells of the periosteum, as well as the osteoblasts and osteocytes contained strong to intense alkaline phosphatase activity. Activity was intense in the osteoid (Plate VIII, 8d ), but decreased as trabeculae developed (early mineralization) (Table lO). Membrane bone contours were distinguishable in stage 35 (9 days) mandibles (Plate VIII, 8e ) due to strong to intense alka­ line phosphatase activity. B. Acid phosphatase activity became strong in bone primordia when they appeared as dense cell condensa­ tions (Plate VIII, 8c) and increased as development advanced (Table 10). Activity was strong in newly formed trabeculae and was particularly intense around the edges. C. ATP dephosphohydrolase activity (Plates X, 10c and XI, lid) was similar to that of acid phosphatase, however, activity did not become as intense around the edges of trabeculae. D. AMP dephosphohydrolase activity was very simi­ lar to that of alkaline phosphatase (Table 10). Strong activity occurred in early bone formation (Plate X, lOd) and increased in intensity in osteoid (Plate XI, lie) and TABLE 9 HISTOCHEMICAL ACTIVITY--BONE DEVELOPMENT Histochemical Supra-Angular Angular Opercular Dentary Reaction Stages Stages Stages Stages30 31 33 35 30 31 33 35 30 31 33 35 30 31 33 35 ALKALINE PHOSPHATASE + + + + + + + + - 4- -t- + + 4- 4- - - + + + + - - + + + ̂ ACID PHOSPHATASE + ++ + + + + + - + + + + + + - - + + + + + - — + + + ATP DEPHOS. + + + + + + + + + + + + + + + - - + + + + - — + + + AMP DEPHOS. + + + + + + -r + - —- 4- -h + + - - + + + - — 4- + + + 0to3 RNA N - + + + N 4- + + N N + N N + ALCIAN BLUE pH 1.14 — — — + - — — + N N N N N N - pH 2.47 + + - - - + + N N - + N N N + PAS + - + + + + + + - — — + + - - - + - — — - KEY; N Negative activity - Trace ac tivity + Moderate activity + + Strong activity + + + Intense activity + + + + Very int ense activity TABLE 10 HISTOCHEMICAL ACTIVITY--SUPRA-ANGULAR BONE DEVELOPMENT Alcian Alcian Location Acid P. Aik. P. RNA ATP De. AMP De . PAS + Blue Blue TEM^ (pH 1.14) (pH 2.4?) MESENCHYME (BEFORE + + + + + + + - - - - OSTEOID) PREOSTOE- BLASTS + + + + OSTEOBLASTS + 4- + + -f- + + + 4- + - - - - OSTEOCYTES + + + + + + 4- + 4-4- - - - - OSTEOID + + •f + + + + T -r + + + + + + + - PERIOSTEUM + + + 4- + - U0J04- + - - - MINERALIZED TRABECULAE + + + + + + + + + + + + TRABECULAE EDGES + + + + + + + + + + + + + + + + + Toluidine blue metachromasia KEY; - Trace activity + Moderate activity ++ Strong activity +++ Intense activity ++++ Very intense activity 84 newly mineralized bone. E. RNA became strong in the cytoplasm of the osteoblasts and osteocytes during osteoid formation (Plate XI, Ilf) and mineralization (Table 10). Osteo­ blasts along trabeculae edges contained intense RNA activ- i ty. F. Intensity of carbohydrate staining increased as trabeculae developed and mineralization occurred (Table 10) (Plate XIII, 13a, 13b, 13c, 13d, and 13e), There was some evidence of toluidine blue metachromasia in mineral­ ized trabeculae of the supra-angular bone (most advanced bone development). In experimental mandibles, ossification appeared to be normal, as indicated by histochemical observations. Done development was retarded 12 to 24 hr. and thus, the initial enzymatic activities were later in making their appearance. Enzymatic intensities were normal when they occurred. While the blastema of the dentary and opercular bones were present in several embryos sacrificed at an estimated stage 35- (9 days), ossification had not begun. The development of the trabeculae in the angular and supra- angular bones was not as extensive as in control mandibles. Bones were generally thick (Plate X, lOe and lOf) and failed to elongate properly. Each bone molded to the contours (Plate VIII, 8f ) of abnormal cartilage and to blunted distal mandibular contours (Plate VIII, 8g). In some 85 otnhryos with severe cartilage bends, the dentary-mento- riiaiid i btilar-gon i a 1 bone primordia were either not separate in the mid-line (Plate VIII, 8g) or were in close juxta­ position. These bones were normally separate in the mid­ line at 9 days of incubation (Plate VIII, 8e ). 86 IM.ATIC VII Acid and Alkaline Phosphatase Activity (A) 7a Normal embryo--stage 24. Acid phosphatase activity is strong in the ectoderm, on either side of the mid-line and in the numerous necrotic cells and macrophages (arrow) found in the mid-region. (x 49) 7b Normal embryo--stage 27+. Alkaline phosphatase is present throughout the ectoderm, cartilage and muscle primordia, and nerve fibers. C--Meckel's cartilage, N--nerve, T--trigeminal "musculature." (x 49) 7c Control embryo— stage 28+--sacrificed 8 hours after experimental embryos were x-irradiated. Acid phos­ phatase activity is shown. The nerve fibers and ecto­ derm are active sites. A number of necrotic cells can be discerned just medial to the distal cartilage tips and along the lateral surface of the cartilage. C-- Meckel's cartilage, N— nerve, P--perichondral cap, T--trigeminal "musculature." (x 49) 7d Same as 7c. (Detail — insert) The nerve fibers are shown passing near the proximal end of the cartilage. Necrotic cells (arrows) are found on both sides of the cartilage. C--Meckel's cartilage, N — nerve. (x 105) 7e Same as 7c. Alkaline phosphatase activity is more in­ tense in the perichondrium than within the cartilage. There is considerable activity in the ectoderm, N-- nerve, P--perichondrium. (x 49) 7f Experimental embryo— estimated stage 29— sacrificed 2 days after x-irradiation. Meckel's cartilage is stunted. Considerable acid phosphatase and necrotic cells are present distally (arrows). C--Meckel's cartilage. (x 49) 8 7 P L A T E VI I nu 7 c 7 d /i 7 e 7 f 88 PLATE VIII Acid and Alkaline Phosphatase Activity (B) 8a Control embryo— stage 31--sacrificed 2 days after experimental embryos were x-irradiated. Acid phos­ phatase is intense in the perichondria1 cap. Trigemi­ nal muscle primordia and nerve fibers have strong activities. C— Meckel's cartilage, N— nerve, P — peri- chondrial cap, T— trigeminal "muscle." 