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Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non-odontogenic but neural crest-derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick-specific probes for Msx-1, Msx-2, and Bmp-4 mRNAs were used as markers for early dental mesenchyme. Our results demonstrated that interactions of mouse odontogenic epithelium with chick mandibular mesenchyme induce early changes in the chick mandibular mesenchyme including the appearance of a translucent zone, cell proliferation, and induction of expression of Msx-1, Msx-2, and Bmp-4, which have been shown to be associated with the formation of dental mesenchyme. In addition, tooth bud-like structures that resemble E13 tooth buds in vivo both morphologically and in their patterns of gene expression formed after 6 days in the heterospecific recombinations. The tooth bud-like structures consist of invaginated mouse mandibular epithelium and condensed chick mandibular mesenchyme expressing high levels of Msx-1 and Bmp-4, but undetectable levels of Msx-2. Unlike the induction of Msx-1, Msx-2, and Bmp-4 in the underlying mesenchyme, which is specific for signals derived from odontogenic epithelium, the induction of a translucent zone and cellular proliferation in the underlying mesenchyme may be related to the growth-promoting potential of embryonic epithelia and not be specific to signals derived from the odontogenic epithelium. Similar to mouse odontogenic epithelium, agarose beads soaked in recombinant BMP-4 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in chick mandibular mesenchyme after 24 hours. These observations together showed that avian mandibular mesenchyme has odontogenic potential that is expressed upon interactions with inductive signals from mouse odontogenic epithelium. Similar to odontogenesis in vivo, formation of dental mesenchyme in chick mandibular mesenchyme is mediated by the activation of Msx-1, Msx-2, and Bmp-4.

The roles of mandibular epithelium in chondrogenesis and growth of mandibular mesenchyme were examined in organ cultures. Epithelium and mesenchyme were separated from the mandibular arches of chick embryos at stages before and after the onset of chondrogenesis in vivo (stages 18-28). Isochronic and heterochronic tissue recombinations were prepared. Removal of the mandibular epithelium resulted in reduced growth of the explants and enhanced chondrogenesis, resulting in increased levels of mRNAs for type II collagen and aggrecan. The presence of mandibular epithelium promoted cell division in loosely arranged undifferentiated tissue from the mandibular mesenchyme and resulted in increased levels of type I collagen mRNA. Enhanced chondrogenesis was also observed in the mesenchyme isolated with basement membrane and isolated mesenchyme grown within Matrigel. These findings suggest that mandibular epithelium has mitogenic and chondrogenic-inhibitory effects on the underlying mesenchyme that are stage independent. Furthermore, the chondrogenic-inhibitory effect of mandibular epithelium on the underlying mesenchymal cells is not mediated by basement membrane.

The purpose of this study was to investigate the pattern of sulphated glycosaminoglycan synthesis during morphogenesis and cytodifferentiation in mouse tooth rudiments and to compare the results with those obtained in another study for salivary gland, a branched organ. Sulphated glycosaminoglycan was labelled by incubating molar rudiments from day 15 of gestation to day 1 post partum in medium containing [35S]-sodium sulphate. The rudiments were washed, homogenized and digested in pronase and then were sequentially digested by chondroitinase ABC and chemically degraded by nitrous acid oxidation. The fractions from each of these procedures were analysed by chromatography on Sephadex G-50 columns. The analysis revealed that, during morphogenesis, levels of chondroitin sulphate increased to a peak of 91% at day 18 and levels of heparan sulphate diminished to 8% during this period. As cytodifferentiation occurred, the level of chondroitin sulphate dropped to 64% and that of heparan sulphate increased to 35%. These results are similar to those reported for rat submaxillary gland, a branching organ. It appears that this pattern of sulphated glycosaminoglycan synthesis is not a unique feature of branching morphogenesis but may be one which marks the transition between morphogenesis and cytodifferentiation in non-branching rudiments as well.

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26–32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondro-genesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heter-ogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage pro-teoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.

