Epithelial influences on membrane bone formation in the maxilla of the embryonic chick

In light of previous evidence that epithelial influences are required for membrane bone formation in certain instances (mandible, Tyler and Hall, '77 a,b; cranium, Benoit and Schowing, '70), the possibility that maxillary epithelium influences membrane bone formation within the maxillary mesenchyme was investigated. Intact maxillary processes, and enzymatically separated epithelial and mesenchymal components of the maxilla were obtained from embryonic chicks of three to five days of incubation (Hamburger and Hamilton ['51] stages (HH) 18-25) were grown individually as grafts on the chorioallantoic membranes of host chick embryos. The histodifferentiation of grafted intact maxillary processes was similar to that in vivo. The histodifferentiation of grafted maxillary mesenchyme depended upon the embryonic age at which the mesenchyme was isolated from its epithelium. In grafts of maxillary mesenchyme isolated from its epithelium at HH 22 or earlier stages, cartilage differentiated, but membrane bone did not form. In grafts of maxillary mesenchyme isolated from its epithelium at HH 23 or later stages, membrane bone formed in addition to cartilage. The results indicate that maxillary mesenchyme requires the presence of epithelium through HH 22 in order for membrane bone to form within the mesenchyme. Cartilage formation within the mesenchyme does not require epithelial influences during the embryonic period tested (HH 18-25). Maxillary epithelium isolated from its mesenchyme and grown as a graft became underlaid by host fibroblasts and achieved a limited degree of differentiation; the epithelium did not induce ectopic membrane bone formation within the host tissue, indicating that the presence of maxillary epithelium is not a sufficient condition for promoting membrane bone formation in normally non-osteogenic tissue.     

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.     

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.     

The role of epithelial coliagen and proteoglycan in the initiation of osteogenesis by avian neural crest cells

Osteogenesis was inhibited when mandibular processes from 3 1/2-day-old embryos were cultured in BUdR, LACA, α, αβ-Dipyridyl, 4-Methylumbelliferone, and 4-Methylumbelliferyl-β-D-glucoside or β-D-xyloside. Mandibular processes were then cultured in the test substances for 3 days, enzymatically separated into their epithelial and ectomesenchymal components, combined with mandibular components from untreated embryos, and either organ-cultured or grafted to chorioallantoic membranes of host embryos. Osteogenesis was inhibited when treated epithelium, but not when treated ectomesenchyme, was present in the tissue recombinations. Analysis of the known action of these inhibitors indicates that proliferation, hydroxylation of collagen, and synthesis of proteoglycans by epithelial cells are all necessary components of this osteogenic epithelial-ectomesenchymal interaction.     

Distribution of Matrix Proteins in Perichondrium and Periosteum During the Incorporation of Meckel's Cartilage into Ossifying Mandible in Midterm Human Fetuses: An Immunohistochemical Study

Immunohistochemical localization of versican and tenascin‐C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin‐C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin‐C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin‐C immunoreactivity in the primary bone marrow was also weak, but tenascin‐C positive areas did not overlap with versican‐positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican‐positive perichondrium and tenascin‐C‐positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin‐C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage. Anat Rec, 297:1208–1217, 2014. © 2014 Wiley Periodicals, Inc.     