8b Same as 8a (adjacent section). Alkaline phosphatase distribution is shown. There is some activity in the perichondrium. Lateral mesenchyme has considerable activity, particularly where the dentary bone will develop. C— Meckel's cartilage, D— dentary primordium. ( X 49 ) 8c Control embryo— stage 31--sacrificed 2 days after exper­ imental embryos were x-irradiated. The proximal end of Meckel's cartilage is shown. Acid phosphatase is intense in the perichondrium and in the supra-angular bone primordium. C— Meckel's cartilage, S— supra- angular bone. (x 49) 8d Same as 8c (adjacent section). Alkaline phosphatase is moderately strong in the perichondrium and in the supra-angular bone. C--Meckel's cartilage, S— supra- angular bone (X 49). 8e Control embryo— stage 35--sacrificed 4 days after experimental embryos were x-irradiated. Alkaline phos­ phatase is mainly confined to the membrane bones. The bones parallel Meckel's cartilage, the outline of which cannot be made out. A--angular, D--dentary, M--mento- mandibular, 0— opercular, and S— supra-angular bones. (x 10) 8f Experimental embryo— estimated stage 34--sacrificed 4 days after x-irradiation. Alkaline phosphatase activity is shown in the abnormally curved membrane bones. The bones have developed to conform with the cartilage curvature. A small section of outlined cartilage is visible. C--Meckel's cartilage. (x 10) 8g Same as 8f. The dentary-mentomandibular bones have fused in the mid-line. (x 49) 89 P L A T E V I I I ' 15. ^ - 8 a 8 b ■ 8 c 8 d V \ l D 01 3 1 V 1 d C6 94 PLATE XI RNA, ATP and AMP Dephosphohydrolase Activity 11a Control embryo--stage 30--sacrificed 2 days after experimental embryos were x-irradiated. RNA activity is shown in the quadrato-jugal and supra-angular bone primordia and in Meckel's cartilage. C--Meckel's c a r ­ tilage, QJ--quadrato-jugal bone, S--supra-angular bone. ( X 49 ) 11b Same as 11a (adjacent section). This section was treated with RNA. Bone primordia can no longer be d i s ­ tinguished. The cytoplasm of the cartilage cells appears as clear areas w i t h i n the cartilage. The remaining cartilage stain is due to the staining of the cartilage m a t r i x with methyl green-pyronin. C — Meckel's c a r ­ tilage. ( X 49) 11c Control embryo--stage 30--sacrificed 2 days after e x p e r ­ imental embryos were x-irradiated. ATP dephosphohydro­ lase activity is shown in the developing proximal " mus­ culature," the quadrato-jugal bone, the quadrate and a small portion of Meckel's cartilage. C--Meckel's cartilage, P — proximal "musculature," Q — quadrate, Q J — quadrato-jugal bone. (x 49) lid Control embryo--stage 35--sacrificed 4 days after e x p e r ­ imental embryos were x-irradiated. ATP d e p h osphohydro­ lase activity is shown in a section of the dentary and opercular bones. C--Meckel's cartilage, D--dentary bone, E— ectoderm, 0 — opercular bone, T — trigeminal "musculature." (x 49) lie Same as lid (adjacent section). AMP dephosphohydro­ lase activity is m a i n l y confined to the dentary and opercular bones. C — Meckel's cartilage, D--dentary bone, E--ectoderm, 0--opercular bone, T--trigeminal "musculature." (x 49) Ilf Same as lid (adjacent section). Some RNA is present in the dentary and opercular bones. RNA in Meckel's cartilage is obscured by the staining of the cartilage matrix. C--Meckel's cartilage, D--dentary bone, E - - ectoderm, 0--opercular bone, T — trigeminal "muscula­ ture." (X 49) 95 P L A T E XI f I l a I I b I I I I d I e I I f 96 PLATE XII Carbohydrate Activity (A) 12a Control embryo— stage 28+--sacrificed 12 hours after experimental embryos were x-irradiated. Toluidine Blue metachromasia is shown in Meckel's cartilage. (x 49) 121) Same as 12a (adjacent section). Metachromatic stain is removed following hyaluronidase treatment. (x 49) 12c Control embryo— stage 29--sacrificed 1 day after experimental embryos w ere x-irradiated, Alcian Blue (pH 2.47) stains the cartilage m a trix and ectoderm. Nuclei are stained. (x 49) 12d Same as 12c (adjacent section). Alcian Blue (pH l.l4) stains the cartilage matrix and the ectoderm. (x 49) 12e Control embryo— stage 27+--sacrificed 4 hours after experimental embryos w ere x-irradiated. Early s t a i n ­ ing of the cartilage primordium is shown (Alcian Blue— pH 2.47). The nuclei are stained, but the mandibular nerve is negative. C— Meckel's cartilage, N--nerve. (x I8 5) 12f Same as 12e. Alcian Blue (pH l.l4) stains the d e v e l ­ oping cartilage matrix. C - - M e c k e l 's cartilage. (x 185) 9 7 P L A T E XII 12 12 b 2 d ■ s /X- I 2 e I 2 f 98 PLATE XIII Carbohydrate Activity (B) 13a Control embryo— stage 35+--sacrificed 4 days after experimental embryos were x-irradiated. Alcian Blue (pH l.l4) stains the cartilage intensely. The supra- angular bone is slightly stained. C— Meckel's car­ tilage, S--supra-angular bone. (x 49) 13b Same as 13a (adjacent section). Alcian Blue (pH 2.47) stains the supra-angular bone and Meckel's cartilage. The stain is stronger in the perichondrium and the outer portion of the cartilage than in the interior. C— Meckel's cartilage, S--supra-angular bone. (x 49) 13 c Same as 13a (adjacent section). Toluidine Blue meta­ chromasia is strong in the cartilage and perichondrium. C--Meckel's cartilage, S— supra-angular bone. (x 49) 13d Same as 13a. Toluidine Blue staining following hyalu­ ronidase treatment indicates that much of the meta­ chromatic staining in the cartilage is due to chondroi- tin sulfate. There is little change in the bone stain. C--Meckel's