Reverse transcription and cDNA amplification (polymerase chain reaction) of total RNA preparations were used to characterize the expression of EGF mRNA in the mandibular arch of day 9–17 mouse embryos. EGF mRNA was present in mandibles at day 9 and 10 but not at days 11–17. Separate RNA preparations from epithelium and mesenchyme at day 10 revealed EGF mRNA in both components.

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of alpha 1(I) collagen in various tissues including odontoblasts.

Previous heterospecific tissue recombinations indicate that mandibular epithelium exerts the first known inductive signal for odontogenesis in mouse embryos. BMP-4 and EGF are two growth factors implicated as signaling molecules mediating the initial inductive epithelial-mesenchymal interactions during odontogenesis. The purpose of the present study was to examine and compare the effects of these growth factors and mouse mandibular epithelium on expression of Msx-1 and Msx-2 genes in molar-forming mesenchyme. Agarose beads soaked in growth factors or pieces of mouse mandibular epithelium (E11) were placed in contact with E11 molar-forming mesenchyme and cultured for 24 h. Whole-mount in situ hybridization analysis revealed that, in contrast to mouse mandibular epithelium and BMP-4-releasing beads, EGF-releasing beads did not induce the expression of Msx-1 and Msx-2 in E11 molar-forming mesenchyme. These observations suggest that whereas BMP-4 may be involved in activation of Msx-1 and Msx-2 in the underlying mesenchyme, EGF may regulate events involved in the formation of dental lamina.

Homeobox-containing genes are thought to be involved in regulating pattern formation in a variety of tissues during embryogenesis. We have examined the expression of the homeobox-related genes Msx-1 and Msx-2 during the development of the chick mandibular arch. Northern blot hybridization indicates that transcripts for both Msx-1 (1.6 Kb) and Msx-2 (3 Kb) are present in the mandibular arch as early as stage 18. The levels of both transcripts in the whole mandible decrease as cartilage is formed in vivo and in vitro. Using in situ hybridization, transcripts of Msx-1 were localized in high amounts to the mesenchyme of the mesial tips of the arches. Msx-2 transcripts were localized in high amounts to medial regions of the arches. Little or no hybridization of either probe was detected in the chondrogenic and myogenic regions of the arches. Transcripts of both genes were also excluded from calcified bone and cartilage. Our results further demonstrate that the mesial tip mesenchyme expressing Msx-1 includes areas of highly proliferative cells and has in vitro chondrogenic potential. The region of mesenchymal cells expressing the Msx-2 gene overlap with areas of developmentally programmed cell death which also contain very few proliferative cells and lack chondrogenic potential in vitro. These results are consistent with the possibility that Msx-1 may be involved in the outgrowth of the mandibular arch and Msx-2 may be involved in both developmentally programmed cell death and delineating the non-chondrogenic region of the medial part of the mandibular arch.

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival eipthelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular martix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibitied enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular martix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.

Development of the mouse embryonic mandible from days 9 to 14 involves tissue interactions in the formation of bone, cartilage, salivary glands and teeth. Growth factors may play an important role in these interactions. Epidermal growth factor (EGF) mRNA expression has been characterized and its presence has been shown to be necessary for odontogenesis. In addition, retinol alters the pattern of dental lamina formation; this effect is correlated with an alteration of the expression of the mRNA for this mitogen (EGF). Transforming growth factor-alpha (TGF alpha) mRNA expression has now been characterized by polymerase chain reaction for this entire period of development (days 9-14). Although the mRNA is present at the same time as EGF (days 9 and 10 only), retinol does not alter the expression of this mitogen as it does EGF. This suggests that retinoids may act to control the proliferative pattern of the dental lamina through EGF expression and not TGF alpha expression, although mRNAs for both mitogens are present at the same time.