Histological Development of the Fused Mandibular Symphysis in the Pig

The development of the mandibular symphysis in late fetal and postnatal pigs, Sus scrofa dom. (n = 17), was studied as a model for the early fusing symphysis of anthropoid primates, including humans. The suture-like ligaments occurring in species that retain a mobile symphysis are not present in the pig. Instead, cartilage is the predominant tissue in the mandibular symphysis prior to fusion. In late fetuses the rostrum of the fused Meckel's cartilages forms a minor posterior component of the symphysis whereas the major component is secondary cartilage, developing bilaterally and joined at the midline with mesenchyme. This remnant of Meckel's cartilage likely fuses with the flanking secondary cartilage. The overall composition of pig symphyseal histology in fetal and infant animals varies regionally and individually. Regions where the paired secondary cartilages abut in the midline resemble double growth plates. Chondrogenic growth in width of the symphysis is likely important in early stages, and central proliferation of mesenchyme is the probable source of new chondrocytes. Laterally, the chondrocytes hypertrophy near the bone fronts and are replaced by alveolar bone. Complete synostosis except for a small cartilage remnant had occurred in one 8-week-old postnatal specimen and all older specimens. Surprisingly, however, the initial phase of symphyseal fusion, observed in a 5-week-old postnatal specimen, involved intramembranous ossification of midline mesenchyme rather than endochondral ossification. Subsequently, fusion progresses rapidly at the anterior and labial aspects of the symphysis, leaving only a small postero-lingual cartilage pad that persists for at least several months. Anat Rec, 302:1372–1388, 2019. © 2018 Wiley Periodicals, Inc.     

Differential changes in cartilage cell proliferation and cell density in the rat craniofacial complex during secondary palate development

During mammalian secondary palate formation sagittal growth of the lower face has been shown to be more rapid than that of the upper face, and the tongue and mandible extend beneath the primary palate. In order to identify factors contributing to this differential growth pattern, cellular and morphologic growth of the major cartilages of the upper and lower facial regions were studied in radioautographic sections labeled with tritiated thymidine. Evaluation of cell-density recordings, labeling indices, and structural dimensions revealed significant differences between Meckel's cartilage in the lower face, and the nasal cartilage and anterior cranial base cartilage in the upper face. After formation of the precartilaginous blastema, labeling indices were high in Meckel's cartilage (20–30%), but very low in the nasal cartilage and the anterior cranial base (0–2%). During secondary palate formation of the volume of Meckel's cartilage increased more rapidly than the other cartilages and its growth was primarily in the sagittal direction. Between days 15 and 17, the increase in the length of Meckel's cartilage (165%) was approximately twice as great as the increase in the combined length of the nasal cartilage and the anterior cranial base (77%). During this period induction of cleft palate with some teratogens has been shown to severely retard growth of Meckel's cartilage and produce mandibular retrognathia that contributes to delayed elevation of the palatal shelves. Therefore, extensive cell proliferation in Meckel's cartilage, during a period of limited proliferation in other craniofacial cartilages, appears to contribute to its rapid growth and its differential sensitivity to growth inhibition.     

Prx1 and Prx2 cooperatively regulate the morphogenesis of the medial region of the mandibular process

Mice lacking both Prx1 and Prx2 display severe abnormalities in the mandible. Our analysis showed that complete loss of Prx gene products leads to growth abnormalities in the mandibular processes evident as early as embryonic day (E) 10.5 associated with changes in the survival of the mesenchyme in the medial region. Changes in the gene expression in the medial and lateral regions were related to gradual loss of a subpopulation of mesenchyme in the medial region expressing eHand. Our analysis also showed that Prx gene products are required for the initiation and maintenance of chondrogenesis and terminal differentiation of the chondrocytes in the caudal and rostral ends of Meckel's cartilage. The fusion of the mandibular processes in the Prx1/Prx2 double mutants is caused by accelerated ossification. These observations together show that, during mandibular morphogenesis, Prx gene products play multiple roles including the cell survival, the region‐specific terminal differentiation of Meckelian chondrocytes and osteogenesis. Developmental Dynamics 238: 2599–2613, 2009. © 2009 Wiley‐Liss, Inc.     

Region- and stage-specific effects of FGFs and BMPs in chick mandibular morphogenesis.