cartilage, S--supra-angular bone. (x 49) Ije Same as 13a. The perichondrium and developing bone is strongly PAS positive. The cartilage matrix is moderately PAS positive. C--Meckel's cartilage, S— supra-angular bone, (x 49) 13f Control embryo— stage 28+--sacrificed 12 hours after experimental embryos were x-irradiated. Alcian Blue (pH l.l4) stains the basement membrane of the ectoderm. B— basement membrane, E— ectoderm, M--mesenchyme. (x 371) 13g Experimental embryo--estimated stage 29--sacrificed 12 hours after x-irradiation. Alcian Blue (pH l.l4) is shown in the basement membrane. B--basement mem­ brane, E — ectoderm, M — mesenchyme. (x 371) 9 9 P L A T E XI I I ' - '■ ■ i t 13 o I 3 b i I 3 c I 3 d I 3 f M ^ ‘ I 3 e 13 g CHAPTER IV DISCUSSION Sensitive Period Results of this study indicate that the embryonic chick mandible is most sensitive to the effects of x— irradiation at 5 days of incubation or approximately stage 27. A dose of 625r permits maximum survival of 5-day embryos with the production of slight to extreme forms of the anomaly, without massive hemorrhaging. Since the same dose produces 0:1 ly slight mandibular reductions in 4 and 6 day embryos, it IS believed that the range of the sensitive period is well delineated, i.e., between the extremes of 4 and 6 days. Higher doses applied at the 4 and 6 day extremes result in more severe reduction, but not with the frequency or severity of those occurring at 5 days at lower doses. Results are consistent with the 5 day irradiation sensitivity of the mandible mentioned by Goff (unpublished data). Boric acid, pilocarpine (Landauer, 1952, 1953) 1 aza- serine (Dagg & Karnofsky, 1955) and eserine sulphate (Agarwal, 1956) had their greatest effect on mandibular development when administered at 4 days of incubation. The difference in stimulus-response time may reflect the time required for 100 101 ,'ibsoipt ion and accumulation of an adequate concenLration ot (lie tcraLogen into the mandible, or to its mode of action, oi' to a combination of these factors. It is entirely pos­ sible that chemical agents and x— irradiation mediate their effects at different points and in different manners. Landauer (1952) found that boric acid effects were almost completely eliminated by the addition of riboflavin. The effects of azaserine were not diminished by the addition of riboflavin (Dagg & Karnofsky, 1955). It seems reasonable that azaserine and boric acid either effect different meta­ bolic pathways or different points in the same pathway as suggested by Dagg and Karnofsky. It is likely that chemicals which cause mandibles to be reduced, but not bent, have a greater effect on the cartilage, while a chemical causing cartilage curvature (eserine sulphate) has a greater effect on surrounding tissues. Anomalous Mandibular Development Goff (1959) noted hemorrhages in the mandibles of x-irradiated chick embryos, drawing attention to the possi­ bility that hemorrhages are causal factors in the production of anomalous mandibular development. Embryos dying acutely, after receiving high (790r) irradiation doses, also have hemorrhages in the brain, eye, limbs, and back. It is to be expected that such hemorrhaging generally results in death. Histological observation of a sampling of embryos x-irradiated at a dose of 750r demonstrate massive hemorrhage 102 in the majority of mandibles. Such blood vessel rupture and flooding of developing tissues obstructs the ability to analyze specific primordial sensitivity. However, mandibles of embryos receiving 625r rarely develop severe hemorrhages, but anomalous development results, including the least and most severe forms. Therefore, hemorrhage as a causal factor in the production of the mandibular anomaly in this study is discounted. Mandibular blisters can not be correlated with anomalous mandibular development. Since a number of control embryos exhibited small clear blisters beneath their mandibles, it is apparent that the posterior ectoderm-mesenchymal connec­ tion of the 5 and 6 day embryonic mandible is very loose, which tends to allow for the accumulation of fluid in an embryo exposed to a teratogenic agent inducing edema as one of its effects. In embryos where severe hemorrhage occurs posterior to Meckel's cartilage, fairly normal development proceeds. It may be that mandibular hemorrhages drain into clear blisters leaving relatively unaltered mandibular pri­ mordia. When mesenchymal necrosis is very severe, anomalous development results, with or without the presence of a blister or hematoma. Grabowski (1964), in the study of the etiology of hypoxia-induced malformations in the chick embryo, observed occasional hematomas underneath the chin without any appreciable effect on mandibular development. 103 Radiation -sensitivity correlates with the amount of cell death in developing mandibular tissues. Judging from the anomalies that occur, the cells of the mesenchyme that are most affected are the preconnective tissue cells, which lie in the lateral mesenchyme. Within the proximal portion of the mesenchyme are migrating bone primordial cells (Jacob­ son & Fell, 1941) which are apparently more resistant, since membrane bones were not specifically affected. Presumptive connective tissue cells make up the bulk of the remaining lateral mesenchyme, with the exception of the dentary bone primordia, which also appears resistant. Bone (quadrate), cartilage and muscle are also present as mesenchymal conden­ sations at the sensitive period, but subsequent development shows them to be less sensitive than the lateral connective tissue. It is only in the more severe anomalies that muscle, bone and cartilage are significantly affected. The variation in sensitivity of connective tissue primordia and that of