Retinoids alter the patterning of murine odontogenesis in vivo and stimulate epithelial proliferation. Because odontogenesis is dependent on proliferation of mandibular epithelium, the effects of retinol on the patterning of odontogenic epithelium were studied. These experiments control for developmental stage, applied retinoid concentration and duration of exposure. Explants exposed for 24 h to 0.1 μg/ml of retinol exhibited enhanced odontogenesis. Day-9 mandibles exposed to retinol at 1–5 μg/ml had altered epithelial patterns consistent with those in previous in vivo experiments, inclucling supernumerary epithelial buds in regions associated with supernumerary incisors in vivo . These changes were associated with a dose-dependent increase in epithelial proliferation and a prolonged expression of epidermal growth factor (EGF) mRNA. Altered expression of EGF mRNA may be responsible for the disrupted pattern of the dental lamina. This is the first report of a retinoid-induced alteration in EGF mRNA expression.

11 This additional skeletogenic potential of the cranial neural crest makes it qualitatively different from trunk neural crest and, in some ways, similar to mesodermal tissue. One skeletal derivative of the cranial neural crest is Meckel's cartilage. Meckel's cartilage is a central rod of cartilage that runs the length of the developing mandible.11 ~14 Initially, the mandibular skeleton consists only of Meckel's cartilage, which presumably functions developmentally as a skeletal element. Later, Meckel's cartilage becomes surrounded by six membranous bones.1113 In birds this cartilage persists into adult life,1115 but in mammals it is only a temporary structure that disappears within the body of the mandible.1116 The role of Meckel's cartilage in mammalian mandibular bone formation and even basic information on the migratory patterns and differentiative fates of mammalian neural crest cells are not well understood.

To study the process of tissue remodeling that occurs during wound healing, radioactive proteoglycan ([35S]-PGS) was used to assay for enzymatic activities present in the extracellular fluid of healing tissue. Mice, wounded by removal of a 2 × 1.5 cm patch of skin from the dorsal surface, were sacrificed after 3 days of healing. Granulation tissue (1 cm2) was removed, spread onto a sterile wire mesh support and placed in the center well of an organ culture dish. To each well was added 1 ml MCDB medium supplemented with 10% fetal calf serum and antibiotics and 5–20 µl of [35S]-PGS (100,000 cpm/10 µl). Medium, removed from the well by aspiration after 24 and 48 h of culture, was boiled 5 min at 100 °C and stored frozen at -20 °C. Alterations of the PGS were assayed with a Sepharose 4B column (1 × 50 cm) which had an excluded and included volume of 17 and 46 ml, respectively. PGS, incubated without cells or with tissues from unwounded animals, eluted at 26 ml. PGS, incubated with granulation tissue and cultured for either 24 or 48 h, eluted from the Sepharose 4B at 29 ml, a 10% increase in elution volume, suggesting that the size or shape of the PGS has been altered by enzymes secreted by the cells of the granulation tissue. In contrast, PGS incubated with tissues from unwounded animals or without granulation tissue showed no changes. These data suggest that enzymatic activities secreted by cells of granulation tissue may be involved in remodeling during healing.

In the Mov13 mouse mutant, transcription of the alpha 1 (1) collagen gene is blocked by a retroviral insert in the first intron. We now report that teeth derived from homozygous embryos produce a dentin layer containing normal amounts of collagen 1. In situ hybridization and RNAase protection experiments indicate that the mutant allele is efficiently transcribed in odontoblasts, in contrast to other cell types. Correct splicing of the primary transcript containing the viral sequence results in a functional alpha 1 (1) collagen mRNA. The absence of a mutagenic effect in odontoblasts, as opposed to fibroblasts, suggests that the retroviral insert interferes with tissue-specific transcriptional control of the alpha 1 (1) collagen gene, most likely by inactivating cell-type-specific cis-acting regulatory elements.

Embryonic mouse tooth germs treated with L-azetidine-2-carboxylic acid cease their development, undergo a regression of the enamel organ, and do not maintain the histological characteristics of the explanted dental organ. On the other hand if procollagen is added exogenously to explants continously treated with L-azetidine, the effects of the inhibitor are not seen and the tissue is maintained. Thus, exogenously supplied procollagen supports morphogenesis in tooth rudiments that are unable to synthesize procollagen.