The mandibular processes are specified as at least two independent functional regions: two large lateral regions where morphogenesis is dependent on fibroblast growth factor (FGF)-8 signaling, and a small medial region where morphogenesis is independent of FGF-8 signaling. To gain insight into signaling pathways that may be involved in morphogenesis of the medial region, we have examined the roles of pathways regulated by FGFs and bone morphogenetic proteins (BMPs) in morphogenesis of the medial and lateral regions of the developing chick mandible. Our results show that, unlike in the lateral region, the proliferation and growth of the mesenchyme in the medial region is dependent on signals derived from the overlying epithelium. We also show that medial and lateral mandibular mesenchyme respond differently to exogenous FGFs and BMPs. FGF-2 and FGF-4 can mimic many of the effects of mandibular epithelium from the medial region, including supporting the expression of Msx genes, outgrowth of the mandibular processes and elongation of Meckel's cartilage. On the other hand, laterally placed FGF beads did not induce ectopic expression of Msx genes and did not affect the growth of the mandibular processes. These functional studies, together with our tissue distribution studies, suggest that FGF-mediated signaling (other than FGF-8), through interactions with FGF receptor-2 and downstream target genes including Msx genes, is part of the signaling pathway that mediates the growth-promoting interactions in the medial region of the developing mandible. Our observations also suggest that BMPs play multiple stage- and region-specific roles in mandibular morphogenesis. In this study, we show that exogenous BMP-7 applied to the lateral region at early stages of development (stage 20) caused apoptosis, ectopic expression of Msx genes, and inhibited outgrowth of the mandibular processes and the formation of Meckel's cartilage. Our additional experiments suggest that the differences between the effects of BMP-7 on lateral mandibular mesenchyme at stage 20 and previously reported results at stage 23 (Wang et al., [1999] Dev. Dyn. 216:320-335) are related to differences in stages of differentiation in that BMP-7 promotes apoptosis in undifferentiated lateral mandibular mesenchyme, whereas it promotes chondrogenesis at later stages of development. We also showed that, unlike mandibular epithelium and medially placed FGF beads, medially placed BMP-7 did not support outgrowth of the isolated mesenchyme and at stage 20 induced the formation of a duplicated rod of cartilage extending from the body of Meckel's cartilage. These observations suggest that BMPs do not play essential roles in growth-promoting interactions in the medial region of the developing mandible. However, BMP-mediated signaling is a part of the signaling pathways regulating chondrogenesis of the mandibular mesenchyme.     

Ift88 is involved in mandibular development

The mandible is a crucial organ in both clinical and biological fields due to the high frequency of congenital anomalies and the significant morphological changes during evolution. Primary cilia play a critical role in many biological processes, including the determination of left/right axis patterning, the regulation of signaling pathways, and the formation of bone and cartilage. Perturbations in the function of primary cilia are known to cause a wide spectrum of human diseases: the ciliopathies. Craniofacial dysmorphologies, including mandibular deformity, are often seen in patients with ciliopathies. Mandibular development is characterized by chondrogenesis and osteogenesis; however, the role of primary cilia in mandibular development is not fully understood. To address this question, we generated mice with mesenchymal deletions of the ciliary protein, Ift88 (Ift88^fl/fl;Wnt1Cre). Ift88^fl/fl;Wnt1Cre mice showed ectopic mandibular bone formation, whereas Ift88 mutant mandible was slightly shortened. Meckel's cartilage was modestly expanded in Ift88^fl/fl;Wnt1Cre mice. The downregulation of Hh signaling was found in most of the mesenchyme of Ift88 mutant mandible. However, mice with a mesenchymal deletion of an essential molecule for Hh signaling activity, Smo (Smo^fl/fl;Wnt1Cre), showed only ectopic mandibular formation, whereas Smo mutant mandible was significantly shortened. Ift88 is thus involved in chondrogenesis and osteogenesis during mandibular development, partially through regulating Sonic hedgehog (Shh) signaling.     