bone, cartilage and muscle condensations is responsible for the variation in the manifestation of mandibular anomalies discussed in the following sections. Cell Death Pattern I When the connective tissue primordia are very lightly affected, the result is the development of smaller, but normally formed mandibles. In such mandibles Meckel's carti­ lage, muscle and bone primordia are only lightly necrotic. Histogenesis of these tissues occurs normally as indicated 104 by histological and histochemical observations. Cell Death Pattern II When the connective tissue primordia are affected to a greater degree and cartilage, muscle and bone primordia are not proportionately more severely damaged, cartilage bending results. Mean lengths of Meckel's cartilage in mandibles with slight reduction (Cell death pattern I) and in mandibles with cartilage bends are very similar. These results indicate that Meckel's cartilage elongates in spite of the reduction of more sensitive mandibular tissues. Histological and histochemical evidence supports the earlier work of Jacobson and Fell (I94l) in showing that the peri­ chondral cap is the main growing point of Meckel's cartilage. Even though the perichondral cap is not severely affected and distal cartilage growth continues, cell death in the perichondral cap results in the growth of a less sturdy distal portion of Meckel's cartilage. The combined factors of the growth of a weakened distal cartilage, within the confines of a mandible of reduced space, results in the mechanical bending of the distal end. The angle of each cartilage bend is dependent upon the degree of mesenchymal sensitivity. As the destruction of presumptive connective tissue increases cartilage bends become more acute. Folding of the distal end of the cartilage is a consequence of extreme mesenchymal necrosis. Histogenesis of Meckel's cartilage appeared normal as indicated by the histological iÜl and histochemical analysis of this study. Comparison of control and experimental cartilage contours indicate that the major bend occurs within the distal 1/3 of Meckel's cartilage. This distal portion of Meckel's cartilage is marked by a curvature in control embryos, which may indicate a weak point in the cartilage. The fact that the major bend occurs more proximally in 4— day embryos demonstrates that the stress point is also dependent upon the incubation age. It is to be expected that cartilage bends would form more proximally when connec­ tive tissue is damaged at an earlier period in development. The trigeminal musculature terminates at the initiation or the apex of the major bend. Muscle growth apparently stopped abruptly at this point in some embryos as indicated by the clumping of developing fibers. Muscle is hindered in its proper elongation, perhaps due to r educ­ tion in mandibular space, or its gro^vth may require normal cartilage elongation. Since muscle primordia are more sensitive than the cartilage, reduction in muscle cells (as a result of cell death) may also be in part responsible for the termination of distal muscle growth. The histo­ chemistry can only be interpreted as normal even though muscle arrangement and size relationships are altered. Bone development (initial ossification) is retarded from 12 hr to 1 day, which is consistent with the overall retardation of the embryo. Trabeculae formations are not 106 a.s advanced as in controls of the same age of incubation. The contours of the membrane bones conform with the abnor­ mally curved or bent cartilage. In addition, membrane bones are short and blunted in their long dimensions, which may be a consequence of the anomalous development of Meckel's cartilage. Jacobson and Fell (I94l) suggested that normal cartilage elongation may be necessary for the proper stretch­ ing and growth of the membrane bones, and this idea is supported by these observations. Otherwise histogenesis, as revealed by histological and histochemical analysis, is normal. The fusion, or near fusion, of the dentary and mentomandibular-gonial bones in the mid-line of the mandible in a number of x-irradiated embryos may be the consequence of both the reduction in distal-lateral connective tissue and the bending of Meckel's cartilage. Both factors can act to bring the bone primordia closer together in the mid- line. Erdmann (1940) indicated that the ment omandibular- gonial bones fuse in the mid—line in 10 day embryos, which is later in development than the observations of this study. The tongue conforms with the shape of the mouth cavity in normal development (Hamilton, I965). Histological observations reveal that the tongue contour in mandibles of cell death pattern II is altered, but conforms with the shape of the lower jaw. Observations in this study do not 1 0 7 reveal bulging or excessive growth. The tongue as a factor in the production of the anomaly is eliminated. Cell Death Pattern III When dense mesenchymal blastemas are severely affected, along with connective tissue primordia, stub mandibles result. The perichondral cap, trigeminal muscle and dentary and mento- mandibular—gonial bone primordia are heavily damaged. With the main growing point of Meckel's cartilage destroyed, along with the bulk of the distal tissue condensations, severely shortened mandibles are expected results. Intense acid phosphatase activity accompanies the severe necrosis in this cell death pattern. The occurrence of massive necrosis in the distal end of the mandible 2 days after x-irradiation in some embryos, suggest that secondary cell deaths occurred, since necrotic cells are generally fairly well removed in the majority of embryos by 2 days after x-irradiation. Another possibility is that necrosis is so severe initially that macrophages are incapable of removing