At 13 days of development the epidermis of mice homozygous for the pupoid foetus (pf/pf) mutation varies in thickness between one and ten cell layers. By 16 days of development cells from the dermis have invaded the epidermis and may be found throughout the epidermis and on its surface. Among these cells are nerve fibres and Schwann cells as well as other unidentified cells. Antibodies directed against fibronectin bind to these abnormal groups of cells in the mutant epidermis and on its surface. A basal lamina, as determined by ultrastructure and by the immuno-fluorescent localization of laminin, was always found at the interface of the mutant epidermis and the invading cell population. By 19 days of development the mutant epidermis is thickened and is permeated by a network of cells including nerve fibres, Schwann cells, blood vessels, and collagen and fibronectin-secreting cells. A basal lamina always separates these groups of invading cells from the epidermal cell population.

Embryonic molars and incisors were dissected from mandibles of 15-day post-fertilization C57BL/10 mouse embryos and were cultured in vitro for six days on agar-solidified Eagle's basal medium. Experimental explants were cultured on medium which was the same as the control except that 50, 75 or 100 microgram/ml tetracycline was added. Treated explants of both incisors and molars were suppressed in development and reduced in size. Enamel organs and dental papillae of all tooth germs subjected to higher tetracycline concentrations were abnormal in structure and differentiation of ameloblasts and odontoblasts was inhibited. Explants treated with higher dosage levels of the drug were more severely affected than those exposed to lower concentrations. Recovery from the suppression induced by tetracycline was observed in explants transferred to control medium for four days of growth following treatment. Differentiated ameloblasts and odontoblasts observed in the recovering tooth germs indicated that the inhibition in development was temporary. The results of this study showed that tetracycline can alter dental development in vitro prior to mineralization. The observed inhibition may be related to a disruption of collagen biosynthesis which is thought to play a role in the controlling epithelial-mesenchymal interaction involved in tooth germ morphogenesis.

First molar tooth germs were dissected from mandibles of mouse embryos 16, 17, 18 and 19 days of gestational age. The tooth rudiments were separated into dental papilla, dental sac and enamel organ after enzymic digestion. These embryonic dental tissues were recombined and grown for 4 weeks in anterior chambers of eyes of homologous adult mice. The removal of the dental sac from recombinations of only dental papillae and enamel organs did not significantly alter the proportion of grafts which formed teeth when compared with those grafts which consisted of all 3 dental tissues. Both groups of recombinants formed teeth in 50–80 per cent of the grafts cultured. The dental papilla and dental sac tissues showed the same inductive activity with the enamel organ with the 16-day-old tissues. However, the dental papilla cells maintained their inductive capacity for a longer period. Whereas 70 per cent of the 16-day-old tissue recombinations of dental sac and enamel organ formed teeth, only 15 per cent of the recombinations of older ages of the same tissues formed teeth.

This study examined the effects of hadacidin, an analogue of L-aspartic acid, on epithelial fusion, breakdown, and mesenchymal contact in explants of fetal mouse palatal shelves. Shelves from 14-day C57BL/6Js were explanted to agar-solidified Eagle's basal medium. Control palates grown in this medium mimicked normal closure; i.e., an epithelial seam formed, broke down, and mesenchymal contact occurred in vitro. Experimental palates were grown in this medium supplemented with 1 or 2 mg/ml hadacidin or with concentrations of L-aspartic acid equal to or in excess of that of hadacidin. Histological observations of explants exposed to 2 mg of hadacidin for 2 days indicated that cells of the marginal epithelia became adherent to form epithelial bands, but that subsequent epithelial breakdown and mesenchymal contact did not occur. The presence of mi to tic figures in the epithelial bands and in the mesenchyme of treated explants indicated that hadacidin inhibited breakdown, and, in addition, that it did not severely suppress or debilitate all cellular activity. This observation supports the fact that explants treated with hadacidin for 2 days, when moved to control medium for an additional 2 days, were able to undergo epithelial-band breakdown and mesenchymal penetration. Preliminary data from explants treated with hadacidin and L-aspartic acid indicated that competitive reversal by the amino acid did not occur consistently in vitro at concentrations of the amino acid 3 times that of hadacidin.

