Epithelial influences on skeletogenesis in the mandible of the embryonic chick

Intact mandibular processes and the enzymatically separated mesenchymal and epithelial components of the mandible from embryonic chicks of 2.5- to 5-day incubation (Hamburger and Hamilton, '51: stages 16-25) were grown individually, either in organ culture or as grafts to the chorioallantoic membranes of host embryos. The differentiation of cultured and grafted intact mandibular processes was histologically normal, but the time of histodifferentiation differed from that in vivo. The histodifferentiation of cultured and grafted mandibular mesenchyme grown isolated from its epithelium depended upon the age of the embryo from which the mesenchyme had been obtained. Intramembranous ossification producing membrane bones of the mandible occurred in mesenchyme isolated from 4.5- to 5-day embryos (HH 24–25), but did not occur in mesenchyme isolated from younger embryos. Cartilage (Meckel's) and subperichondrial bone in the articular process of Meckel's cartilage differentiated in mesenchyme isolated from embryos of all age groups tested (HH 16–25). Mandibular mesenchyme, therefore, requires the presence of epithelium until 4.5 days of incubation if the membrane bones of the mandible are to differentiate; if epithelial influences are required for Meckel's cartilage and subperichondrial bone formation, they are not required beyond 2.5 days of incubation. Mandibular epithelium isolated from its mesenchyme became layers of squamous cells in culture; but when grafted onto the chorioallantoic membrane, the epithelium became underlain by host fibroblasts and differentiated into a stratified squamous epithelium. Mandibular epithelium, therefore, is capable of differentiation in the presence of foreign fibroblasts derived from the chorioallantoic membrane.     

Inhibition of membrane bone formation by vitamin A in the embryonic chick mandible

Embryonic chick mandibles (Hamburger and Hamilton [HH] stages 17-25) were cultured in the presence of various concentrations of vitamin A to determine the effect of hypervitaminosis A on membrane bone formation. In normal development, the mandible differentiates a centrally located Meckel's cartilage surrounded by membrane bones. Mandibles cultured without added vitamin A differentiated normally, though the timing of differentiation was retarded from that in ovo. Treatment with vitamin A interfered with skeletogenesis to varying degrees depending upon the initial age of the explant and the concentration of vitamin A. At low concentrations of vitamin A (1 microgram/ml), neither cartilage nor membrane bone formed in young explants (HH stage 17), whereas cartilage formed in 78% and membrane bone in 11% of older explants (HH stage 25). Higher concentrations of vitamin A (2-4 micrograms/ml) inhibited membrane bone formation in all explants, and 4 micrograms/ml of vitamin A inhibited chondrogenesis in most (88%) of the older explants. To determine whether tissue interactions influence this effect of vitamin A on skeletogenesis, mandibular mesenchyme was separated from its epithelium and treated with vitamin A. Under normal culture conditions, isolated mesenchyme (HH stage 25) differentiated both cartilage and membrane bone. Hypervitaminosis A inhibited membrane bone formation in the isolated mesenchyme at all levels tested (1-4 micrograms/ml) and inhibited chondrogenesis at levels 2-4 micrograms/ml. Hence, vitamin A can act directly upon the mesenchyme to inhibit both membrane bone formation and chondrogenesis, but its action is mitigated by the presence of the epithelium.     

Development of the frontal bone and cranial meninges in the embryonic chick: An experimental study of tissue interactions

The frontal region of the embryonic chick was studied to determine whether epithelial influences are necessary for frontal bone development. The frontal bone is a membrane bone, of neural crest and head mesodermal origin, which develops within mesenchyme sandwiched between two epithelia, neural ectoderm and epidermis. Rudiments were treated enzymatically to separate epithelial and mesenchymal tissues. Frontal mesenchyme then was grown as chorioallantoic membrane grafts either in the presence or absence of neural ectoderm and/or epidermis. The results indicate that neural ectoderm, though required during early stages of development to induce frontal bone development (Schowing, 1968), is not required during later stages (HH 22–30, the stages tested in this study) for osteogenesis. Epidermis, however, was shown to be required for frontal bone development during the stages tested. Frontal mesenchyme formed bone when epidermis was present on the outer aspect of the mesenchyme, and did not form bone when the epidermis had been removed prior to grafting, whether or not neural ectoderm was present. This dependence upon epidermis continues beyond the onset of meningeal differentiation. Once the outer ectomeninx-dermis is distinguishable from the inner endomeninx, osteogenic capabilities are confined to the ectomeninx-dermis layer. Furthermore, the ectomeninx-dermis layer attached to epidermis is able to form membrane bone in the absence of the endomeninx and neural ectoderm. The endomeninx, though normally nonchondrogenic, was shown to be capable of forming cartilage when the neural ectoderm is removed. Neural ectoderm, therefore, may have an inhibitory effect on chondrogenesis in the endomeninx.     