all of the dead cells by this time. Results are similar, in some respects, to those reported by Skalko (I965) in his study of the effect of Co^^ radiation on development and DNA synthesis in the rat embryo. DNA synthesis was reduced, as compared to controls, by both I351" and 270r, in the 1st 12 hr post radiation, but greater reduction in DNA synthesis occurred following 270r, the tera­ togenic dose. However, by 2 4 hr after radiation DNA synthesis 108 rates were the same in the controls and both sets of experi­ mental embryos. Skalko hypothesized that embryos receiving 13'3r were capable of apparently complete recovery, while the damage was so great initially in embryos receiving 270r that complete recovery was not possible, despite the fact that a greater increase in DNA synthesis had occurred in these embryos. It is apparent in this study that the variety of anomalies resulting from teratogenic doses is due, in part, to differences in sensitivity between embryos and their ability to recover from the radiation damage. Malformations in cell death pattern XI result due to the differential sensitivity of the mandibular tissues. Mechanical bending of the cartilage is a result of the differential sensitivity of presumptive connective tissue and cartilage. In cell death pattern III radiation damage is initially so severe in bone, muscle and cartilage primordia that compensation is not possible. Hicks (1954) in analyzing radiation damage in embryonic mice and rats suggested that malformations repre­ sented a balance between damage and the capacity of any given anlage or developing zone to repair. Argyris, (I968) in a review on the growth of the tissues of the skin induced by damage of various kinds, indicated that the stimulation of growth of each of the tissues is probably specific, i.e. requires damage of the 109 tissue itself for the induction of its proliferation. The greater DNA synthesis observed by Skalko (I965) in rat embryos following radiation at a teratogenic dose may be the result of a similar phenomenon in embryonic tissues. DNA synthesis studies on chick embryos following a wide range of doses of x-irradiation may shed light on this question. While maxilla reduction is slight in comparison to that of the mandible, the tendency for a significant in­ crease in reduction at 6 days of incubation indicates greater sensitivity at this time. The maxilla develops later than the mandible during embryonic development, thus accounting for its greater sensitivity at 6 days. Cornification occurs in mandibles of experimental embryos allowed to develop beyond the usual time of sacri­ fice, i.e., 15, 16 days of incubation. Since mandibles exhibiting all extremes of the anomaly develop cornified mandibles, it is apparent that the cornification of the epidermis is not prevented by x-irradiation. Normal Cell Death The role of cell death in normal morphogenesis of the chick embryo is incompletely known. Basic to this report is the resolution of normally occurring cell death in the developing mandible. Local cell deaths occur during embryonic processes such as folding, évagination, invagina­ tion, part separation, fusion. Lumen formation, ossification n o iind the ingrowth of tissue (Gliicksmann, 1950). Necrosis observed in the mid—line of 3 and 4 day mandibles in this study verifies the results of Jacobson and Fell (1941). They concluded that the necrotic cells provided room for ingrowing myogenic cells. Necrotic cells present in the mesenchyme distal—medial to Meckel's cartilage up to stage 30 (7 days) may provide space for the distal elongation of Meckel's cartilage. This seems likely since muscle primordia are located medial to the cartilage primordia from 4 days on during development. Strong acid phosphatase activity in this region correlates with the cell deaths in this area. It is possible that these necrotic cells are the lingering result of the more massive cell death occur­ ring at 3 and 4 days, but their specific location and persistence in the area makes this seem unlikely. Necrotic cells observed in the proximal osteogenic center are in agreement with the earlier results of Jacob­ son and Fell (I94l). They indicated that the dead cells may divide the osteogenic center into 3 parts, thus sepa­ rating the supra-angular, angular and opercular primordia. Cell deaths in developing spinal ganglia of the chicken embryo have been well documented (review, Glucks- mann, 1950). As presumptive neuroblasts and spongioblasts die, cells that would become Schwann cells for the ensheath- ment of fibers of such cells, may degenerate because there is no stimulation for their differentiation. This may be Ill responsible for some of the cell deaths observed along the mandibular nerve fibers in this study. Normal Histogenesis Bone Initial ossification occurs at 7 days for the supra- angular, 7 1/2 to 8 days for the angular, 8 days for the opercular and 8 to 9 days for the dentary. The ossification sequence in this study follows, fairly closely, the time scale of Erdmann (1940). The proximal-distal appearance of membrane bones correlates with the distal migration of primordia determined by Jacobson and Fell (l94l). The activities of alkaline phosphatase and RNA observed in this study are in agreement with activity pat­ terns for bone reported in a review by Cabrini (196I). Carbohydrate staining results agree with those reported for bone in a review by Pritchard (1956). Strong ATP dephosphohydrolase activity in osteo­ blasts, osteocytes and developing trabeculae indicate that this enzymatic activity is involved in the formation of membrane bone. Bourne (review, 1956) stated that ATP was a component of preosseous cartilage. It was indicated that ATP may or may not provide phosphate used in