First and second branchial arches were dissected from mouse embryos of 9-13 days gestational age. The epithelial and mesenchymal components were separated after enzymic digestion. Scanning electron microscopy did not reveal a dental lamina along the dental arches before day 12, after which the lamina was formed locally in the incisor and molar regions. There was no epithelial down-growth in the diastema region. Heterotypic recombinations of mandibular arch and second branchial arch tissues showed that early mandibular arch epithelia, before day 12, have odontogenic potential and can elicit the formation of a dental papilla in non-odontogenic, neural-crest-derived mesenchymal cells of the second arch. However, the mandibular mesenchyme must interact with mandibular epithelium in order to have the competence to induce teeth in non-odontogenic epithelium.

Dipyridyl inhibits the morphogenesis of tooth germs, but the germs recover when transferred to control medium. In this study, the effect of dipyridyl on basement membrane was investigated in vitro. The basal lamina was always present, but the subjacent collagen fibrils disappeared in the presence of dipyridyl and reappeared during recovery in control medium.

The inhibitory action of tetracycline on the development of embryonic mouse incisors cultured in vitro was examined. Explants exposed to tetracycline were severely inhibited in development. In contrast, tooth germs cultured in the presence of both tetracycline and iron escaped inhibition and attained a stage of development which compared favorably with the controls.

The development of specialized epithelial derivatives in mammalian skin such as hair and vibrissae has not been subjected to detailed experimental analysis. Chase (1) and Rawles (2) suggested that the development of hair and of vibrissae involved an inductive interaction between the epidermis and dermis of developing skin. This suggestion was based mainly on certain similarities between the developmental anatomy of hair and vibrissae, and that of feathers.

Pelage and vibrissal follicle development was examined in experimental combinations of embryonic epithelial and mesodermal tissues isolated from the snout, dorsum and plantar surfaces of the foot plate. Following separation of the tissues by cold tryptic digestion, experimental and control explants were grafted to the anterior chambers of homologous adult eyes. When mesodermal tissues from follicle-bearing areas are combined with glabrous epithelium from the foot plate, hair follicles and sebaceous glands are induced. Complete and harmonius development occurs in the intraocular grafts. Experimental exchanges of tissues between the two hair-bearing regions indicated that epithelium from the snout and dorsum can participate with homologous and heterologous follicle-bearing mesoderm to produce hair follicles. These data provide conclusive evidence for the inductive role of the dermal papilla during the ontogeny of pelage and vibrissae follicles.

Studies of epithelio-mesenchymal interactions during embryonic organogenesis have led to a number of conclusions regarding the nature of cellular and tissue differentiation (McLoughlin, 1963; Grobstein, 1967). For example, the importance of both the epithelium and the mesenchyme and the dependence of some systems on a limited number of specific mesenchymal tissues have been pointed out (Hilfer, 1968). Intimately connected with the analysis of the factors that elicit differentiation during such interactions is the question of structural specificity of the differentiated structure. Is the directive for the final form of the structure resident in the epithelium, in the mesoderm, or in both? Can a seemingly stable epithelium undergo transformation to a more labile state and respond to a new interaction with the result that a new epithelial structure is formed (Billingham & Silvers, 1963, 1968)?

