Skeletal morphogenesis in the urodele skull: II. Effect of developmental stage in thyroid hormone-induced remodeling

This study investigates the effect of developmental stage on thyroid hormone (TH)-mediated remodeling in the skeletal tissues of hemidactyliine plethodontid urodeles. Rate of morphogenesis was quantified in 17 metamorphic tissues for three different size-age classes of Eurycea bislineata larvae immersed in a metamorphic dosage of T₄. Extent of morphogenesis after a 3-week immersion was also quantified in these tissues plus four larval ones for the full size range of E. bislineata larvae and for less complete size ranges of E. wilderae, E. longicauda guttolineata, Gyrinophilus porphyriticus, and Pseudotriton ruber larvae. Although all tissues respond more slowly with decreasing size/age, two tissue-specific effects are evident in all species. Larval ossifications are less inducible than metamorphic ossifications, and progressive metamorphic events are more retarded and, in some cases, more prone to abnormal morphogenesis than regressive ones. The first effect agrees with the prediction that tissues that naturally remodel at metamorphosis are more responsive to a metamorphic dosage of TH than those that respond at a larval stage and lower TH. The second effect agrees with the prediction that progressive morphogenesis is more likely to be impaired at small size than regressive morphogenesis, although the frequent discrepancies between individuals of similar size implicate developmental age more than size in this effect. Collectively, these two effects provide only equivocal support for the hypothesis that direct development in plethodontids evolved via precocious TH activity. However, the unexpected transition from ceratobranchial replacement to ceratobranchial shortening in medium-sized larvae suggests that the former pathway requires a longer period of cell specification at low TH. Since ancestral plethodontids appear to have been distinguished by an exceptionally long larval period with exceptionally low TH activity, this developmental prerequisite may in turn be partly responsible for their singular evolution of ceratobranchial replacement. © 1995 Wiley-Liss, Inc.     

Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

Background

The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.

Methodology/Principal Findings

We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13.

Conclusion

A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.     

Skull development during anuran metamorphosis: III. Role of thyroid hormone in chondrogenesis

Metamorphosis of cranial cartilages in anuran amphibians constitutes one of the most dramatic and extensive ontogenetic transformations in vertebrates. We quantitatively examined the role of thyroid hormone (3,3',5-triiodo-L-thyronine; T3) in mediating gross aspects of this morphological repatterning in the skull of the Oriental fire-bellied toad, Bombina orientalis. T3 was administered via plastic (Elvax) micropellets in three treatment dosages (2.5, 0.25, and 0.025 microgram) and one control dosage (0 microgram) to tadpoles of three Gosner developmental stages--28/29, 30/31, and 32/33; tadpoles were recoved up to 8 d (treatment and control dosages) or 14 d (control dosage) later. Response of larval cartilages to exogenous T3 was dosage dependent but not implant-stage dependent; chondrogenic tissues that participate in metamorphic transformation are competent to respond to T3 well before they normally do. Metamorphic effects of T3 were visible within 2 d; in most treatment groups, the normal metamorphic sequence was two-thirds complete after 8 d. While T3 also induced precocious ossification, the normal temporal relation between bone formation and cartilage transformation was dissociated in experimental groups. Morphological integration between cartilage and bone during cranial metamorphosis is at least partly the result of each tissue responding independently to endocrine factors.     

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.     

Variation and timing of the cranial ossification sequence of the oriental fire-bellied toad,Bombina orientalis (Amphibia,Discoglossidae)

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.     

Apoptosis in amphibian organs during metamorphosis

During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis.     

Ossification of the human fetal ilium.

Ossification of the ilium is similar to that of a long bone. It possesses three cartilaginous epiphyses and one cartilaginous process. Moreover, it undergoes peculiar osteoclastic resorption, comparable with that of the cranium bones. Asymmetrical ossification of the ilium, haversian bone remodelling and apposition of chondroid tissue posterosuperiorly to the acetabulum most probably emphasize the importance of mechanical factors in the morphogenesis of the hip bone during fetal life.     