the formation of bone salt, but may be concerned in the production of energy for the breakdown of glycogen. It seems likely that the high levels of ATP dephosphohydrolase in osteoblasts 112 and osteoid of the membrane bones of this study demonstl a ( (' dephosphorylation of ATP is important to the mineralization of membrane bones. Results of this study suggest that AMP dephospho­ hydrolase activity may be correlated with some morphogenetic process in the proximal and distal-lateral osteogenic centers. The first appearance of (stage 26) activity occurs in the proximal mesenchyme, adjacent to the ectoderm, and suggests an ectodermal-mesenchymal interaction (e.g., tertiary induc­ tion) prior to the formation of the proximal portions of the membrane bones. As development proceeds AMP dephospho­ hydrolase activity becomes widespread in the proximal mesen­ chyme and as the quadrato-jugal bone of the upper jaw forms, followed by the supra-angular, angular and opercular bones in turn, activity becomes very intense within each primordium. AMP dephosphohydrolase activity may be related to a specific morphogenetic property of the mandible, i.e., induction of membrane bone formation. As development continues, AMP dephosphohydrolase activity proceeds distally (following the first occurrence) in the mesenchyme adjacent to the ectoderm. A continuing ectoderm-mesenchymal interaction may accompany the distal migration of the primordia. The appearance of strong activity in the distal-lateral mesenchyme prior to the intense activity that appears shortly thereafter in the ment omandibular-gonial and dentary bones may indicate an ectoderm-mesenchymal 113 interaction similar to that suggested proximally. Very intense levels of AMP dephosphohydrolase activity in osteoid, mineralized bone, osteoblasts and osteocytes indicates its continuous involvement well along in the bone formation process. It would be worthwhile observing the AMP dephospho­ hydrolase activity in the developing membrane bones of the upper beak as well as in the remainder of the skull. Results of this study suggest that a similar wave of activity is at least involved in the formation of the quadrato-jugal bone. If additional study still indicates AMP dephosphohydrolase involvement in membrane bone formation, it would be valuable to continue this investigation employing other methods such as tissue exchange, transplantation, and tissue culture. Cartilage Cell condensations (perichondral cap) noted at the distal tips of Meckel's cartilage are growth centers for cartilage elongation (Jacobson & Fell, 19^1). The activity of acid phosphatase, RNA and ATP dephosphohydrolase verify the distal growth center. Cytoplasmic basophilia and the intensity of carbohydrate staining indicates a proximal- distal pattern of chondrification, which was also the conclu­ sion of Jacobson & Fell (19^1). The distribution of acid mucopolysaccharides in the cartilage matrix was similar to that previously described for the chick embryo (Rolle, 196? and Hall, 1968). The failure i l 4 of Meckel's cartilage to stain positively for kerato—sulphate is in agreement with Muir (1964). Muir indicated the presence of kerato-sulphate after the 15th day in the cartilage of the chick embryo. The appearance of alkaline phosphatase in early carti­ lage formation is not in complete agreement with the results Milaire (1963) obtained in studying the mouse limb bud. Milaire noted considerable ATP and AMP dephosphohydrolase, as well as acid phosphatase in the developing perichondrium of, but not in, preosseous cartilage. These enzymatic activities are observed in the perichondrium and cartilage proper in this study. The fact that Meckel's cartilage grows interstitially as well as appositionally and does not ca Ic1fy may account for its difference in enzymatic distribu­ tion. AMP dephosphohydrolase activity was only barely present Ln Meckel's cartilage during chondrification. It has been reported (review, Cabrini, I96I) that RNA is localized in nearly all cartilage cells, which was the case in this study. Cabrini pointed out that it was only after calcification (which was not observed in Meckel's carti­ lage) had occurred that RNA activity diminished. Muscle No description of the embryological development of lower jaw musculature in the chicken embryo is available. The transverse trigeminal muscle may correspond to the mylo- hydoideus muscle of the duck, Anas platyryhnchos (Kallius, 1901), which runs to a tendon in the mid—lino of the lower jaw. In the duck, the definitive position for the muscle is no fnrther forward (distal) than the mid—point of the mandible. It is evident from this study that the transverse trigeminal muscle in the chicken will also have a subterminal location. The longitudinal trigeminal muscle may correspond to the geniohyoid muscle of the duck, also described by Kallius, which runs medial-lateral to Meckel's cartilage and attaches near the apex of the lower jaw. In a review by Beckett and Bourne (I96O) it was pointed out that work carried out on the histochemical devel­ opment of embryonic muscle was almost nil. The histochemistry of adult avian muscle has been studied in considerable detail (review, George & Berger, I966). Enzymatic localizations are described for particular structures within adult muscle fibers (sarcotubules, mitochondria, myofibrils). While study of cellular detail was not attempted in the present work, it is shown that acid phosphatase and ATP dephosphohydrolase activity are present in developing fibers. The former was described in a review (Beckett and Bourne, I96O) as being present in chick muscle only in early stages of differentiation from the meso­ derm. Such results are not indicated in this study. ATP and ATP dephosphohydrolase