Comparative ossification sequence and skeletal development of the postcranium of palaeognathous birds (Aves: Palaeognathae)

Palaeognaths constitute one of the most basal lineages of extant birds, and are also one of the most morphologically diverse avian orders. Their skeletal development is relatively unknown, in spite of their important phylogenetic position. Here, we compare the development of the postcranial skeleton in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana) and elegant crested‐tinamou (Eudromia elegans), focusing on ossification. All of these taxa are characterized by element loss in the appendicular skeleton, but there are several developmental mechanisms through which this loss occurs, including failure to chondrify, failure to ossify and fusion of cartilages prior to ossification. Further evidence is presented here to support a reduction in size of skeletal elements resulting in a delay in the timing of ossification. This study provides an important first look at the timing and sequence of postcranial ossification in palaeognathous birds, and discusses the influence of changes in the pattern of skeletal development on morphological evolution.     

Development of the ethmoidal structures of the endocranium in Discoglossus pictus (Anura: Discoglossidae)

We use histological techniques and computer‐aided three‐dimensional reconstructions made from serial histological sections to describe the ontogeny of the ethmoidal endocranium of discoglossid frog Discoglossus pictus. We identify a pattern of development for the suprarostral cartilage that differs from previous findings and probably represents the ancestral anuran pattern. The nasal cartilages, including the inferior prenasal cartilage, are de novo adult structures. The only larva‐derived structures of the adult nasal capsules are the posterior aspects of the solum nasi and septum nasi. We also identify patterns of development for the ethmoid plate and postnasal wall that occur during early in ontogenesis. These patterns are associated with development events during metamorphic climax. The pattern of timing of chondrification of the anterior nasal cartilages more closely coincides with that of the neobatrachian species than that recorded for the pelobatid frog Spea. In addition, this study supports a sister taxon relationship between Discoglossus and Alytes. J. Morphol. 271:1078‐1093, 2010. © 2010 Wiley‐Liss, Inc.     

Postembryonic ontogeny of the spadefoot toad,Scaphiopus intermontanus (Anura: Pelobatidae): Skeletal morphology

Postembryonic skeletal ontogeny of the pelobatid frog Scaphiopus intermontanus is described based on a developmental series of cleared-and-stained, whole-mount specimens. The focus is on laboratory-reared individuals fed a herbivorous diet as larvae. Although there is variation in the timing of ossification of individual skeletal elements relative to developmental stages based on external morphological criteria, the sequence of skeletal development generally is conservative. Compared with its close relative, S. bombifrons, ossifications that occur during prometamorphosis tend to be slightly delayed in S. intermontanus; however, cranial bones that ossify during late metamorphic climax in S. intermontanus are delayed until postmetamorphosis in S. bombifrons. The differences in timing between the two species are consistent, however, with differences observed between two developmental series of S. intermontanus raised at two different temperatures. Noteworthy features of skeletal development in S. intermontanus include: 1) presence of palatine ossifications that form from independent centers of ossification and soon fuse with the postnarial portion of the vomers to form the compound vomeropalatine bones; 2) compound sphenethmoid that may arise from four or more endochondral centers of ossification and one dorsal, dermal center of ossification; and 3) presence of transverse processes and vestigal prezygapophyses on the first postsacral vertebra. The morphology of the larval orbitohyoideus and interhyoideus muscles is compared. The record of skeletal ontogeny and muscle morphology presented herein for the herbivorous larval morph can serve as a baseline for comparisons with the ontogeny of the carnivorous larval morph of Scaphiopus. J. Morphol. 238:179–244, 1998. © 1998 Wiley-Liss, Inc.     

Skeletogenesis and sequence heterochrony in rodent evolution, with particular emphasis on the African striped mouse, Rhabdomys pumilio (Mammalia)

Data documenting skeletal development in rodents, the most species-rich ‘order’ of mammals, are at present restricted to a few model species, a shortcoming that hinders exploration of the morphological and ecological diversification of the group. In this study we provide the most comprehensive sampling of rodent ossification sequences to date, with the aim of exploring whether heterochrony is ubiquitous in rodent evolution at the onset of skeletal formation. The onset of ossification in 17 cranial elements and 24 postcranial elements was examined for eight muroid and caviomorph rodent species. New data are provided for two non-model species. For one of these, the African striped mouse, Rhabdomys pumilio, sampling was extended by studying 53 autopodial elements and examining intraspecific variation. The Parsimov method of studying sequence heterochrony was used to explore the role that changes in developmental timing play in early skeletal formation. Few heterochronies were found to diagnose the muroid and caviomorph clades, suggesting conserved patterning in skeletal development. Mechanisms leading to the generation of the wide range of morphological diversity encapsulated within Rodentia may be restricted to later periods in development than those studied in this work. Documentation of skeletogenesis in Rhabdomys indicates that intraspecifc variation in ossification sequence pattern is present, though not extensive. Our study suggests that sequence heterochrony is neither pivotal nor prevalent during early skeletal formation in rodents.     