Expression and regulation of the LIM homeodomain gene L3/Lhx8 suggests a role in upper lip development of the chick embryo

LIM-homeodomain (Lhx) genes constitute a gene family that plays critical roles in the control of pattern formation and cell type specification. We have identified a chicken L3/Lhx8 gene, which was widely expressed in the craniofacial region. Whole-mount in situ hybridization showed that L3/Lhx8 mRNA was expressed from stage 15--31 HH in overlapping domains of the maxillary process. Frozen sections revealed these signals in the mesenchyme underneath the epithelium. To determine whether the expression of L3/Lhx8 in the maxillary primordia required signals from the overlying oral epithelium, maxillary processes of stage 23 HH chick embryos were transplanted into the limb bud, in which the mesenchyme was grown in the presence or absence of oral epithelium. The maxillary mesenchyme with epithelium showed significant levels of L3/Lhx8 gene expression. In contrast, no expression of L3/Lhx8 was detected in the epithelium-free mesenchyme. To further explore signaling molecule(s) responsible for Lhx induction, a bead, soaked in either Fgf-8b or TGF-β3, was implanted into an epithelium-free mesenchymal graft. Both TGF-β3 and Fgf-8b beads induced expressions of L3/Lhx8 in epithelium-free mesenchymal grafts. Our data suggest that the L3/Lhx8 gene contributes to epithelial mesenchymal interaction in facial morphogenesis and that Fgf-8b and TGF-β3 were, at least in part, responsible for the Lhx expression in the maxillary process.     

Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme in vitro

Separation and recombination experiments, employing a variety of tissue configurations in organ culture, were performed to determine the extent to which the epithelium of the maxillary process influences the viability of the underlying mesenchyme during organogenesis. The results of these studies indicated that the viability of mesenchyme of the maxillary process of early stage embryos was severely impaired when separated from the overlying epithelium. The influence of epithelium on the viability of mesenchyme was stage dependent; that is, the requirement for the presence of epithelium for the maintenance of the viability of mesenchyme became progressively less pronounced at older developmental stages. The response of mesenchyme to the presence of recombined epithelium resulted in the appearance of a delimited zone of influence extending, within specific boundaries, from the epithelial-mesenchymal interface. Preliminary data from homotypic (maxillary epithelium-maxillary mesenchyme), heterotypic (limb apical ectodermal ridge-maxillary mesenchyme) and heterochronic (stage 28 epithelium-stage 22 mesenchyme) recombination experiments indicated that viability of mesenchyme could be achieved only through direct epithelial-mesenchymal contact which allowed restoration of normal morphological relationships at the interface of the two tissues.     

Role of Sonic hedgehog in patterning of tracheal-bronchial cartilage and the peripheral lung.

Sonic hedgehog (Shh) was conditionally deleted in respiratory epithelial cells of the embryonic lung in vivo. Deletion of Shh before embryonic day (E) 13.5 resulted in respiratory failure at birth. While lobulation was not perturbed, the lungs were hypoplastic, with reduced branching of peripheral lung tubules, evident from E13.5. Smooth muscle and endothelial cells were absent or reduced, the latter in relationship to the loss of peripheral lung parenchyma. Tracheal-bronchial ring abnormalities occurred when Shh was deleted between E8.5 and E12.5. Deletion of Shh later in gestation (after E13.5) caused mild abrogation of peripheral branching morphogenesis but did not disrupt tracheal-bronchial development. Defects in branching morphogenesis and vascularization seen in Shh null mutant (Shh(-/-)) mice were substantially corrected when SHH was ectopically expressed in the respiratory epithelium; however, peripheral expression of SHH failed to correct cartilage abnormalities in the trachea and bronchi, indicating a spatial requirement for SHH expression near sites of cartilage formation. Expression of SHH by the respiratory epithelium plays an important role in the patterning of tracheal-bronchial mesenchyme required for formation of cartilage rings in conducting airways. SHH regulates branching morphogenesis and influences differentiation of the peripheral lung mesenchyme required for formation of bronchial and vascular smooth muscle.     

