Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation

Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T(3)) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T(3) increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T(3) was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.     

Efficacy test of Polycan, a beta-glucan originated from Aureobasidium pullulans SM-2001, on anterior cruciate ligament transection and partial medial meniscectomy-induced-osteoarthritis rats

The object of this study was to assess the efficacy of Polycan from Aureobasidium pullulans SM-2001, which is composed mostly of beta-1,3-1,6-glucan, on osteoarthritis (OA)-induced by anterior cruciate ligament transection and partial medial meniscectomy (ACLT&PMM). Three different dosages of Polycan (85, 42.5, and 21.25 mg/kg) were orally administered once a day for 84 days to male rats a week after ACLT&PMM surgery. Changes in the circumference and maximum extension angle of each knee, and in cartilage histopathology were assessed using Mankin scores 12 weeks after Polycan administration. In addition, cartilage proliferation was evaluated using bromodeoxyuridine (BrdU). As the result of ACLT&PMM, classic OA was induced with increases in maximum extension angles, edematous knees changes, and capsule thickness, as well as decreases in chondrocyte proliferation, cartilages degenerative changes, and loss of articular cartilage. However, these changes (except for capsule thickness) were markedly inhibited in all Polycan- and diclofenac sodium-treated groups compared with OA control. Although diclofenac sodium did not influence BrdU uptake, BrdU-immunoreactive cells were increased with all dosages of Polycan, which means that Polycan treatment induced proliferation of chondrocytes in the surface articular cartilage of the tibia and femur. The results obtained in this study suggest that 84 days of continuous oral treatment of three different dosages of Polycan led to lesser degrees of articular stiffness and histological cartilage damage compared with OA controls 91 days after OA inducement, suggesting that the optimal Polycan dosage to treat OA is 42.5 mg/kg based on the present study.     

Cartilage on the move: Cartilage lineage tracing during tadpole metamorphosis

The reorganization of cranial cartilages during tadpole metamorphosis is a set of complex processes. The fates of larval cartilage‐forming cells (chondrocytes) and sources of adult chondrocytes are largely unknown. Individual larval cranial cartilages may either degenerate or remodel, while many adult cartilages appear to form de novo during metamorphosis. Determining the extent to which adult chondrocytes/cartilages are derived from larval chondrocytes during metamorphosis requires new techniques in chondrocyte lineage tracing. We have developed two transgenic systems to label cartilage cells throughout the body with fluorescent proteins. One system strongly labels early tadpole cartilages only. The other system inducibly labels forming cartilages at any developmental stage. We examined cartilages of the skull (viscero‐ and neurocranium), and identified larval cartilages that either resorb or remodel into adult cartilages. Our data show that the adult otic capsules, tecti anterius and posterius, hyale, and portions of Meckel's cartilage are derived from larval chondrocytes. Our data also suggest that most adult cartilages form de novo, though we cannot rule out the potential for extreme larval chondrocyte proliferation or de‐ and re‐differentiation, which could dilute our fluorescent protein signal. The transgenic lineage tracing strategies developed here are the first examples of inducible, skeleton‐specific, lineage tracing in Xenopus.     

An analysis of the influence of thyroid hormone on the synthesis of proteins in the tail fin of bullfrog tadpoles

By incubation of explants of tail fin from tadpoles of Rana catesbeiana in a solution of 35S-methionine for 4 h, newly synthesized proteins were labeled isotopically. After separation by two-dimensional polyacrylamide gel electrophoresis, those proteins were visualized by fluorography. Exposure of explants to culture medium containing thyroxine (T4) (150 nM) increased the incorporation of 35S-methionine into several proteins with 48 h. Effects of T4 on the relative abundance of two of these newly synthesized proteins were detected after 8 h of hormonal treatment. Very similar patterns of newly synthesized proteins were observed when proteins from explants of tail fin removed from tadpoles at metamorphic climax and immediately incubated with 35S-methionine were compared with proteins produced in fin derived from premetamorphic animals. These results are interpreted to indicate that both treatment of explants with T4 and elevation of endogenous levels of thyroid hormones during spontaneous metamorphosis increased the relative rates of synthesis of several identical proteins. The potential involvement of those proteins in early phases of metamorphic action which eventually lead to cell death and resorption is discussed.     