The response of embryonic mouse dental epithelium and mesoderm to tissues of ectopic origin was examined. Isolated molar or incisor mesoderm was confronted with epithelium isolated from the plantar surface of the embryonic mouse foot plate or from the snout. Harmoniously structured teeth were formed from the foot epithelium and incisor or molar mesoderm. These data are interpreted as an unequivocal demonstration of the inductive role of the dental mesenchyme. Teeth were absent in confrontations of dental mesenchyme and snout epithelium. The presence of hair follicles in these explants is described and discussed with reference to other integumental epithelio-mesenchymal interactions. Dental epithelium forms keratinizing surface-like epithelium and invading bands of epithelium in association with foot mesoderm; definitive structures are not formed. On the other hand, when incisor or molar epithelium is associated with snout mesoderm, hair follicles are seen in addition to keratinizing surface-like epithelial configurations. The roles of the epithelial and mesenchymal tissues and the nature of epithelio-mesenchymal interactions in the developing mouse integument are discussed.

Previous studies of adrenocortical activity during sleep deprivation revealed unchanged or lowered plasma 17-hydroxycorticosteroid (17-OHCS) levels and either unchanged, lowered, or elevated urine 17-OHCS values, depending on the subject and the length of the deprivation period. In the present study, plasma 17-OHCS, urine 17-OHCS, and urine vanillymandelic acid (VMA) were measured during 205 hr. of wakefulness, the rationale being that an extended period of sleep deprivation might result in more definitive physiological responses. Plasma 17-OHCS in all 4 subjects decreased to a low point at about 90 hr., then increased to peak values at about 170 hr. Urine 17-OHCS values tended to reflect changes in plasma 17-OHCS levels. Urine VMA excretion varied considerably among the 4 subjects. The results suggest that prolonged sleep deprivation per se results in only mild, if any, specific activation of the pituitary-adrenocortical axis and in variable increases of catecholamine biosynthesis. Differing patterns of physiological activity may occur among sleep-deprived subjects.

The ability of fragments of incisor enamel organ and lip-furrow epithelium from 15-and 16-day old embryonic mice to regulate into harmonious tooth constructions is described. The cervical loop and upper half portions of the incisor enamel organ were confronted with incisor or molar dental papillae. Similar combinations were made from lip-furrow epithelium and incisor or molar papillae. The cultures were grown in the anterior chambers of homologous host eyes. The epithelial fragments from the incisor enamel organ when associated with the dental papillae reconstruct teeth typical in all respects; enamel and dentin matrices are deposited. Lip-furrow epithelium arises from the oral epithelium and is temporally and spatially related to the incisor dental epithelium proper. This ectopic epithelium was confronted by incisor and molar papillae. Harmonious teeth developed in these explants. It is concluded that the ability of the dental papillae to elicit new cytodifferentiative and biochemical syntheses from the lip-furrow epithelium indicates that the dental papillae act inductively during tooth ontogeny. The shape of the teeth reconstructed from enamel organ fragments and lip-furrow epithelium were incisiform or molariform in response to the incisor or molar dental papillae. These data confirm the conclusion that the structural specificity for tooth shape resides in the dental papilla.

SYNOPSIS The development of hair teeth and feathers is leviewed in the context ot experimental manipulation of dermal epideimal interactions The inductive role of the del mis is described, and the consequences of altering the ages and sources of the interacting tissue components is demonstrated The importance of examining the developmental capibilities of these tissues after disruption of the dermal epidermal inteiface as well as in intact explants is discussed In addition the role of the basement membrane m establishing and stabilizing integumental denvatrves and the role of collagen synthesis and depositionis examined

During its development the epidermis of the pf pf mutant mouse is invaded by cells from the underlying dermis. These invading cells establish a network of cells including fibroblasts, endothelial cells, and nerve fibers, throughout the epidermis. Subsequent to these events the keratohyalin protein, filaggrin, is drastically reduced and keratinization fails to occur. Heterotypic tissue recombinations indicate that the pf gene is not expressed in the skin. After simply grafting whole mutant dorsal skin, filaggrin synthesis is initiated and an orderly process of epidermal differentiation is achieved. These results suggest that the pf gene acts systemically and that the failure of epidermal differentiation in the mutant occurs secondary to abnormal epidermal organization.

