The cellular basis of cartilage growth and shape change in larval and metamorphosing Xenopus frogs.

As the first and sometimes only skeletal tissue to appear, cartilage plays a fundamental role in the development and evolution of vertebrate body shapes. This is especially true for amphibians whose largely cartilaginous feeding skeleton exhibits unparalleled ontogenetic and phylogenetic diversification as a consequence of metamorphosis. Fully understanding the evolutionary history, evolvability and regenerative potential of cartilage requires in-depth analysis of how chondrocytes drive growth and shape change. This study is a cell-level description of the larval growth and postembryonic shape change of major cartilages of the feeding skeleton of a metamorphosing amphibian. Histology and immunohistochemistry are used to describe and quantify patterns and trends in chondrocyte size, shape, division, death, and arrangement, and in percent matrix from hatchling to froglet for the lower jaw, hyoid and branchial arch cartilages of Xenopus laevis. The results are interpreted and integrated into programs of cell behaviors that account for the larval growth and histology, and metamorphic remodeling of each element. These programs provide a baseline for investigating hormone-mediated remodeling, cartilage regeneration, and intrinsic shape regulating mechanisms. These programs also contain four features not previously described in vertebrates: hypertrophied chondrocytes being rejuvenated by rapid cell cycling to a prechondrogenic size and shape; chondrocytes dividing and rearranging to reshape a cartilage; cartilage that lacks a perichondrium and grows at single-cell dimensions; and an adult cartilage forming de novo in the center of a resorbing larval one. Also, the unexpected superimposition of cell behaviors for shape change onto ones for larval growth and the unprecedented exploitation of very large and small cell sizes provide new directions for investigating the development and evolution of skeletal shape and metamorphic ontogenies.

How thyroid hormones and their inhibitors affect cartilage growth and shape in the frog Xenopus laevis.

Understanding how skeleton changes shape in ontogeny is fundamental to understanding how its shape diversifies in phylogeny. Amphibians pose a special case because their jaw and throat skeleton consists of cartilages that are dramatically reshaped midway through life to support new feeding and breathing styles. Although amphibian metamorphosis is commonly studied by immersing larvae in thyroid hormones (TH), how individual cartilages respond to TH is poorly understood. This study documents the effects of larval stage and TH type (T4 vs. T3), dose and deprivation on the size, shape and morphogenesis of the lower jaw and ceratohyal cartilages in the frog Xenopus laevis. It uses thyroid inhibitors to isolate the effects of each hormone at specific concentrations. It also deconstructs the TH responses into the effects on individual dimensions, and uses measures of percent change to eliminate the effects of body size and growth rate variation. As stage increases, T4 and T3 responses become increasingly similar to each other and to natural remodeling; the differences at low and intermediate stages result largely from abnormal responses to T3. Most notably, the beak-like lower jaw commonly observed at the lowest stage in other studies results largely from arrested growth of cartilage. TH responses are superimposed upon the growth typical for each stage so that cartilages can attain postmetamorphic shapes through dimensional changes that exceed those of natural metamorphosis. Using thyroid inhibitors alters the outcome of TH-induced remodeling, and T4 has almost the same capacity to induce metamorphic shape changes as T3. The results have implications for understanding how the starting shapes of larval elements affect morphogenesis, how chondrocytes behave to change cartilage shape, and how intracellular processing of TH might contribute to interspecific differences in shape change. Also, the data on animal mortality and which stages and doses most closely replicate natural remodeling have practical value for researchers who treat Xenopus tadpoles with TH.

Deconstructing cartilage shape and size into contributions from embryogenesis, metamorphosis, and tadpole and frog growth.