Skeletal morphogenesis in the urodele skull: III. Effect of hormone dosage in TH-induced remodeling

This study examines the dosage dependency of thyroid hormone (TH)-mediated remodelling in the cranial skeleton of the hemidactyliine plethodontid urodele, Eurycea bislineata. One set of experiments quantifies morphogenetic responses in 21 tissues for four size-age classes of larvae immersed in four different T₄ concentrations. A second set varies both the period and concentration of T₄ treatment to evaluate the effect of different TH profiles on adult tissue shape. The tissues surveyed in this study exhibit a 100-fold range in TH sensitivity. Those in regressive morphogenesis have tissue-specific sensitivities which correlate with the timing of their remodelling in natural development: bone resorption is more sentitive than cartilage resorption and is initiated earlier in metamorphosis. In contrast, the TH sensitivities of tissues in progressive morphogenesis vary within each tissue type and even within some tissues, and they do not correlate with timing in natural development. Some explanation for this discrepancy is offered by the constant spatial and temporal relationships between nasal cartilage and dermal bone, which suggest that some TH-mediated ossification may additionally require induction by cartilage. Also, the failure of nasolacrimal duct morphogenesis at all but the lowest dosage correlates with the inductdion of integumentary changes that may preclude duct formation. Variable T₄ treatments produce no effect upon the adult skull, other than loss of the nasolacrimal duct and/or foramen. These results have two developmental implicatons. First, the dosage dependencies of the nasolacrimal duct, ossification sequences, and cranial remodelling patterns all support a TH profile with exceptionally low levels at larval stages and at least a 100-fold increase at metamorphosis. Second, a small change in the rate of TH activity has the potential to effect a large-scale rearranggement and restructuring of TH-dependent remodelling. The lack of such transformations in metamorphic plethodontids suggests that TH activity is highly conserved in this group. © 1995 Wiley-Liss, Inc.     

Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.     

Development and tissue origins of the mammalian cranial base

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the “New Head”.     

Evolutionary innovation in the vertebrate jaw: A derived morphology in anuran tadpoles and its possible developmental origin

The mouthparts of anuran tadpoles are highly derived compared to those of caecilians or salamanders. The suprarostral cartilages support the tadpole's upper beak; the infrarostral cartilages support the lower beak. Both supra- and infrarostral cartilages are absent in other vertebrates. These differences reflect the evolutionary origin of a derived feeding mode in anuran tadpoles. We suggest that these unique cartilages stem from the evolution of new articulations within preexisting cartilages, rather than novel cartilage condensations. We propose testing this hypothesis through a search for similarities in the development of the suprarostral and infrarostral cartilage articulations and of the primary jaw joint. In Xenopus, the gene zax is expressed in a region corresponding to the infrarostral cartilage. This gene is related to the bapx1-gene, which regulates jaw joint development. Further investigation of these genes, as well as other genes with joint-related functions, in anuran craniofacial development may provide a connection between the morphological diversity seen in the vertebrate head and the corresponding diversity in genetic regulatory processes. We believe that the evolution of larval jaws in anurans may shed light on the general evolutionary mechanisms of how new articulations, not only in the jaw region, could have arisen in the vertebrate skull.     

Development and morphology of rostral cartilages in batoid fishes (Ghondrichthyes: Batoidea), with comments on homology within vertebrates