Origin of the torus mandibularis: An embryological hypothesis

Torus mandibularis, a well‐known protuberance in the dental field, has been defined as a hyperostosis in the lingual aspect of the body of the mandible above the mylohyoid line. However, the origin of the torus mandibularis has not yet been clarified. The aim of this study was to provide a better understanding on the origin of the torus in view of the specific development of Meckel's cartilage at the site corresponding to the adult torus. A total of 40 mid‐term human fetuses at 7–16 weeks of gestation were examined. The 10–13 weeks stage corresponded to the critical period in which Meckel's cartilage with endochondral ossification underwent a bending at the beginning of the intramandibular course. At the level of mental foramen, which was located between the deciduous canine and the first deciduous molar germs, the medial lamina of the mandible protruded medially to reach Meckel's cartilage. Thus, the medial lamina covered the posterior and superior aspect of the bending Meckel's cartilage just above the attachment of the developing mylohyoid muscle (i.e., in the oral cavity). We considered a bony prominence, which composed the protruding medial lamina and the bending Meckel's cartilage as the fetal origin of the torus mandibularis. A new theory is proposed for the origin of the torus mandibularis based on the existence of an anlage formed during the development of the mandible, variable in morphology and size, but always constant. Clin. Anat. 26:944–952, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of parathyroid hormone-related peptide (PthrP) and its receptor (PTH1R) during the histogenesis of cartilage and bone in the chicken mandibular process

The purpose of this study was to examine the expression and actions of parathyroid hormone-related protein (PTHrP) when skeletal histogenesis occurs in the chicken mandible. Prior to the appearance of skeletal tissues, PTHrP and PTH1R were co-expressed by cells in the ectoderm, skeletal muscle, peripheral nerve and mesenchyme. Hyaline cartilage was first observed at HH stage 27 when many but not all chondroblasts expressed PTHrP and PTH1R. By stage 34, PTHrP and PTH1R were not detected in chondrocytes but were expressed in the perichondrium. Alkaline phosphatase (AP)-positive preosteoblasts and woven bone appeared at stages 31 and 34, respectively. Preosteoblasts, osteoblasts and osteocytes co-expressed PTHrP and PTH1R. Treatment with chicken PTHrP (1-36) increased cAMP in mesenchyme from stage 26 embryos. Continuous exposure to chicken PTHrP (1-36) for 14 days increased cartilage nodule number and decreased AP while intermittent exposure did not affect cartilage nodule number and increased AP in cultures of stage 26 mesenchymal cells. Adding a neutralizing anti-PTHrP antibody to the cultures reduced cartilage nodule number and did not affect AP. These findings show that PTHrP and PTH1R are co-expressed by extraskeletal and skeletal cells before and during skeletal tissue histogenesis, and that PTHrP may influence skeletal tissue histogenesis by affecting the differentiation of mandibular mesenchymal cells into chondroblasts and osteoblasts.     

A morphometric analysis of craniofacial growth and changes in spatial relations during secondary palatal development in human embryos and fetuses

Staged human embryos and fetuses in the Carnegie Embryological Collection were morphometrically analyzed to show craniofacial dimensions and changes in spatial relations, and to identify patterns that would reflect normal developmental events during palatal formation. Normal embryos aged 7–8 weeks postconception (Streeter-O'Rahilly stages 19–23) and fetuses aged 9–10 weeks postconception, in eight groups with mean crownrump (CR) lengths of 18–49 mm, were studied with cephalometric methods developed for histologic sections. In the 4-week period studied, facial dimensions increased predominantly in the sagittal plane with extensive changes in length (depth) and height, but limited changes in width. Growth of the mandible was more rapid than the nasomaxillary complex, and the length of Meckel's cartilage exceeded the length of the oronasal cavity at the time of horizontal movement of the shelves during stage 23. Simultaneously with shelf elevation, the upper craniofacial complex lifted, and the tongue and Meckel's cartilage extended forward beneath the primary palate. Analysis of spatial relations in the oronasal cavity showed that the palatomaxillary processes became separated from the tongue-mandibular complex as the head extended, and the tongue became positioned forward with growth of Meckel's cartilage. As the head position extended by 35°, the cranial base angulation was unchanged and the primary palate maintained a 90° position to the posterior cranial base. However, the sagittal position of the maxilla relative to the anterior cranial base increased by 20° between stages 19 and 23. In the late embryonic and early fetal periods, the mean cranial 128° and the mean maxillary position angulation of approximately 34° were similar to the angulations previously shown to be present later prenatally and postnatally. The results suggest that human patterns of cranial base angulation and maxillary position to the cranial base develop during the late embryonic period when the chondrocranium and Meckel's cartilage form the primary skeleton.     