are universally present in biological systems. The intense ATP dephosphohydrolase activity found in developing trigeminal and hyoid muscle fibers may be correlated with developing functional ability of the 116 musculature. AMP dephosphohydrolase activity is not out­ standing in early muscle development, but it has been demon­ strated in adult pigeon pectoralis muscle fibers (review, George & Berger, 1966). In this same review, glycogen was mentioned as a component of avian skeletal muscle. No conclusive state­ ments may be made from this study concerning the presence of glycogen, but it may be that some of the strong PAS+ staining is attributable to glycogen. Observations of this study indicate that alkaline phosphatase is present in early forming muscle masses, but not appreciably in later development (fiber formation). These results are consistent with those reviewed by Beckett & Bourne (196O) to the effect that alkaline phosphatase had been noted in the fibrils of inyotomes of the 5 mm human embryo, but not in later fetuses. The connective tissue staining of the developing skeletal muscle indicates the formation of sulphated and carboxylated mucopolysaccharides (Lev & Spicer, 1964). Comparison with Congenital Anomalies Most congenital mandibular anomalies are not adequately described in the Literature to permit an interpretation on the basis of observations in this study. The descriptions of the following anomalies at least allow general speculation. In the congenital anomaly in which the lower beak is a mere vestige (Marble, Harpers & Hammers, 1944) it seems reasonable to assume that the growing point for Meckel's 117 cartilage is disrupted. It may be speculated that the distal mesenchyme, including proximal bone and muscle con- densations are also affected. The failure of connective tissues to develop property may be the primary factor involved in the produc­ tion of the congenitally reduced mandibles described by McGibbon (1946). The fact that extreme forms of the anomaly resulted in the bending of the cartilage may indi­ cate a more severe connective tissue reduction and that a gradation is involved in the genetic effect. It can be assumed with considerable assurance that the congenital effects are in operation during the 4th and particularly the 5til day of incubation in the production of both of these malformations. General Comments While the histochemical tests employed in this study, with the exception of acid phosphatase, did not reveal significant metabolic alterations in early histo­ genesis, it is realized that such an effect is not ruled out. The fact that the products of the breakdown of induced necrotic cells are added to the environment of developing tissues indicates a change in the chemical composition of this environment. That cell death is primarily involved in the production of the mandibular anomaly is not ques­ tioned, but a positive statement can not be made indicating that biochemical changes are not involved. CHAPTER V SUMMARY AND CONCLUSIONS 1. The sensitive period for the induction of reduced mandibles in chick embryos, with x-irradiation, was deter­ mined to be 5 days of incubation or stage 27- 2. Blister formation could not be correlated with mandibular reduction. 3- A possible correlation between nerve growth and cell death of the surrounding tissue is suggested. Cell deaths around the distal ends of Meckel's cartilage through the 7th day may aid distal cartilage elongation. Necrosis in the mid-line of 3 day mandibles and in the proximal osteogenic centers were in agreement with cell death sites determined by Jacobson and Fell (I94l). 4. Variations in mandibular anomalies can be correlated with the incidence and frequency of cell deaths. It is apparent in this study, that the variety of anomalies resulting from teratogenic doses is due, in part, to dif­ ferences in sensitivity between embryos and their ability to recover from the radiation damage. 5. Smaller, but normally proportioned mandibles devel­ oped as a result of a weak response to irradiation. Connective 118 119 tissue was lightly affected and the overall mandibular response was uniformly distributed. 6 . Mandibular cartilage bends representing an inter­ mediate response, resulted from differentially sensitive connective tissue primordia. The development of the less sensitive primordia of bone, cartilage and muscle was modi­ fied by the insufficiency of connective tissue. 7- The primordia of all the tissues were severely affected in a disto-proximal order in the most severe form of the anomaly. Extremely shortened mandibles resulted. 8 . The overall growth of the x-irradiated embryos was retarded. Estimated stages for experimental embryos aver­ aged 12 lir to 1 day behind those of the controls. 9 . Cc'irtilage formation (or chondrification) was not altered in experimental embryos as indicated by the histo- chemicaJ tests. 10. Bone development in experimental embryos (initial ossification) was retarded from 12 hr to 1 day. LI. The bones of the experimental embryos conformed to the abnormally curved and or bent cartilage. Membrane bones were short and blunted in their long dimensions. Histochemistry was normal. 12. AMP dephosphohydrolase mesenchymal activity was cor­ related with the activity of the osteogenic centers in preparation for bone development; an induction mechanism may be involved. LITERATURE CITED Agarwal, I. P. (1956) Morphogenetic effects of eserine sulphate. jJ. Anitn. Morph. Physiol. 