A test of positional properties of avian wing-bud mesoderm

Supernumerary wing structures are readily produced by grafting pieces of wing-bud mesoderm into different locations of host wing buds, but the mechanism underlying their formation remains obscure. The major aim of this study was to examine the ability of posterior quail wing-bud mesoderm, cultured in vitro long enough to lose ZPA (zone of polarizing activity) activity, to stimulate or participate in the formation of supernumerary structures when grafted into anterior slits of host chick wing buds. Small pieces of anterior and posterior quail wing-bud mesoderm (HH stages 21-23) were placed in in vitro culture for up to 3 days. After 2 days, ZPA activity of cultured mesoderm was lost. After the grafting of 2- to 3-day cultured anterior quail wing-bud mesoderm into posterior slits of host chick wing-buds, a consistently high percentage (70%-90%) of grafts result in formation of supernumerary cartilage; in this experiment, however, only a low percentage of grafts resulted in supernumerary cartilage when 2- to 3-day cultured posterior mesoderm was grafted into anterior slits. Taken with controls, these results show that positional differences exist between cultured anterior and posterior wing-bud mesoderm. Serial-section analysis of numerous operated wings has shown several patterns of contribution to supernumerary structures by cells of graft and host. Single supernumerary digits induced by grafts of ZPA mesoderm into anterior slits were normally composed entirely of host cells, but graft cells regularly contributed to skeletal elements of more complex supernumerary structures. Cartilage rods produced by anterior-to-posterior grafts were composed mostly of graft cells, but cartilage nodules and the bases of some rods were often mosaics of chick and quail cells. The results support the proposition that mesodermal cells of the quail wing-bud possess a form of anteroposterior positional memory, but its nature and the means by which the memory of grafted cells interacts with host mesoderm are still not clear.     

Thymic epithelium tolerizes chickens to embryonic graft of quail bursa of Fabricius

We demonstrated previously that isotopic and isochronic grafts of the quail bursa of Fabricius rudiment performed at 5 days of incubation (E5) into chick embryos resulted in the development of a chimeric bursa whose chick host B lymphocytes and accessory cells differentiated in a foreign, quail epithelial environment. Such animals reject their grafted bursa by the age of 2-3 weeks post-hatching (1,2). Isotopic embryonic grafts of the thymus epitheliomesenchymal anlagen from the quail donor of the bursal rudiment were carried out at E4.5 (before their colonization by hemopoietic precursor cells), following partial or complete host thymectomy. The quail thymic epithelial stroma was accepted and invaded by chick hemopoietic precursor cells that further differentiated into lymphocytes and dendritic cells. Tolerance of the foreign bursa was induced in such thymobursal chimeras. This demonstrates that the thymic epithelium has the capacity to induce tolerance of xenogeneic rudiments when both grafts are implanted at early stages of embryonic development. We also report on the production of two birds in which removal of the chick host thymus was complete thus generating chimeras in which host T and B lymphocytes differentiated in a completely xenogeneic epithelial environment.     

Upper beak truncation in chicken embryos with the cleft primary palate mutation is due to an epithelial defect in the frontonasal mass.

In this study, we used the chicken mutant strain known as cleft primary palate (cpp) to study the mechanisms of beak outgrowth. cpp mutants have complete truncation of the upper beak with normal development of the lower beak. Based on structural analysis and grafts of facial prominences, we localized the defect to the frontonasal mass and its derivatives. Several explanations that would account for the outgrowth defect were investigated, including abnormal expression of genes in the frontonasal epithelium, intrinsic defects in epithelium and/or mesenchyme defects in epithelial-mesenchymal signalling, a localized decrease in cell proliferation or a localized increase in programmed cell death. One of the genes expressed in the frontonasal epithelial growth zone, Fgf8, failed to down-regulate and was maintained for at least 48 hr beyond the time when down-regulation normally occurs. Recombination experiments further illustrated that the frontonasal mass epithelium was abnormal in the cpp mutants, whereas mutant mesenchyme was capable of normal outgrowth when combined with wild-type epithelium. Cell proliferation was not decreased in mutant embryos nor was cell death initially increased. Later, at stages 31-32, when the prenasal cartilage begins directed outgrowth, there was an increase in cell death within the mutant upper but not lower beak cartilage. The cpp beak truncation, therefore, is due to an epithelial defect in the frontonasal mass that is coincident with a failure to down-regulate expression of Fgf8.     

Meckel's cartilage in the human embryo and fetus

This work studied the development of the ventral part of Meckel's cartilage in a series of human embryos (classified in stages) and fetuses. These stages appeared particularly important: stage 16, appearance of Meckel's cartilage; stage 20, beginning of membranous ossification of mandible; and stage 23, end of the embryonic period (8th week). The primitive bony nodule which develops from the embryonic mesenchyme appears as a double bony layer forming a groove containing the neurovascular bundle, into which the dental lamina is also invaginated. It was concluded that during the fetal period, the cartilage participates in the formation of the body of the mandible in an area close to the mental foramen via endochondral ossification. The cartilage disappears in parallel with the development of ossification by the sixth month. © 1994 Wiley-Liss, Inc.     