The rostral cartilages of batoid fishes were examined to elucidate their development, morphology and homology. Comparison of a variety of rostral cartilages among elasmobranchs with other groups of vertebrates shows that rostral cartilages originate embryologically from the trabecula and/or lamina orbitonasalis. Because different morphogenetic patterns of the derivatives of the two embryonic cartilages give rise to a wide variety of forms of rostral cartilages even within elasmobranchs, and because morphogenesis involves complex interactions among participating structures in the ethmo-orbital area, we put forward conceptual and empirical discussions to elucidate the homology of the rostral cartilages in batoid fishes. With six assumptions given in this study and based on recent discussions of biological and historical homology, our discussions centre on: (1) recognition of complex interactions of participating biological entities in development and evolution; (2) elucidation of a set of interacting biological and evolutionary factors to define a given morphological structure; (3) assessment of causal explanations for similarities or differences between homologous structures by determining genetic, epigenetic and evolutionary factors. Examples of conceptual approaches are given to make the approaches testable. Although a paucity of knowledge of rostral cartilage formation is the major obstacle to thorough analysis of the conceptual framework, several tentative conclusions are made on the homology of rostral cartilages that will hopefully attract more research on development and evolution in vertebrate morphology. These are: (1) the rostral cartilage in each group of vertebrates examined can be defined by both developmentally associated and adult structural attributes, yet such data do not allow us to assess homology of a variety of forms of rostral cartilages at higher taxonomic categories; (2) the entire rostral cartilage in elasmobranchs is formed by the contribution of the embryonic trabecula and lamina orbitonasalis. The status of the development and homology of the rostral cartilage in holocephalans remains uncertain; (3) there is no simple picture of evolution of rostral cartilages among three putative monophyletic assemblages of elasmobranchs, galeomorphs, squaloids (possibly plus Squatina, Chlamydoselachus and hexanchoids as the orbitostylic group) and batoid fishes. It is highly likely that rostral cartilages in each subgroup or subgroups of these assemblages may be of phylogenetic significance but that it may not serve as a basis to unite these assemblages into much higher assemblages; (4) the tripodal rostral cartilage is unique in form in the group including some carcharhinoid and lamnoid sharks. The status of the analogous tripodal cartilage in some squaloids remains uncertain. The unfused tripodal cartilage of the electric ray Narke is interpreted as developmentally equivalent to, but not homologous with, the unfused or fused ones in the sharks; (5) the rostral cartilage in the electric ray Torpedo is uniquely formed because of its embryonic origin solely from the ventro-medial part of the lamina orbitonasalis, but it is regarded as homologous with the rostral cartilages which are formed by the trabecula and other components of the lamina orbitonasalis in other batoid fishes; (6) the cornu trabecula contributes to the formation of the ventral stem of the rostral cartilage at least in elasmobranchs, especially to a particular set of rostral cartilages, i.e. the tripodal rostral cartilage in the shark Scyliorhinus and dorso-ventrally flattened rostral shaft in the narcinidid electric rays; (7) there is a unique form of a rostral shaft with rostral appendix in skates and probably guitarfishes; (8) there is no rostral cartilage in adult benthic stingrays, pelagic stingrays Dasyatis violacea and Myliobatidae, although it is present in embryonic stages; (9) there is a unique form of the rostral cartilage as a rostral projection from the dorso-lateral part of the lamina orbitonasalis in pelagic stingrays Rhinopteridae and Mobulidae, which together with part of the pectoral fins, forms a pair of cephalic fins; (10) different developmental mechanisms may be responsible for the absence or loss of rostral cartilages in different groups, i.e. absence of the cartilage derived from the medial area of the trabecula in Torpedo vs absence of the rostral cartilage in benthic stingrays; (11) the rostral cartilages in some placental mammals (cetaceans and sirenians) arise only from the medial area of the trabecula because monotreme and placental mammals do not form the trabecula cranii; (12) some actinopterygians and sacropterygians possess a rostral cartilage which originates only from the medial area of the trabecula. One scombroid group, including Sardini and Thunnini, Scomberomorus, Acanthocybium, Istiophoridae and Xiphias, possesses a unique larval beak composed of the rostral cartilage, ethmoid cartilage and premaxillar bone. The development and homology of other rostral cartilages remain to be further elucidated; (13) urodeles possess a medial rostral process whose anlage is probably developmentally equivalent to that in batoid fishes but the occurrence in urodeles is either atavistic or unique (autapomorphic); (14) the upper jaw of tadpoles is unique in possessing the suprarostral cartilage; the anlage of the cartilage is probably developmentally equivalent to the outgrowth of the cornu trabecula in batoid fishes.     

Nasal capsular cartilage is required for rat transpalatal suture morphogenesis

In the cranial vault, suture morphogenesis occurs when the growing cranial bones approximate and overlap or abut one another. Patency of developing sutures is regulated by the underlying dura mater. Once cranial sutures form, bone growth proceeds from the sutures in response to growth signals from the rapidly expanding neurocranium. Facial sutures do not develop in contact with the dura mater. It was therefore hypothesized that facial suture morphogenesis and bone growth from facial sutures are regulated by tissues with an equivalent role to the dura mater. The present study was designed to test this hypothesis by characterizing the morphology and growth factor expression in developing transpalatal (TP) sutures and their surrounding tissues, and then assessing the role of the overlying nasal capsular (NC) cartilages in maintaining suture patency. TP sutures develop as overlapping sutures, similar to cranial coronal sutures, and expression of Tgf-betas in TP sutures was similar to their distribution in cranial coronal sutures. To establish whether NC cartilages play a role in regulating TP suture morphogenesis, fetal rat TP sutures were cultured with associated attached NC cartilages or with NC cartilages removed. Sutures cultured for upward of 5 days with intact NC cartilages remained patent and maintained their cellular and fibrous components. However, in the absence of NC cartilages, the cellular nature of the sutures was not maintained and they became progressively acellular, with bony bridging across the suture. This finding is similar to that for cranial vault sutures cultured in the absence of dura mater, indicating that NC cartilages play an equivalent role to dura mater in maintaining the patency of developing sutures. These studies indicate that tissue interactions likely regulate morphogenesis of all cranial and facial sutures.     

MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)-dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of "ghost" cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.     

Effects of triiodothyronine treatments on body and organ growth and the development of immune function in dwarf chickens

The effects of triiodothyronine (T3) treatments on general body growth, long bone growth, primary lymphoid organ development, antibody production, and serum growth hormone (GH) and thyroid hormone levels were examined in two dwarf strains (sex-linked dwarf--SLD, and autosomal dwarf--ADW) and in a normal-growing control strain (K) of White Leghorn chickens. One-day-old male chicks from each of these strains were assigned to either an untreated control group or to one of the groups receiving a T3 supplement ranging from 0.01 to 1.0 ppm. General body growth and long bone growth were significantly (P less than 0.05) stimulated only within the SLD strain by the intermediate T3 dosages. The 1.0-ppm T3 dosage level resulted in depressed body weights within both the K and ADW strains but produced no significant changes within the SLD strain. Thymic growth was significantly stimulated due to treatments of 0.1 ppm T3 in the SLD strain (P less than 0.05) and 1.0 ppm T3 in both the SLD and ADW strains (P less than 0.001 and P less than 0.05, respectively). Bursal growth was significantly depressed (P less than 0.05) at all T3 dosage levels within the SLD strain while 0.01 and 0.1-ppm T3 treatments resulted in significant bursal growth stimulation in the K and ADW strains, respectively. Concomitant with the depressed bursal growth, antibody production was significantly depressed (P less than 0.05) within the SLD strain at the 1.0-ppm T3 dosage level. Antibody production was not significantly affected by any of the T3 treatments within the control K or ADW strains. Serum T3 levels were significantly increased in all strains by the T3 supplementation but thyroxine (T4) serum levels were affected only within the SLD strain. The 0.01-ppm T3 treatment resulted in a significant increase (P less than 0.05) in serum T4 levels within this strain and treatment group. The only increase (P less than 0.05) in GH levels due to T3 treatments occurred within the same SLD treatment group. The higher T3 treatments resulted in serum GH levels being severely depressed (P less than 0.01) in all strains.     

Hormonal stimulation of avian embryonic cartilage growth in vitro: Histologic and ultrastructural features

Summary We studied the histologic and ultrastructural features of embryonic chick cartilage after the cartilage had been incubated in serum-free medium that contained hormones and growth factors known to stimulate in vitro cartilage growth. Pelvic cartilages from 9 d chick embryos were incubated in BGJ_b (Fitton-Jackson modification) medium alone (control) or medium containing one of the following:N ⁶ monobutyryl cyclic AMP 0.5 mM, forskolin 100 μM, triiodothyronine (T₃) 10 nM, insulin 45 nM, or somatomedin C 0.67 nM. At the end of 3 d of incubation the cartilages were fixed in buffered formalin. Significant growth (increases in size, wet and dry weight) was seen with each treatment group. N⁶-Monobutyryl cAMP treated cartilage had an increased number of flattened immature chondrocytes with large nuclei and prominent nucleoli. The histologic and ultrastructural features of forskolin treated cartilage were indistinguishable from N⁶-monobutyryl cAMP treatment. The T₃ treated cartilage contained large hypertrophic chondrocytes with prominent lacunar typical of mature cartilage. T₃ Treated cartilage had considerable vacuole formation and dilated endoplasmic reticulum. Insulin and somatomedin treated cartilage had histologic appearance similar to control cartilage. Thus, the effects of various hormones on embryonic cartilage growth in vitro can be separated as to whether growth is the result of chondrocytic hyperplasia (cyclic AMP mediated), chondrocytic hypertrophy with maturation (T₃), or a combination of both hyperplasia and hypertrophy (insulin and somatomedin-C). This study was supported by a grant (K08 AM 01021-01) from the National Institute of Arthritis, Metabolism, Diabetes, and Kidney Diseases, Bethesda, MD, and a Basil O'Connor Starter Grant (0-400) from the March of Dimes.     

Plasticity and constraints on feeding kinematics in anuran larvae.

