Library

Notes: Avian embryos can be completely paralyzed by injection of neuromuscular-blocking agents. We used a single injection of decamethonium iodide to paralyze embryos at 7, 8, or 10 days of incubation and analyzed the growth of individual bones (clavicle, mandible, ulna, femur, tibia, humerus) and of individual muscles that act upon some of those bones (clavicular and sternal heads of m. pectoralis, and mm. biceps brachii, depressor mandibulae, pseudotemporalis, and adductor externus). Growth of the bones is not equally affected by paralysis. Only 27% of clavicular growth (by mass) but 77% of mandibular growth occurred in paralyzed embryos, whereas the four long bones exhibited 52-63% of their normal growth. Analysis of muscle weight, fiber length and physiological cross-sectional area (weight/fiber length) indicate that there was greater reduction of the muscles acting on the limbs than of those acting on the mandible, i.e., diminished growth of the skeleton is correlated with reduced muscular activity. Specific retardation of clavicular growth is due to fusion of sternal rudiments and collapse of the thorax, as well as virtual absence of the musculature that normally attaches to the clavicle. We discuss these results in the light of intrinsic and extrinsic factors governing growth of tne embryonic skeleton. Paralysis reduces skeletal growth by reducing both the movements taking place in ovo, and the loads imposed on the bones by muscle contraction, changes that represent alterations in the mechanical environment of the skeleton.

Notes: The sequence of appearance of the 17 different skull bones in the oriental fire-bellied toad, Bombina orientalis, is described. Data are based primarily on samples of ten or 11 laboratory-reared specimens of each of 11 Gosner developmental stages (36-46) representing middle through late metamorphosis. Ossification commences as early as stage 37 (hind limb with all five toes distinct), but the full complement of adult bones is not attained until stage 46 (metamorphosis complete). Number of bones present at intermediate stages is poorly correlated with external morphology. As many as four Gosner developmental stages elapse before a given bone is present in all specimens following the stage at which it may first appear. The modal ossification sequence is frontoparietal, exoccipital, parasphenoid, septomaxilla, premaxilla, vomer, nasal, maxilla, angulosplenial, dentary, squamosal, quadratojugal, pterygoid, prootic, interfrontal, sphenethmoid, and mentomeckelian. Most specimens are consistent with this sequence, despite the poor correlation between cranial ossification and external development as assayed by Gosner stage.The timing of cranial ossification in Bombina orientalis differs in many respects from that described for two other, distantly related anurans, the leopard frog (Rana pipiens) and the western toad (Bufo boreas). These include the total number and sequence of appearance of bones, and the timing of ossification relative to the development of external morphology. Interspecific variation may reflect differences in the timing of the tissue interactions known to underlie skeletal differentiation and evolution.

Notes: The identification, spatial relationships, and sequences of development of the cartilaginous and bony elements of the chondrocranium, osteocranium, and splanchnocranium in the medaka, Oryzias latipes, are described here for the first time. The development of the cartilaginous head skeleton commences at stage 29 and is essentially complete by stage 35 (hatching). The parasphenoid bone and two pairs of branchiostegals are present at this stage and several other replacement and dermal bones begin to appear shortly thereafter. Development of the osteocranium and ossification of the splanchnocranium continue throughout the larval and juvenile phases and are essentially complete at sexual maturity at approximately 3 months (at 25°C), at which time the fish range in length between 25 and 30 mm.The description of the adult head skeleton of O. latipes is compared to those of O. melastigma, O. luzonesis, and other Oryzias spp. previously described and a redesignation of the relationships between certain elements in the adult head skeleton is proposed, based on the developmental data presented. Furthermore, the value of the medaka as a model teleost to study the embryological origins of, and in particular, the neural crest contributions to, the cranial and visceral skeleton is outlined based on certain characteristics of the medaka's life history traits. These include the ease of obtaining embryos for which the exact time of fertilization is known (without sacrificing any brood stock) and the relatively rapid development of the chondrocranium, which is nearly complete at hatching, a process which can occur in as short a time as 6 days (at 34°C). The usefulness of the ontogenetic data obtainable from further studies into the embryonic origins of head and visceral skeletal elements revealed in the present study, is briefly discussed.