Mineralization processes in demineralized bone matrix grafts in human maxillary sinus floor elevations.

For reconstruction of the severely resorbed lateral maxilla for dental implant placement, one of the successful procedures is to elevate the maxillary sinus floor by implanting demineralized bone matrix (DBM). We studied bone formation in DBM grafts in the lateral maxilla in humans by means of histology and histomorphometry. Six months after grafting, at the time of dental implantation biopsies were taken from the grafted areas of seven patients. All biopsies contained mineralized matrix (MM) in the grafted area. At close inspection, three types of mineralization were found. First, lamellar biomineralization was seen in and near the maxillary host bone. Second, remineralization was observed in some particles that probably had not been completely demineralized. In the area connecting the graft and host bone, where woven bone was formed against DBM particles, a third mechanism was detected. In this case many dotlike foci of remineralization appeared close to the bone-DBM interface. The remineralized DBM and woven bone were both subsequently remodeled. Bone formation was most active in the area adjoining the maxillary host bone. We conclude that in human sinus floor elevation, allogenic DBM increases mineralized tissue volume by osteoconduction that is supported by the remineralization processes. Osteoinduction by this material seems questionable.     

Bladder tissue formation from cultured bladder urothelium.

Tissue recombination is a powerful method to evaluate the paracrine-signaling events that orchestrate the development of organs using the in vivo environment of a host rodent. Studies have reported the successful generation of primary cultures of rodent bladder urothelium, but none have reported their use to recapitulate bladder tissue with tissue recombination. We propose that primary cultured bladder urothelium, when recombined with inductive embryonic bladder mesenchyme, will form bladder tissue in a recombination model. Adult rat bladders were isolated and urothelium obtained. Sheets of bladder urothelium were re-suspended in collagen and maintained in tissue culture. After expansion (>20 passages), the urothelium was recombined with embryonic day-14 mouse bladder mesenchyme, then grafted beneath the renal capsule of immunocompromised mouse hosts. Grafts were harvested after 28 days. Control grafts were performed with bladder mesenchyme alone, cultured bladder urothelium alone, and collagen matrix alone. Final tissues were evaluated with staining and immunohistochemistry (H&E, Gomori's trichrome, broad-spectrum uroplakin, and smooth muscle actin alpha and gamma). Immunocytochemistry on cultured urothelium for broad-spectrum keratin, vimentin, and broad-spectrum uroplakin confirmed pure populations, void of mesenchymal contaminants. Staining of recombinant grafts demonstrated bladder tissue with mature urothelium and stromal differentiation. Control tissues were void of bladder tissue formation. We have successfully demonstrated that a chimeric bladder is formed from primary cultured bladder urothelium recombined with embryonic bladder mesenchyme. This is a powerful new tool for investigating the molecular mechanisms of bladder development and disease. Future applications may include the in vitro genetic manipulation of urothelium and examining those effects on growth and development in an in vivo environment.     

Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro

The thymus is surrounded by a thin layer of mesenchyme and the epithelial-mesenchymal interaction is known to be essential for the thymus development. To clarify the roles of mesenchyme in the thymus lobule formation that occurs around embryonic days 14–15 in vivo, we set up a three-dimensional organ culture system. The epithelium of embryonic day 13 thymic primordium was separated from the mesenchyme and cultured in Matrigel (reconstituted basement membrane). Addition of the mesenchyme to a chamber separated by a membrane filter induced the lobule formation of the thymic epithelium in vitro. We found that epidermal growth factor (EGF) can replace the mesenchyme for lobulation of the embryonic thymus in vitro. Among other growth factors tested, only transforming growth factor (TGF)-α was as effective as EGF, in agreement with the fact that EGF and TGF-α bind to the same receptor. These results suggest that EGF or its family members may be involved in morphogenesis and differentiation of the thymus gland epithelium, although we cannot exclude the possibility that other unknown factors are required in vivo.     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiate response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.     