Tadpoles of the majority of anuran species have tiny, anatomically complex mouths. In most species the larval jaws are keratinized sheaths (beaks) overlying infrarostral cartilages. Surrounding the beak is a flexible oral disc and transverse rows of small, keratinized denticles. We used high-speed videography (250, 500 and 1000 frames per second) of Rana catesbeiana tadpoles to observe the kinematics of these mouthparts in feeding and breathing. Tadpoles can protract and retract their jaws as well as make them wider and narrower with each gape cycle. We demonstrate that during air-breathing, movement of the oral disc helps surfacing tadpoles to capture air quickly by preventing water from coming into the mouth. For our feeding study, we observed tadpoles as they grazed on both clean and algal covered glass surfaces. As the jaws close, the lower beak narrows to a greater degree when it encounters resistance. The denticle rows are used to both anchor the mouth and rasp surfaces during feeding. The hyperkinetic mouth parts of tadpoles permit grazing on non-planar surfaces of variable resistance. A trade-off in having such mobile jaws is loss of stability; no generalized tadpoles can generate great forces with their jaws, which would be necessary to subdue and dismember large tough prey. The feeding system of tadpoles is built out of soft tissues (such as cartilage and keratin) that can be shed (the keratinized sheaths) or remodeled (the underlying infrarostral cartilage) quickly, thus facilitating metamorphosis.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.     

Effects of developmental and growth history on metamorphosis in the gray treefrog, Hyla versicolor (Amphibia, Anura)

In ecological models, the timing of amphibian metamorphosis is dependent upon rate of larval growth, e.g., tadpoles that experience a decrease in growth rate can initiate metamorphosis early. Recent authors have suggested that this plasticity may be lost at some point during the larval period. We tested this hypothesis by exposing groups of tadpoles of the gray treefrog, Hyla versicolor, to different growth schedules. In endocrine models, metamorphosis is dependent on thyroxine levels and thyroxine is antagonized by prolactin (amphibian larval growth hormone), consistent with the idea that a rapidly growing tadpole can delay metamorphosis. Thus, we also manipulated the rate of development by supplementing or maintaining natural thyroxine levels for half of the tadpoles in each growth treatment. All tadpoles that received thyroxine supplements metamorphosed at the same time regardless of growth history. They also metamorphosed earlier than tadpoles not treated with thyroxine. Tadpoles not given thyroxine supplements metamorphosed at different times: those growing rapidly during day 15-34 metamorphosed earlier than tadpoles growing slowly. Growth rate before day 15 and after day 34 had no effect on metamorphic timing. The difference in larval period between these rapidly growing tadpoles and their sisters given thyroxine treatments was less than the same comparison for tadpoles that grew slowly during the same period. This apparent prolactin/thyroxine antagonism did not exist after day 34. These results are consistent with the hypothesis of a loss of plasticity in metamorphic timing.     

Central CO2 chemoreception in developing bullfrogs: anomalous response to acetazolamide.

Central CO(2) chemoreception and the role of carbonic anhydrase were assessed in brain stems from Rana catesbeiana tadpoles and frogs. Buccal and lung rhythms were recorded from cranial nerve VII and spinal nerve II during normocapnia and hypercapnia before and after treatment with 25 microM acetazolamide. The lung response to acetazolamide mimicked the hypercapnic response in early-stage and midstage metamorphic tadpoles and frogs. In late-stage tadpoles, acetazolamide actually inhibited hypercapnic responses. Acetazolamide and hypercapnia decreased the buccal frequency but had no effect on the buccal duty cycle. Carbonic anhydrase activity was present in the brain stem in every developmental stage. Thus more frequent lung ventilation and concomitantly less frequent buccal ventilation comprised the hypercapnic response, but the response to acetazolamide was not consistent during metamorphosis. Therefore, acetazolamide is not a useful tool for central CO(2) chemoreceptor studies in this species. The reversal of the effect of acetazolamide in late-stage metamorphosis may reflect reorganization of central chemosensory processes during the final transition from aquatic to aerial respiration.     

tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Zebrafish tgfβ3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfβ3 in head skeletal formation, we knocked down tgfβ3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfβ3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfβ3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfβ3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfβ3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfβ3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfβ3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfβ3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfβ3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfβ3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfβ3 protein levels in zebrafish.     

Development of the suprarostral plate of Pipoid Frogs

The rostral region of nonpipoid tadpoles has two sets of cartilages, the cornua trabeculae and the suprarostral cartilages, whereas the rostral region in pipoid larvae is occupied by a single and continuous cartilage, the suprarostral plate. The homology of this region in pipoid and nonpipoids tadpoles has been controversial. We examined the early formation and development of the suprarostral plate using serially cross‐sectioned specimens of Rhinophrynus, Xenopus, and Hymenochirus. We conclude that the cartilaginous structures present in the rostral area of pipoid and nonpipoid larvae are homologous. Furthermore, we found two different developmental patterns among pipoid larvae. The chondrocranium of Hymenochirus boettgeri is described and illustrated to understand its developmental pattern and because of its uniqueness among pipoid chondrocrania. J. Morphol. 240:143–153, 1999. © 1999 Wiley‐Liss, Inc.