Notes: In anuran amphibians, cranial bones typically first form at metamorphosis when they rapidly invest or replace the cartilaginous larval skull. We describe early development of the first three bones to form in the Oriental fire-bellied toad, Bombina orientalis - the parasphenoid, the frontoparietal, and the exoccipital - based on examination of serial sections. Each of these bones is fully differentiated by Gosner stage 31 (hindlimb in paddle stage) during premetamorphosis. This is at least six Gosner developmental stages before they are first visible in whole-mount preparations at the beginning of prometamorphosis. Thus, developmental events that precede and mediate the initial differentiation of these cranial osteogenic sites occur very early in metamorphosis - a period generally considered to lack significant morphological change. Subsequent development of these centers at later stages primarily reflects cell proliferation and calcified matrix deposition, possibly in response to increased circulating levels of thyroid hormone which are characteristic of later metamorphic stages. Interspecific differences in the timing of cranial ossification may reflect one or both of these phases of bone development. These results may qualify the use of whole-mount preparations for inferring the sequence and absolute timing of cranial ossification in amphibians.

Notes: Haematoxylin, Alcian Blue-Chlorantine Fast Red (ABCR) and the Ralis osteoid-specific stain were employed to closely follow the histogenesis of the tibia of the embryonic chick so as to provide an accurate description of the onset of ossification.An overview of the major cytological events preceding osteogenesis in the tibia was obtained from hindlimbs of embryos of H. H. (Hamburger and Hamilton, '51) stages 16-26 (2.5-5 days of incubation) stained with ABCR. A description of the cytological changes in the periosteum as it develops from the perichondrium and an analysis of the timing of the onset of osteoid deposition was obtained from the tibiae of accurately aged and staged embryos of H. H. stages 28-32 (5.5-8 days). These tibiae were stained specifically for the detection of osteoid:the freshly-secreted, unmineralized product of fully-differentiated osteoblasts. The perichondrium transformed into a bi-layered periosteum at H. H. late stage 29 (6.5 days) while osteoid was first detected adjacent to the hypertrophic cartilage of H. H. stage 30 (6.5-7 days) tibial diaphyses.These results, correlated with the immunoflourescent studies of Von der Mark et al. ('76a,b), which revealed the presence of Type I (bone-type) collagen-synthesizing cells in the perichondria of tibiae from embryos of H. H. stage 28 (5.5-6 days), demonstrated that the onset of determination of cells for osteogenesis and the cytodifferentiation of the periosteum are not temporally coupled.

Notes: Among vertebrates, some teleosts are unique in having bone which lacks osteocytes embedded in the matrix. The fate of cells that secrete the matrix of these acellular bones has not been investigated thoroughly. Histological and fluorescent microscopic analysis of the vertebral bone of Oryzias latipes demonstrated that acellularity is not a secondary appearance of an early cellular bone during ontogeny. Vertebral bone is devoid of cells embedded in the matrix throughout development. Cells that secrete bone matrix do not become trapped in their own secretion. Instead, they always remain as a surface layer over the outer surface of the bone. Fluorescent microscopic visualization of tetracycline injected into growing fish demonstrated that bone was only deposited by osteoblasts lining the outer surface of the bone; no deposition of bone took place on the inner surface.