FGF9 can induce endochondral ossification in cranial mesenchyme

Background  

The flat bones of the skull (i.e., the frontal and parietal bones) normally form through intramembranous ossification. At these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. This type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone.     

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.     

Development of the cranium and paired fins in Betta splendens (Teleostei: Percomorpha): Intraspecific variation and interspecific comparisons

The development of the chondrocranium and the relative timing of ossification of the osteocranium is described in the teleost fish Betta splendens from a large series of cleared and differentially stained specimens. General trends in ossification patterns are examined from developmental, phylogenetic, and functional contexts. As in many other vertebrates, dermal bones form before cartilage bones. Ossification sequence conforms to functional need in a very general way, but there are many inconsistencies in the details of order. For example, some bones that are directly involved in feeding ossify no earlier than bones more indirectly involved. Comparisons of ossification sequence within specific cranial regions are made among Betta splendens, Oryzias latipes (Atherinomorpha), and Barbus barbus (Ostariophysi) within a phylogenetic framework. Many evolutionary changes in relative sequence of ossification are evident within regions among these taxa, yet many other sequences are conserved. The logistic difficulty of comparing entire cranial ossification sequences (vs. regional sequences) makes evident the need for new methods for identifying and quantifying sequence changes. Intraspecific variation in order of ossification is described for the first time in teleost fishes. To the extent that ossification sequence varies intraspecifically, conclusions drawn from previous interspecific comparisons are compromised. Understanding the importance of changes in ossification order within and among taxa will require experimental, functional, and evolutionary work. © 1996 Wiley-Liss, Inc.     

Skull development during anuran metamorphosis: I. Early development of the first three bones to form--the exoccipital, the parasphenoid, and the frontoparietal

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.     

Decreased cyclin-dependent kinase activity promotes thyroid hormone-dependent tail regression in <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

The thyroid hormone (TH), 3,5,3′-triiodothyronine (T₃), is an important regulator of diverse cellular processes including cell proliferation, differentiation, and apoptosis, with increasing evidence that the modulation of the phosphoproteome is an important factor in the TH-mediated response. However, little is understood regarding the mechanisms whereby phosphorylation may contribute to T₃-mediated cellular outcomes during development. The cyclin-dependent kinases (Cdks) and mitogen-activated protein kinases (MAPK/ERK) have been implicated in TH signaling in mammalian cells. In this study, we have investigated, in frogs, the possible role that these kinases may have in the promotion of tail regression during tadpole metamorphosis, an important postembryonic process that is completely TH-dependent. Cdk2 steady state levels and activity increase in the tail concurrent with progression through the growth phase of metamorphosis, followed by a precipitous decrease coinciding with tail regression. Cyclin-A-associated kinase activity also follows a similar trend except that its associated kinase activity is maintained longer before a decrease in activity. Protein steady state levels of ERK1 and ERK2 remain relatively constant, and their kinase activities do not decrease until much later during tail regression. Tail tips cultured in serum-free medium in the presence of T₃ undergo regression, which is accelerated by coincubation with a specific Cdk2 inhibitor. Coincubation with PD098059, a MAPK inhibitor, has no effect. Thus, T₃-dependent tail regression does not require MAPKs, but a decrease in Cdk2 activity promotes tail regression. This work was supported by a NSERC operating grant, NSERC University Faculty Award, and Michael Smith Foundationfor Health Research Scholar Award.     