Relationships between ectoderm and skeletal morphogenesis in the chick embryo limb bud

Summary When in chick embryos (H.-H. stages 22 to 25) a variously large area of ectoderm with the subjacent mesodermal layer external to the superficial vessel network, loosened from the dorsal face of the wing bud is rotated 180° in situ, or a similar ecto- and mesodermal sheet isolated from the dorsal face of the leg bud is grafted, in normal of 180° reversed orientation, onto the dorsal face of the wing bud, no changes in the normal developmental pattern of the wing skeleton ensue. As the grafted tissue, which apparently does not contain prospective chondrogenic cells, develops as a flat implant, the normal geometry of the ectodermal hull is not altered: therefore, the biomechanical conditions and the polarized growth of the skeletogenous mesenchyme of the wing bud, which seem to be controlled by the enveloping epithelium, remain practically unchanged.Morphological alterations of the skeletal pieces of the wing and formation of ectopic cartilage follow instead the implantation on the dorsal face of the wing bud, in normal or 180° reversed orientationm of an ecto- and mesodermal sheet similar to the one mentioned above but containing also a varying amount of the mesenchyme lying beneath the superficial vessel network.     

Tooth induction and temporal patterning in palatal epithelium of fetal mice

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.     

Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin βA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin βA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin βA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFβ family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.     

Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler.

Parathyroid hormone (PTH)-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR) play an essential role in controlling growth plate development. The aim of the present study was to use the deer antler as a model to determine whether PTHrP and PPR may also have a function in regulating cartilage and bone regeneration in an adult mammal. Antlers are the only mammalian appendages that are able to undergo repeated cycles of regeneration, and their growth from a blastema involves a modified endochondral process. Immunohistochemistry was used to establish sites of localization of PTHrP and PPR in antlers at different stages of development. The pattern of Indian Hedgehog (IHH) and transforming growth factor-beta1 (TGF beta1) distribution was also investigated, because PTHrP expression in the developing limb is regulated by IHH and during embryonic growth plate formation TGF beta1 acts upstream of PTHrP to regulate the rate of chondrocyte differentiation. In the antler blastema (<10 days of development), PTHrP, PPR, and TGF beta1 were localized in epidermis, dermis, regenerating epithelium, and in mesenchymal cells but IHH expression was not detected. In the rapidly growing antler (weeks 4-8 of development), PTHrP, PPR, and TGF beta1 were localized in skin, perichondrium, undifferentiated mesenchyme, recently differentiated chondrocytes, and in perivascular cells in cartilage but not in fully differentiated hyperytrophic chondrocytes. IHH was restricted to recently differentiated chondrocytes and to perivascular cells in cartilage. In mineralized cartilage and bone, PTHrP, PPR, IHH, and TGF beta1 were immunolocalized in perivascular cells and differentiated osteoblasts. PTHrP and PPR were also present in the periosteum. TGF beta1 in vitro stimulated PTHrP synthesis by cells from blastema, perichondrium, and cartilage. The findings of this study suggest that molecules which regulate embryonic skeletal development and postnatal epiphyseal growth may also control blastema formation, chondrogenesis, and bone formation in the regenerating deer antler. This finding is further evidence that developmental signaling pathways are recapitulated during adult mammalian bone regeneration.     

Ultrastructure of the developing stomach in human embryos

Summary Ultrastructural development of the stomach was studied by light, scanning electron and transmission electron microscopy, using 19 human embryos at Carnegie stages from 14 to 23 (6.8–28.0 mm in crown-rump length, 5 to 8 weeks of gestation). The precise time of appearance of differentiated characteristic structures was examined electron microscopically. The first gastric pit, with radially arranged epithelial cells beneath which the basement membrane bulged into the mesenchyme, was observed on the lesser curvature at stage 22. Although the mesenchymal condensation which would develop into the inner circular muscle layer appeared at stage 18 onward, cytoplasmic myofibrils were not observed until stage 22. Nerve fibers were first observed at stage 16, and at later stages they gathered into bundles to form a nerve plexus external to the developing inner circular muscle layer. On the basis of accurate timing of the appearance and the mode of development of these structures, possible relations between developing gastric layers were discussed. Histocytochemically, glycogen or other carbohydrates were demonstrated in the cytoplasm of the gastric epithelium throughout the stages examined. These carbohydrates were localized mainly in vacuole-like spaces in the basal part of the epithelial cells. This subcellular localization, and the amount of carbohydrate, did not change significantly during the observed embryonic period. In the serosa, carbohydrates were not detected at stages 14 and 15, but observed consistently within the vacuoles in the cytoplasm from stage 17 onward. No other layer of the embryonic stomach had detectable carbohydrates. These observations suggest that carbohydrates in the gastric epithelium at an early developmental stage are not directly related to the developing mucin secretory activity of the epithelium, but may serve as an energy source for cell growth and differentiation of the epithelium and/or for mesenchyme-epithelial interactions.