Understanding skeletal diversification involves knowing not only how skeletal rudiments are shaped embryonically, but also how skeletal shape changes throughout life. The pharyngeal arch (PA) skeleton of metamorphosing amphibians persists largely as cartilage and undergoes two phases of development (embryogenesis and metamorphosis) and two phases of growth (larval and post-metamorphic). Though embryogenesis and metamorphosis produce species-specific features of PA cartilage shape, the extents to which shape and size change during growth and metamorphosis remain unaddressed. This study uses allometric equations and thin-plate spline, relative warp and elliptic Fourier analyses to describe shape and size trajectories for the ventral PA cartilages of the frog Xenopus laevis in tadpole and frog growth and metamorphosis. Cartilage sizes scale negatively with body size in both growth phases and cartilage shapes scale isometrically or close to it. This implies that most species-specific aspects of cartilage shape arise in embryogenesis and metamorphosis. Contributions from growth are limited to minor changes in lower jaw (LJ) curvature that produce relative gape narrowing and widening in tadpoles and frogs, respectively, and most cartilages becoming relatively thinner. Metamorphosis involves previously unreported decreases in cartilage size as well as changes in cartilage shape. The LJ becomes slightly longer, narrower and more curved, and the adult ceratohyal emerges from deep within the resorbing tadpole ceratohyal. This contrast in shape and size changes suggests a fundamental difference in the underlying cellular pathways. The observation that variation in PA cartilage shape decreases with tadpole growth supports the hypothesis that isometric growth is required for the metamorphic remodeling of PA cartilages. It also supports the existence of shape-regulating mechanisms that are specific to PA cartilages and that resist local adaptation and phenotypic plasticity.

Caging, but not air deprivation, slows tadpole growth and development in the amphibian Xenopus laevis.

Xenopus laevis tadpoles raised in submerged cages in normoxic water develop more slowly than tadpoles raised with access to air. This study distinguishes between the effects of being caged and being deprived access to air on development and growth. Tadpoles were raised in high and low density control tanks and in cages in the same tank that were either completely submerged or with the top exposed to air. Experiments were repeated with the cages in different positions relative to the air stones and with and without the water flow from air stones supplemented with a pump. Whereas caging tadpoles has a large effect on their development and growth, additionally depriving them of air has a small effect and this effect can be removed by optimizing water flow through the cage. The effect of caging, though significant in this study, is small compared to the variation in growth and developmental rates that is commonly encountered within and among controls in lab studies. Caging effects can also be diminished by optimizing rearing conditions and/or having exceptionally vigorous tadpoles. The effects of air deprivation and caging thus pose less of a problem for experimenting on air-deprived (AD) and air-restored Xenopus tadpoles than their inherent variability in growth and developmental rates and their susceptibility to growth and developmental arrest. Further, the effect of air deprivation in this air-breathing amphibian does not pose a conflict with evolutionary hypotheses for lung loss involving lengthening of the larval period and delay in the onset of air breathing.

The importance of cartilage to amphibian development and evolution.

The duality of amphibians is epitomized by their pharyngeal arch skeletons, the larval and adult morphologies of which enable very different feeding and breathing behaviors in aquatic and terrestrial life. To accomplish this duality, amphibian pharyngeal arch skeletons undergo two periods of patterning: embryogenesis and metamorphosis, and two periods of growth: larval and postmetamorphic. Their extreme ontogenetic variation, however, is coupled with relatively limited phylogenetic variation. I propose that amphibians face an evolutionary tradeoff between their ontogenetic and phylogenetic diversification that stems from the need to grow and transform the pharyngeal arch skeleton in cartilage rather than bone. Cartilage differs fundamentally from bone in its histology, function, development and growth. Cartilage is also the first skeletal tissue to form embryonically and provides more cellular pathways for shape change than bone. This article combines morphological, histological and experimental perspectives to explore how pharyngeal arch cartilage shape is controlled in amphibian embryogenesis, growth and metamorphosis, and how amphibian skeletal ontogenies are impacted by using cartilage to evolve a complex life cycle and in evolving away from a complex life cycle.

Plasticity of lung development in the amphibian, Xenopus laevis.

Contrary to previous studies, we found that Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD) and air-restored (AR) tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution.

Integrating ecology and developmental biology to explain the timing of frog metamorphosis.

Amphibian metamorphosis has long intrigued ecologists and developmental biologists, yet the two research programs have progressed separately and toward different goals. Plasticity in metamorphic timing has profound effects on fitness, which has prompted ecologists to develop and test models for predicting how environmental factors affect the size and age of metamorphosis. These models rely upon untested assumptions about the mechanisms for regulating growth and development. Whereas developmental biologists explicitly investigate these mechanisms at the hormonal and genetic levels, they largely ignore the role of environmental input. Recent developments in our understanding of the molecular biology of frog metamorphosis are revealing how these two research programs could be integrated. Here, I review these developments to test ecologists' assumptions about frog metamorphosis, and to present strategies for both research fields to investigate the mechanistic basis of metamorphic plasticity.