The epithelial root sheath (ERS) was isolated from first molars of 5-day-old post-natal CD-1 mice using trypsin. After isolation, ERS cells remained viable in vitro and immunohistochemical examination of cultures confirmed the epithelial phenotype and the absence of mesenchymal contamination. Recombinants of isolated ERS and dental papilla resulted in odontoblast differentiation within cells of the dental papilla, and the formation of root-like fragments of dentine after 2 weeks of intra-ocular grafting. These findings indicate the inductive influence of the ERS on dental papilla cells.

This chapter deals with the tissue interactions in developing skin, teeth, and related ectodermal derivatives. Epithelial-mesenchymal interactions that take place between labile epithelia and mesenchyme are the inductive interactions that occur after primary induction of the nervous system and the axial skeleton. These interactions are responsible for the differentiation of the major organ systems of vertebrate embryos.

The investigative approaches of experimental teratology and experimental embryology do not differ fundamentally in their strategies. In fact the only difference between the two disciplines may well be their points of departure; that is whether or not the production and understanding of the development of abnormal or normal structure is the major focus of the investigation. Yet, workers in the two disciplines often have failed to incorporate or specifically relate to each other’s basic advances. It is the purpose of this chapter to examine advances in experimental embryology of epithelio-mesenchymal interactions with a view toward relating these recent data to teratological problems. There have been a number of advances in the understanding of the mechanisms involved in the development of epithelial derivatives of the integument (haif, glands, etc.) and oral mucosa (teeth, glands, etc.) and in epithelio-mesenchymal interactions involved in development of skeletal derivatives (limbs, palate, cranial bones). The overriding theme of epithelio-mesenchymal interactions encompasses all of the most often studied experimental models in teratological research as well as in experimental embryology.

The present study examined the effect of aging on epithelium and on its ability to respond to an inductive stimulus provided by murine dental papillae. In fetal CD-1 mice, 15- to 17-day molar mesenchyme was combined with 15- to 19-day epithelium from the secondary palates. Enamel organs were separated from the dental papillae, and palatal epithelium was peeled away from its underlying mesenchyme after treatment with 1% trypsin. Recombinants of epithelium and papillae were initially cultured on a solidified complex medium for 24 hr followed by an additional 10-14 days of intraocular explanation. Control specimens consisted of isolated molar papillae. Nineteen of 88 isochronal, heterotypic recombinations formed teeth. None of the 46 heterochronal, heterotypic grafts of 18- and 19-day palatal epithelium combined with 15- to 17-day molar papillae-produced teeth. Instead, keratin-filled epithelial cysts and bone spicules were formed. Isolated control molar papillae often formed bone in the intraocular sites but did not form teeth or contain epithelium. These results show that palatal epithelium is first restricted to its developmental pathway at 18 days of gestation. Younger epithelium can convert to functional ameloblasts that secrete enamel protein. In addition to the change in gene expression, normal tooth morphology is attained. The loss of competence of the palatal epithelium at 18 days gestation coincided with the acquisition of stratum corneum and the attainment of the fully differentiated state. The oral surface of palatal epithelium appears to be determined histogenically and morphogenically at 18 days of gestation in mice.

A number of synthetic, possibly naturally occurring, psychotogenic indolic compounds have been of interest to investigators for many years.1In addition, there have been a large number of studies covered in recent reviews on "abnormalities" of indole compound excretion in various mental disorders, including: phenylketonuria, Hartnup's disease, pellagra, Huntington's chorea, porphyria, and both the affective and schizophrenic psychoses.2-4The inconsistencies of findings, particularly in the area of psychoses in the face of continued studies finding significant differences, suggested to us the possibility that extraneous variables might have been operating in a systematic way to produce these results. In a series of studies undertaken to explore some of these, we have demonstrated the importance of controlling for urine volume, general dietary habits, amount of tryptophan in the diet, the time of sample collection and bowel flora. In addition, while studying the potential role of nonspecific stress factors, we have

SummaryPsychological, physiological, and biochemical studies were conducted on 4 healthy young-adult males while they underwent 120 hr. of sleep deprivation. The subjects quickly Formed a group and dealt with this contrived stress in much the same way that groups cope with stressful environments. Al

Tooth germs grown on Eagle's medium solidified with agar will differentiate in vitro. Normal development was suppressed by beta-2-thienylalanine (1 or 2 millimols per liter), but it proceeded when phenylalanine and beta-2-thienylalanine were present in equimolar concentrations. This effect may have been due to interference with protein metabolism in the dermal papillar cells.