Larval and metamorphic skeletal development in the fast-developing frog Pyxicephalus adspersus (Anura, Ranidae)

Pyxicephalus adspersus , is exceptional among living frogs. Embryonic development, larval phase, and metamorphosis can be completed in 17 days at a temperature of 29°C. The metamorphosis only takes 5 days. The present study shows that, despite the unusually short larval phase in P. adspersus, the state of skeletal differentiation reached at the end of metamorphosis is similar to that of other frog species. There is no shift of cranial bone formation postmetamorphosis as could have been expected and is known from other species. The majority of compared species are particularly similar in the sequence of bone formation in the postcranial skeleton. However, there are clear differences among species in the timing of these events relative to the larval growth trajectory, absolute time, and certain developmental markers, such as external limb differentiation. For example, skeletogenesis and externally visible limb differentiation are only loosely integrated. Interspecific comparisons show that, in P. adspersus, the early onset of skeletal ossification is an unusual feature among frogs. Freshly metamorphosed froglets of P. adspersus are already distinct from comparable stages of other species in having strong jaws, fang-like teeth, and a squamosal-maxilla contact. The latter stabilizes the maxillary arcade and the suspensorium and might relate to the ability to catch and swallow very large vigorous prey, such as siblings, shortly after metamorphosis. The presence of a complete set of dermatocranial elements and postmetamorphic ossification of only the sphenethmoid and operculum are considered plesiomorphic features, whereas the much less completely ossified skulls of metamorphosed froglets, particularly in Bufo and Hamptophryne, are likely apo- morphic developmental traits within the Anura. Accepted: 11 January 1999     

Measurement and regulation of thyroidal status in teleost fish

Summary We have reviewed the stages in teleost thyroid function and its regulation, from the initial biosynthesis of the TH to their eventual interaction with putative receptors.TH biosynthesis depends on an adequate plasma iodide level, determined partly by dietary iodide and partly by active branchial iodide uptake from the water, Pulse-injected radioiodide can be used to evaluate thyroidal iodide uptake, aspects of TH biosynthesis and TH thyroidal secretion. However, owing to variable plasma iodide levels, care is required in interpretating these parameters. TH biosynthesis, thyroglobulin properties and intrathyroidal secretion mechanisms have received limited recent attention. Histological indices of thyroid tissue changes, while useful in many situations, do not always correlate with more direct estimates of thyroidal secretion and can be misleading.Thyroid function is regulated by the hypothalamo-pituitary-thyroid axis, but neither the identities of the hypothalamic factors nor a reliable immunoassay for TSH have been established. Currently, activity of the hypothalamic-pituitary axis is usually determined by pituitary thyrotrope histological appearance or bioassay of pituitary TSH. Plasma free T₄ feeds back at both the pituitary and hypothalamic levels and inhibits TSH release. Thyroidal T₄ secretory activity is presumably adjusted to maintain a constant plasma T₄level according to physiologic state.Plasma T₄ is probably the most commonly used index of thyroidal status. However, (1) T₄ is probably not the active form of TH, (2) the T₄ plasma level may be influenced by the binding properties of plasma proteins, and (3) the T₄ concentration alone makes no provision for the rate of T₄ turnover in plasma. The most practical way to measure thyroidal T₄SR is to determine plasma T₄DR, and assuming steady-state conditions, equate it to T₄SR. The T₄DR is determined from kinetic studies employing^*T₄, which also enable estimates of sizes of vascular and extravascular T₄ pools and their rates of exchange. Excretion of T₄ or its derivatives in urine or bile can be determined also. A high proportion of T₄ is enzymatically monodeiodinated in liver and other tissues, generating T₃ for local (intracellular) and vascular systemic compartments.Bothin vivo andin vitro methods have been used to quantify T₄ deiodinase activity, which is highly responsive to physiologic state and environmental variables. T₃ production is inhibited by a moderate T₃ excess indicating an autoregulatory system, whereby tissue T₃ levels are maintained at a set-point appropriate for a particular physiologic state. The rate of T₃ production provides an informative measure of thyroidal status in a given tissue. However, other pathways also contribute to the maintenance of T₃ homeostasis at a particular set-point. These include the rate of T₃ degradation to 3,3-T₂, the rate of T₄ substrate diversion to rT₃ (an inactive isomer) and by the excretion of parent compounds or conjugates in bile and urine. Potential losses across branchial or integumentary surfaces have yet to be evaluated.The most fundamental measure of thyroidal status is represented by the amount of T₃ saturably bound to receptors/nucleus for the cell type of interest. This is estimated most accurately in double isotope studies in which T₃ contributions from both vascular and intracellular compartments are evaluated. Less satisfactory but meaningful indices of T₃ availability to receptor sites may be obtained from the plasma T₃ (or free T₃) level and from the tissue T₃ level. The former is appropriate if the cell type in question obtains its T₃ primarily from plasma; the latter should be measured if the cell type derives its T₃ mainly through intracellular deiodinase activity. If the proportion of vascular T₃/intracellular T₃ bound to receptors is known, it may indicate the degree of receptor activation. However, even cytosolic T₃ levels may not vary in proportion to nuclear T₃ levels.Differences in thyroidal function between teleosts and homeotherms can be attributed to distinctive strategies in iodide economy and to fundamental differences in control of thyroidal status. Owing to more certain iodide availability (branchial iodide pump and plasma iodide-binding proteins), teleosts are probably more liberal in their iodide use and have less efficient mechanisms for recovery and retention of hormonal iodide than homeotherms. Also, primary control of teleost thyroidal function appears peripheral. It is the finely regulated conversion of T₄ to T₃ in tissues which may largely determine the T₄ secretion rate. Thus, T₄, as a prohormone, may be produced more to satisfy the substrate needs for T₄ conversion rather than to drive T₃ production. Because TH are mainly implicated in tissue- or cell-specific processes involved in development, growth and reproduction in teleosts, it may be advantageous for their thyroidal status to be determined locally through T₄-to-T₃ deiodination. In homeotherms, primary control is mainly central through the hypothalamic-pituitary axis, which regulates thyroidal secretion of T₄ and significant amounts of T₃. The level of T₄ (free T₄) is believed to drive the production of T₃ in most peripheral tissues. Because TH are extensively involved in the systemically integrated adjustment of basal metabolic rate in homeotherms, it may have been advantageous to evolve a system leaning towards central control by the hypothalamus, the brain centre associated with thermoregulation.     