Notes: A series of studies by Edgeworth demonstrated that cranial muscles of gnathostome fishes are embryologically of somitic origin, originating from the mandibular, hyoid, branchial, epibranchial, and hypobranchial muscle plates. Recent experimental studies using quail-chick chimeras support Edgeworth's view on the developmental origin of cranial muscles. One of his findings, the existence of the premyogenic condensation constrictor dorsalis in teleost fishes, has also been confirmed by molecular developmental studies. Therefore, developmental mechanisms for patterning of cranial muscles, as described and implicated by Edgeworth, may serve as structural entities or regulatory phenomena responsible for developmental and evolutionary changes. With Edgeworth's and other studies as background, muscles in the ventral gill arch region of batoid fishes are analyzed and compared with those of other gnathostome fishes. The spiracularis is regarded as homologous at least within batoid fishes, but its status within elasmobranchs remains unclear; developmental modifications of the spiracularis proper are evident in some batoid fishes and in several shark groups. The peculiar ventral extension of the spiracularis in electric rays and some stingrays may represent convergence, probably facilitating ventilation and/or feeding in both groups. The evolutionary origin of the “internus” and “externus” remains uncertain, despite the fact that a variety of forms of the constrictor superficiales ventrales in batoid fishes indicates an actual medio-ventral extension of the “externus.” The intermandibularis is probably present only in electric rays. The “X” muscle occurs only in electric rays and is considered to be Edgeworth's intermandibularis profundus. Its association with the adductor mandibular complex in narkinidid and narcinidid electric rays may relate to its functional role in lower jaw movement. Contrary to common belief, in most batoid fishes as well as some sharks, muscles that originate from the branchial muscle plate and extend medially in the ventral gill arches do exist: the medial extension of the interbranchiales in most batoid fishes and some sharks and the “Y” muscle in the pelagic stingrays Myliobatos and Rhinoptera. The latter is another example of the medial extension of the “internus.” Whether the interbranchiales and “Y” muscle are homologous within elasmobranchs and whether homologous with the obliques ventrales and/or transversi ventrales of osteichthyan fishes await further research. Four hypobranchial muscles are recognized in batoid fishes: the coracomandibularis, coracohyoideus, coracoarcualis, and coracohyomandibularis. The coracohyoideus is discrete from the coracoarcualis; its complete structural separation from the latter occurs in several groups of batoid fishes. The sternohyoideus of osteichthyan fishes is regarded as a partially developed, continuous bundle of muscle whose counterpart in chondrichthyan fishes appears to be the fully developed rectus cervicus in holocephalans and the squaloid shark Isistius. The coracoarculais is, therefore, present structurally and possibly functionally as a discrete muscle only in elasmobranchs. Although the coracohyomandibularis has been regarded as unique in batoid fishes, the first coracobranchialis in the sawshark Pristiophorus may represent the coracohyomandibularis. The conceptual frameworks and results of the development and evolution of cranial muscles presented here emphasize the importance of molecular and experimental embryological studies and integration of these areas with comparative anatomical and functional studies. Edgeworth's contributions remain as a remarkable achievement in muscle biology. © 1992 Wiley-Liss, Inc.

Notes: Despite intensive and ingenious investigation, the origins and ultimate fate of the osteoclast remain shrouded in mystery. This brief review evaluates some of the recent experimental approaches used in the study of the osteoclast, especially whether they form from intra- or extra-skeletal progenitor cells, whether from the same osteoprogenitor cell as the osteoblast, and whether, once formed, they may modulate to osteoblasts.That osteoprogenitor cells can, and do, become osteoclasts is well founded, as is the conclusion that such progenitor cells originate as blood-borne, extraskeletal cells. Evidence that sessile, intra-skeletal, progenitor cells can form osteoclasts is less direct. There is good evidence that osteoclasts both shed and take-up nuclei, but no direct evidence that nuclear shedding is accompanied by death of the osteoclast, and no direct evidence for the fate of the shed nuclei. Whether the same osteoprogenitor cell can produce either an osteoblast or an osteoclast also remains an open question.

Notes: The L-proline analog, L-azetidine-2-carboxylic acid, (LACA) was injected into embryonated eggs of the common fowl, Gallus domesticus at daily doses of 350 μg/egg on one or several days between 8 and 12 days of incubation. Treatment at nine-days of incubation preferentially retarded embryonic growth to the twelfth day but recovery of growth rate occurred by 15 days of incubation. Relationships between growth and LACA-inhibited aspects of collagenogenesis are discussed.The earliest aged embryos from which isolated stem cells from membrane bones will form secondary cartilage is ten days of incubation. Secondary chondro-genesis on the quadratojugal, a membrane bone of the skull, was inhibited by treatment of whole embryos with LACA at nine days of incubation but not by treatment at eight days. We concluded that an event involving collagen began at nine days of incubation, was blocked by LACA, and was part of the process of chondrogenic determination of these stem cells. Addition of LACA to the medium in which already determined stem cells from the quadratojugal were cultured prevented expression of the chondrogenic phenotype. This proline analog is then a useful probe for events relating both to determination and to expression of the differentiated state, and allows conclusions to be drawn regarding the role of col-lagenogenesis in these events.

Notes: Whether neural crest cells from the avian embryo are determined for chondrogenesis before they begin their migration away from the neural tube (i.e., before H. H. stages 8.5-9) was investigated by establishing neural folds from embryos of H. H. stages 5-11 either in organ culture, or as grafts to the chorioallantoic membranes of host embryos. Cartilage differentiated from neural folds taken from embryos of H. H. stages 5-7 but not from those taken from older embryos. This stage specific pattern was reversed when the tissue adjacent to the neural tube was grafted to the chorioallantoic membrane. Cartilage only formed from tissues isolated later than H. H. stage 8; i.e., when these adjacent tissues contain neural crest cells. We concluded that neural crest cells are determined for chondrogenesis while still in the neural tube and before their migration to the face and head. This is in contrast to the situation in the only other group which has been examined, the urodele amphibians.