A study in which four healthy adult males underwent 205 hours of sleep deprivation indicated that although they suffered transient ego disruptive phenomena, they did not appear to undergo psychopathological reactions extending beyond the period of sleep deprivation. Detailed psychological, physiological, and biochemical findings are reported.

Introduction This is a study of psychological, physiological, and biochemical variables associated with starvation. Although laboratory experiments on rats 27-30 indicate that these animals respond to acute starvation with adrenalcortical activation, there is reason to question whether this occurs in humans and other animals. Keys 10 reports that starving man has a low metabolic rate, bradycardia, hypotension, hypothermia, and great muscular weakness. A starving man typically states he is depressed and his facies suggest depression more than emaciation. He becomes increasingly quiet, somber, apathetic, and slow in motion. Although he reports increasing irritability, there is little overt indication of it. The starving man conserves energy in every possible way. Severe neurotic symptoms developed in the Minnesota Experiment 11 in which 35 "normal" young men were starved. There was a full return to normal on nutritional rehabilitation. In the Minnesota Experiment neither

This is a report of studies of the sexual behavior of the ARL Colony chimpanzees from July, 1966, through August, 1967. The ARL Colony has an innovation designated the DPS Chimpanzee Consortium which permits the containment of social groups of chimpanzees on 30 acres of New Mexico desert. The solicitation, mounting and copulatory behavior of the consortium animals was similar in form to that reported in field studies. However, the incidence of both copulatory and masturbatory behavior was much higher than in the wild. In spite of this overall high incidence of copulatory behavior, three of the five adult males, who were cage-reared and presumably sex-segregated until maturity, were not observed to copulate when they had the opportunity to do so. However, they seemed to show no reduction in sexual drive. Two masturbated frequently and all three practiced perverse sexual acts. Both adolescent and juvenile males were interested in estrous females and actively solicited, mounted and copulated with them frequently. Three of the four adult consortium females tended to avoid copulation. However, this reluctance of adult females to copulate could be overcome by a large aggressive male and all of the adult consortium females became pregnant. It is hypothesized that feral-born but cage-reared, sex-segregated chimpanzees are capable of normal mating behavior if they are given an opportunity to experiment and learn before a critical period occurring during late adolescence and early adulthood. An adult who has passed this critical period is either unable to learn to copulate or to orient adequately toward an estrous female, although the sexual drive is manifested in masturbation and other ways. There was also a higher incidence of nonsexual mounting and presenting by consortium animals of both sexes than has been reported in the wild. The older females frequently “mothered” small juveniles, but most noteworthy was the fact that many male chimpanzees also interacted with small juveniles in a maternal-like manner. Unlike wild females, the consortium females frequently displayed aggressive dominance behavior toward males as well as other females. The consortium females were also the frequent victims of male aggression, which has been rarely noted in the field. Hypotheses to explain these apparent gender role shifts are offered.

Summary A longitudinal study of three cases of ulcerative colitis is presented. Particular emphasis is placed on a differential response to varying stressful situations. If the patient responded to a stress with anxiety or anger, no effect on the colitic process was noticed. If the anger stimulated conscious or unconscious guilt, the patients were apt to complain of headaches. The stress situations which triggered episodes of ulcerative colitis were those in which the patients experienced a real or fantasied object loss and which they were unprepared to accept or work through. Preceding or concomitant with the colitic process was an atypical passive inactive depression which can be considered as a morbid form of grief. The bearing of these findings of the pathogenesis of ulcerative colitis and their implications in the problem of specificity are discussed.