3, 63-?4. Argyris, T. S. (1968) Growth induced by damage. Advances in Morphogenesis, 7i 1-4]. Darka, T. & Anderson, J. (I963) Histochemistry, Theory, Practice and Bibliography, pp. 92-105. New York, Hoeber. Beckett, E. B. & Bourne, G. H. (196O) Histochemistry of developing skeletal and cardiac muscle. Structure and Function of Muscle. pp. 87-109, New York, Academic Press. Bellairs, R. (I961) Cell death in chick embryos as studied by electron microscopy. Anat. 95, 54-6], Bensley, S. H. (1934) On the presence, properties and distribution of the intercellular ground substance of loose connective tissue. Anat. Rec. 60, 93-109- Bourne, G. H. (1956) Phosphatase and bone. 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X-Rayed Hours Temp Ave. Temp. Ave. Rel. Hum.Range °C ° F % la 5/6/68 56 24-27 3 24 51:3.0 Ib 100.010.556 21-25+ 15 24 le 56 22-24+ 28 24 lia 5/24 68 28-29- 15 24 100.2lO.7 55:1.0 Ilb 68 27-28 27 24 Ile 68 26+-27 34 24 CrTo\ Illa 6/12 68 26-27 24 100.llo.4 54:5.0 Illb 68 26-27 15 24 IIIc 68 26-27 1515 24 IVa 6/l4 66 26-27 8 20 100.4:0.4 56:7.0 9 24 IVb 66 26-27 8 20 24 IVc 66 926-27 8 20 9 24 V 6/27 108 29-29 r l6 24 100.0:0.5 51:3.0 TABLE A (CONTINUED) Exp. Date No. stage Storage Incubation No. Eggs X-Rayed Hours Temp. Ave. Temp. Ave. Rel. Hum. Range °C «F % VI 7/20/68 48 20+-24. 16 24 IOO.0I0.5 54:4.0 VII 12/9/69 48 23-24+ 36 18 100.210.3 50:2.0 13 24 VIII 7/11/68 204 26-27 16 24 100.3:0.2 54:1.0 IX 7/20 175 26-27 16 24 100.0:0.5 5411.0 H X 8/5/69 54 27 19 10 100.1:0.2 54:2.0 •lNO] 8 24 48 27 19 10 20 24 XI 8/13 52 24-27 5 20 100.0:0 .5 54:3.052 26-27- 37 10 13 24 XII 8/13 72 25-26 - -- 99.9-0.2 50:1.0 XIII 12/9 72 27-28- 11 18 12 24 100.2:0.3 50:2.0 TABLE B DATA ON FERTILITY, VIABILITY AND SURVIVING EMBRYOS {%) Exp. Inviable Dead Living Dead Living Infertile Before Controls Controls X-Rayed X-Rayed Staged Total No. Exp. (End )a (End)a (End)a (End Controls Accident No. Eggs I 3.57 2.37 0.59 12.5 35.1 25.6 19.04 1.18 168 II 2.94 0.98 1.47 16.1 4l. 1 20. 6 16.6 0.00 204 III 0.98 0.98 1.47 22.06 22.06 40.69 11.76 0.00 204 IV 1.50 3.03 I.01 17.17 32.83 35.35 9.09 0.00 198 V 1.96 1.47 1.85 9.26 15.74 28. 70 11.11 26.85 108 VI 0.87 I.31 0.00 22.9 6.25 43.7 27.1 0.00 48 to 00 VII 10.4 6.25 0.00 20. 7 6.2 39.3 16.5 0.00 48 VIII 3.92 4.4l 0.49 39.20 0.49 43.62 7.84 0.00 204 IX 0.87 1.31 1.14 42.8 15.4 29.7 10.28 0.57 175 X 3.92 1.96 1.96 25.48 9.80 45.08 11.76 0.00 102 XI 5.77 2.88 0.96 22.15 3.84 51.84 11.50 0.96 io4 XII 2.78 0.00 0.00 25.0 4.16 54.18 13.9 0.00 72 XIII 11.1 4.2 0.00 19.4 8.8 45.8 11.1 0.00 72 (End) - end of experiment TABLE C HISTOLOGICAL AND HISTOCHEMICAL STUDY Exp. Dose Stage Staged Prior to No . Embryos No. EmbryosNo. (r) X-Rayed Exp. X-Rayed Survived % Controls Sur vived % VIII 625 27 16 90 89 98.8 81 80 98.7 IX 750 27 18 79 52 65.8 77 75 97.4 Xa 625 27 6 32 28 87. 5 16 16 100.0 to \0 Xb 625 27 + 6 24 18 75.0 12 10 83.3 XIa 625 26 + 6 31 29 93.5 12 12 100.0 Xlb 625 26 + 6 27 25 92.6 12 11 91.7 XII 625 26 10 39 36 92.3 18 18 100.0 XIII 650 27- 8 39 33 84.6 l4 14 100.0 TABLE D SERIAL SACRIFICE DATA FOR EXPERIMENTS VIII AND IX Time No. Embryos StagesPost X-Rayed Controls X-Rayed ControlsX-Rayed (Estimated) 30 MIN. 4 3 27, 27+, (2)28 (2)27, 27+ 2 HOURS 3 2 27-, 27, 27+ 27, 28 4 HOURS 4 2 27-, 27, (2)28- 27, 27+ 8 HOURS 7 2 (2)27+, 28-, 27-, 28- (3 )28, 28+ 12 HOURS 8 2 27+, (2 )28-, 28, 28+ O (5)28 1 DAY 6 2 (2)28+, (4)29- (2)29 2 DAYS 3 2 (2 )30-, 31- 31, 32- 3 DAYS 3 2 31, (2)32 33, 34 4 DAYS 3 2 (3)35 (2)35+ Mandibles were embedded in paraffin and sectioned through the length of the mandible at 10 microns. All embryos were stained in Harris' Hematoxylin and Eosin. TABLE E SERIAL SACRIFICE DATA FOR EXPERIMENT VIII Time Stages Post No . EmbryosX-Rayed Controls X-Rayed X-Rayed (Estimated) Controls 4 HOURS 3 2 26+, 27, 28- 27, 27+ 8 HOURS 3 2 28-, (2)28 27, 28- 12 HOURS 2 1 (2)28 28 1 DAY 2 2 (2 )28+ (2)29 2 DAYS 2 2 (2 )30- (2)31 Mandibles were embedded in paraffin and sectioned sagitally at 10 microns Sections were stained in Harris' Hematoxylin and Eosin. TABLE F SERIAL SACRIFICE DATA FOR EXPERIMENT X Time Post No. Embryos Stages X-Rayed Controls X-Rayed X-Rayed Controls (Estimated) 30 MIN. 4 2 (2 )27-, 27+, 28+ (2)27 2 HOURS 4 1 26+, (3)27- 27 4 HOURS 4 2 (2)26, 27, 28 (2)28 8 HOURS 4 2 (2)28, (2)29 (2)28* 12 HOURS 4 1 (3)28, 29- 28 to 1 DAY 4 1 (3)28+, 29- 29- 2 DAYS 4 1 28, 29-, (2)29 31- 3 DAYS 2 (2)33 3.5 DAYS 1 1 34- 34 4 DAYS 2 1 34, 35 35 Mandibles were frozen and sectioned with a Cryostat. Acid and alkaline phosphatase reactions were run on alternate sections of each embryo. TABLE G SERIAL SACRIFICE DATA FOR EXPERIMENT XI Time No. Embryos StagesPost X-Rayed X-Rayed Controls X-Rayed Controls (Estimated) 30 MIN. 5 2 27, 27-, (3)27 (2 )27- 2 HOURS 4 2 26, (2 )27-, 28 26, 27 4 HOURS 4 2 26, 26+, (2)27 26, 27+ 8 HOURS 5 2 27, 27+, 28-, (2)28 (2 )28- COCO 12 HOURS 5 2 (5)28 28, 28+ 1 DAY 4 2 28, (2 )28+, 29- (2)29+ 2 DAYS 4 2 29, (2)30-, 30 30, 31 3 DAYS 4 2 32, (2 )32+, 33 33+, 34- 4 DAYS 4 2 34-, 34+, (2)35- 35, 35- Embryos were frozen and sectioned with a Cryostat. Four reactions were run (alternate sections) on each embryo. ATP and AMP dephosphohydrolase, DNA-RNA and DNA were tested for their localizations. TABLE H SERIAL SACRIFICE DATA FOR EXPERIMENTS XII AND XIII Time Stages Post No. EmbryosX-Rayed Controls X-Rayed ControlsX-Rayed (Estimated) 3-6 HOURS 11 5 27-, 27, (2)27+, 28 26+, 27- 27, 27+, 28 12 HOURS 6 4 (2 )28+, (4)29- (4)28+ 1 DAY 5 2 28+, (3)29-, 29 (2)29 2 DAYS 3 1 30, 31-, 31 31 + 3 DAYS 3 3 32-, 32, 33 33+ , 34-, 34 4 DAYS 4 3 33+, 35-, 35 (2)35, 35- Mandibles were frozen and sectioned with a Cryostat. The following reac­ tions were run (alternate sections) on each embryo: PAS; Toluidine blue, Hyal- uronidase treatment followed by Ibluidine blue; Alcian blue in an acetate buffer (pH 2.4?) and Alcian blue in an acetate buffer (pH l.l4). Several embryos in the youngest group and in the oldest group were treated with Alcian blue in an acetate buffer (pH 5.7) containing .4M MgCl^ and .9M MgCl^.