James Hanken

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development

Abstract Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression – the zootype. Haeckel’s drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage – thought to correspond to a conserved stage – show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel’s drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species.

Chapter 22 Whole-Mount Staining of Xenopus and Other Vertebrates

Publisher Summary This chapter discusses whole-mount staining of xenopus and other vertebrates. Whole-mount staining makes the analysis of normal and experimentally manipulated embryos much simpler. It can be used in the assay of cellular differentiation in induction and tissue recombination experiments. It should be possible not only to assay for the indication of specific tissues, but to characterize the three-dimensional relationships between the tissue types. Whole-mount staining greatly simplifies the characterization of the expressions patterns of exogenous DNAs. Similarly, the effects of injected antibodies, antisense reagents, or the ecotopic expression of specific molecules on development can be analyzed rapidly. The prerequisite for any whole-mount analysis is that one is able to see through the specimen. This means either that the specimen must be naturally transparent, a rare feature among higher metazoans, or that it must be possible to “clear” it. Clearing generally involves two steps: extracting material from the specimen, then matching the refractive index of the bulk of the specimen remaining, thereby rendering it transparent. Primary considerations in selecting a clearing agent are how closely it matches the refractive index of the specimen, its inherent toxicity, and its compatibility with the staining reagents used.

Cranial Neural-Crest Migration and Chondrogenic Fate in the Oriental Fire-Bellied Toad Bombina orientalis: Defining the Ancestral Pattern of Head Development in Anuran Amphibians

We assess cranial neural‐crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural‐crest cells begin migrating before neural‐fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural‐crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural‐crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conservative. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity.

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.

Life history and morphological evolution

Morphological evolution is influenced by a wide variety of processes, at levels that likely range from molecules to communities, or even ecosystems. The relative contributions of these processes and other biological properties to morphological evolution in individual lineages, and their ultimate role in mediating the evolutionary success of these groups, remain fundamental problems in evolutionary biology (e.g., Miiller and Wagner, 1991; Nitecki, 1990; Roth and Wake, 1989). One underappreciated feature, at least with respect to its potential role in the evolution of morphology, is life history. Organisms with a complex life history comprising a series of discrete, free-living stages might be expected to possess more morphological adaptations than taxa with simple life histories, especially when successive stages occupy radically different environments. Also, the genetic and developmental mechanisms that underlie the sequential appearance of distinct phenotypes might be expected to allow, if not actually facilitate, morphological change at one stage without correlated effects at others to an extent not possible with a simpler life history (Elinson, 1990). On the other hand, the presence of fully differentiated, functional structures at one stage might be expected to constrain the morphology of structures that form earlier or later (D. Wake and Roth, 1989). Yet, ideas such as these remain largely unexplored in most groups. Here I briefly review aspects of the relation between life history and morphological evolution in Recent amphibians, an especially good group for such an analysis. Many taxa retain the ancestral complex life history, which comprises discrete aquatic larval and terrestrial adult stages bridged by a sharply defined transition, or metamorphosis (Fig. IA). In addition, there is a remarkable array of derived life history modes that can be used to test evolutionary hypotheses and illustrate phylogenetic patterns. When examined in this way, a complex life history can be seen to provide substantial opportunities for morphological evolution in these vertebrates. These opportunites are realized through a variety of evolutionary

Evolution and development of the vertebrate skull: The role of pattern formation

The vertebrate skull is anatomically complex and phylogenetically diverse; it presents unique opportunities to examine the role of developmental processes in evolutionary change. Previous studies have largely examined phylogenetic trends in tissue composition or change in the timing of developmental events (heterochrony). Additional important insights may be gained if skull evolution and development are viewed from the standpoint of pattern formation. Contemporary models of pattern formation offer the possibility of linking developmental mechanisms of cranial morphogenesis from the level of genes, through cell biology, to adult form.

Larvae in Amphibian Development and Evolution

Publisher Summary This chapter describes the development and evolution of amphibian larvae. The free-living, aquatic larva is an ancient feature in amphibians. A complex, biphasic life history is phylogenetically widespread among recent taxa and is generally accepted as the primitive condition for each of the three living orders. Variation in developmental timing is a virtually ubiquitous phenomenon in amphibian evolution; it has played a pivotal role in the diversification of morphology, physiology, and ecology in both larvae and adults. The evolution of larvae fundamentally involves modifications in development. Changes in developmental patterns, especially those concerned with the timing of developmental events, that is, heterochrony are well documented. The mechanistic bases of these and other evolutionary changes in developmental pattern, however, generally remain poorly known. Both kinds of information are needed to reliably define the range of evolutionary opportunities and constraints conferred by the ancestral, metamorphic life history, as well as to assess the extent to which these opportunities and constraints vary among different amphibian lineages.

Limb development and evolution: a frog embryo with no apical ectodermal ridge (AER)

The treefrog Eleutherodactylus coqui is a direct developer — it has no tadpole stage. The limb buds develop earlier than in metamorphosing species (indirect developers, such as Xenopus laevis). Previous molecular studies suggest that at least some mechanisms of limb development in E. coqui are similar to those of other vertebrates and we wished to see how limb morphogenesis in this species compares with that in other vertebrates. We found that the hind limb buds are larger and more advanced than the forelimbs at all stages examined, thus differing from the typical amniote pattern. The limb buds were also small compared to those in the chick. Scanning and transmission electron microscopy showed that although the apical ectoderm is thickened, there was no apical ectodermal ridge (AER). In addition, the limb buds lacked the dorsoventral flattening seen in many amniotes. These findings could suggest a mechanical function for the AER in maintaining dorsoventral flattening, although not all data are consistent with this view. Removal of distal ectoderm from E. coqui hindlimb buds does not stop outgrowth, although it does produce anterior defects in the skeletal pattern. The defects are less severe when the excisions are performed earlier. These results contrast with the chick, in which AER excision leads to loss of distal structures. We suggest that an AER was present in the common ancestor of anurans and amniotes and has been lost in at least some direct developers including E. coqui.

Jaw muscle development as evidence for embryonic repatterning in direct–developing frogs

The Puerto Rican direct–developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval–specific features never form inE. coqui; embryonic muscle primordia initially assume an abbreviated, mid–metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval–to–adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic ‘repatterning’ inEleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.

Skull development during anuran metamorphosis

SummaryWe examined the role of thyroid hormone (TH) in mediating cranial ossification during metamorphosis in the Oriental fire-bellied toad, Bombina orientalis. Exogenous T3 (3,3′,5-triiodo-L-thyronine) was administered in three treatment dosages (0.025, 0.25, and 2.5 μg) plus a control dosage via plastic micropellets implanted within the dermis of tadpoles of three Gosner developmental stages: 28/29, 30/31, 32/33. Tadpoles were recovered after 2, 4, 6, and 8 d, and scored for the presence of three bones —median parasphenoid and paired frontoparietals and exoccipitals—as seen in cleared-and-stained, whole-mount preparations. T3 induced precocious ossification in both a stage-dependent and a dosage-dependent manner; stage dependence corresponded precisely with the degree of osteogenic differentiation at the time of hormone administration. Precocious ossification thus was due to the T3-promoted growth and calcified matrix deposition of these centers. Differential TH sensitivity among osteogenic sites may underlie both the temporal cranial ossification sequences characteristic of metamorphosing amphibians as well as sequence differences commonly observed among taxa.