ADAMTSL1 and mandibular prognathism

Mandibular prognathism is characterized by a prognathic or prominent mandible. The objective of this study was to find the gene responsible for mandibular prognathism. Whole exome sequencing analysis of a Thai family (family 1) identified the ADAMTSL1 c.176C>A variant as the potential defect. We cross-checked our exome data of 215 people for rare variants in ADAMTSL1 and found that the c.670C>G variant was associated with mandibular prognathism in families 2 and 4. Mutation analysis of ADAMTSL1 in 79 unrelated patients revealed the c.670C>G variant was also found in family 3. We hypothesize that mutations in ADAMTSL1 cause failure to cleave aggrecan in the condylar cartilage, and that leads to overgrowth of the mandible. Adamtsl1 is strongly expressed in the condensed mesenchymal cells of the mouse condyle, but not at the cartilage of the long bones. This explains why the patients with ADAMTSL1 mutations had abnormal mandibles but normal long bones. This is the first report that mutations in ADAMTSL1 are responsible for the pathogenesis of mandibular prognathism.     

Stage-specific expression patterns of alkaline phosphatase during development of the first arch skeleton in inbred C57BL/6 mouse embryos

Timing and pattern of expression of alkaline phosphatase was examined during early differentiation of the 1st arch skeleton in inbred C57BL/6 mice. Embryos were recovered between 10 and 18 d of gestation and staged using a detailed staging table of craniofacial development prior to histochemical examination. Expression of alkaline phosphatase is initiated at stage 20.2 in the plasma membrane of mesenchymal cells in the distal region of the first arch. Expression is strongest in osteoid (unmineralised bone matrix) and presumptive periosteum at stage 21.32. Mineralisation begins at stage E23. Expression is present in the mineralised bone matrix. Secondary cartilages form in the condylar and angular processes by stage M24. The cartilaginous cells and surrounding cells in the processes are all alkaline phosphatase-positive and surrounded by the common periosteum, suggesting that progenitor cells of the processes, dentary ramus and secondary cartilages all originate from a common pool. Nonhypertrophied chondrocytes of Meckel's cartilage express alkaline phosphatase at stage M23. Expression in these chondrocytes is preceded by the expression in their adjacent perichondrium. This is true of chondrocytes in all other cranial cartilages examined. 3-D reconstruction of expression in Meckel's cartilage also revealed that the chondrocytes of Meckel's cartilage which express alkaline phosphatase and the matrix of which undergoes mineralisation are those surrounded by the alkaline phosphatase-positive dentary ramus. By stage 25, coincident with mineralisation in the distal section of Meckel's cartilage, most chondrocytes are strongly positive. The perichondria of malleus and incus cartilages express alkaline phosphatase at stage M24. Nonhypertrophied chondrocytes along these perichondria also express alkaline phosphatase. Superficial and deep cells in the dental laminae of incisor and 1st molar teeth become alkaline phosphatase-positive at the bud stage, stages 21.16 and 21.32, respectively. Dental papillae are negative until stage M24 when alkaline phosphatase expression begins in the dental papillae and follicles of the incisor teeth and the dental follicles of the 1st molar teeth. The dental papillae of the 1st molar teeth express alkaline phosphatase at stage 25. Expression in the dental papillae and follicles appears to coincide with cellular differentiation of follicle from papilla. The presumptive squamosal, ectotympanic and gonial membrane bones, lingual oral epithelial cells connected to the dental laminae of the incisor teeth, hair follicle papillae and sheath and surrounding dermis all express alkaline phosphatase in a stage-specific manner.     

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.     

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.