Twisted story of eye migration in flatfish

Early molecular markers for flatfish metamorphosis and eye migration must be linked to the ethmoid region, the earliest part of the flatfish cranium to change, as well as chondral and dermal ossification processes. Serial sections, morphological landmarks, and stereology were used to determine where and when the remodeling of tissues and asymmetry occurs in the head region of metamorphosing Atlantic halibut, Hippoglossus hippoglossus. Not all parts of the head remodel or migrate, and those that do may be asynchronous. Normal metamorphosis limits the torsion of the Atlantic halibut head to the anterior part of the neurocranium and excludes the tip of the snout and the general jaw area. The first cranial structure displaying eye migration-related asymmetric development is the paraethmoid part of the ethmoid cartilage. In early eye migration the medial frontal process moves apace with the eyes, whereas near completion the migrating eye moves significantly closer to the frontal process. Structures of the jaw remain mostly symmetrical, with the exception of the adductor mandibulae muscle and the bone maxillare, which are larger on the abocular than on the ocular side, the muscle occupying the space vacated by the migration of the eye. Thus, normal eye migration involves a series of temperospatially linked events. In juveniles lacking eye migration (arrested metamorphosis), the dermal bone, the prefrontal, does not develop. The two abnormal paraethmoids develop symmetrically as two plate-like structures curving anteriorly, whereas normal elongate fused paraethmoids curve at their posterior. The abocular side retrorbital vesicles are largest in volume only after the completion of normal eye migration. Factors involved in completion of normal metamorphosis and eye migration in flatfish affect chondral and dermal ossification signals in the ethmoid group, as well as remodeling of the mineralized frontal, a series of linked events not involving the entire neurocranium.