Functional roles for the compartmentalization of the subcutaneous lymphatic sacs in anuran amphibians

Compliance of the subcutaneous lymph sacs of the hindlimbs increases from distal to proximal, as does limb segment mass (and presumably rate of lymph formation), for the semiaquatic bullfrog Rana catesbeiana and the cane toad Bufo marinus but not the aquatic clawed toad Xenopus laevis. Subcutaneous lymph-sac compliances vary interspecifically. The distal-to-proximal increase in lymph-sac compliance and estimates of lymph formation rate in the various hindlimb segments indicate that partitioning of hindlimb subcutaneous lymphatic sacs establishes a differential decrease in the intra-lymph-sac pressure for R. catesbeiana and B. marinus. These pressure differentials constitute a "compliance pump" that drives distal-to-proximal intersac lymph flow. The compliance pump alone explains lymphatic return for the aquatic frog X. laevis but does not explain how lymph would reach the dorsally located lymph hearts for terrestrial anurans, so we hypothesize that skeletal muscle pumps return lymph from the femoral and pubic lymph sacs to the lymph heart. This is a fundamentally different role of the subcutaneous lymph-sac system than has been previously proposed. We suggest that the more proximal subcutaneous lymph sacs are important for fluid storage because they have a relatively high compliance.     

Development and ciliation of the palate in two frogs, Bombina and Xenopus; a comparative study

The morphological changes that occur during metamorphosis in the palates of two types of anuran larvae (a discoglossid, Bombina orientalis, and a pipid, Xenopus laevis) are compared. In B. orientalis the structural changes are accompanied by the ciliation of the palate epithelium. Ciliation begins in the anterior region of the palate and continues in a posterior direction throughout metamorphosis. By contrast, the palate of X. laevis never becomes ciliated during its development. Instead, two ciliated grooves develop between the choanae (nasal openings) and the esophageal opening. The grooves transport mucus and trapped objects out of the internal nares and toward the esophagus. These grooves are compared to similar structures on the palate of adult B. orientalis. The timing and pattern of ciliogenesis during metamorphosis in each of these anurans is also described relative to well-established staging series for external frog development. We show that the onset and location of ciliogenesis are consistent and predictable in these anurans and, therefore, make the frog palate an excellent system for the study of ciliogenesis.     

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.     

Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.     

The amphibian globin gene repertoire as revealed by the Xenopus genome

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named 'globin Y') which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin alpha and beta genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.     

Development of the dendrobatid frog Colostethus machalilla

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.     

Description of the adult skeleton and developmental osteology of the hyperossified horned frog,Ceratophrys cornuta (Anura: Leptodactylidae)

The adult skeleton and tadpole chondrocranium of the leptodcatylid frog, Ceratophrys cornuta (Ceratophryinae), are described in detail, including the ontogenetic development of the chondrocanium and the ossification sequence of the skeleton. The chondrocranium of the carnivorous larvae is unique in lacking a frontoparietal fontanelle and possessing a complete dorsal roof of cartilage. Furthermore, the chondrocranium is extremely robust, particularly those elements involved in the feeding mechanism; these include large palatoquadrate cartilages, stout Meckel's, supra- and infrarostral cartilages, and short, wide, cornua trabeculae. The chondrocranium of C. cornuta resembles that described for Ceratophrys cranwelli, but differs from the chondrocrania reported for the species of Lepidobatrachus. The large adult skull is hyperossified; most elements are fused into a single unit, and nearly all dermal elements are ornamented, casqued, and co-ossified. Calcification is present in nearly every cartilaginous element of the skeleton in larger (older) adults. Several osteological characters previously used in ceratophryine systematics, such as the otic ramus of the squamosal and the columella, are reassessed. Contrary to previous reports, the ossified, dorsal dermal shield above the vertebral column in many ceratophryine anurans is absent in C. cornuta. With few exceptions, the ossification sequence relative to metamorphosis is consistent with those that are known for other anurans. The squamosal arises from three distinct centers of ossification, including an otic element. The frontoparietal arises from two centers of ossification that fuse early in development. A robust postorbital arch is formed primarily by the otic flange of the frontoparietal, which articulates laterally with the medial border of the otic ramus of the squamosal. Changes in the timing of development, or heterochrony, are involved with the evolution of the unusual skull and skeleton of ceratophryine frogs. J Morphol 232:169–206, 1997. © 1997 Wiley-Liss, Inc.     

Regulated expression of the X. tropicalis connexin43 promoter

The spatio-temporal expression pattern of the connexin43 gene during Xenopus development has been described (Van der Heyden et al. 2001). To further investigate the regulation and function of connexin43 (Cx43) in amphibians, we have isolated the gene from Xenopus tropicalis (Xt) and determined its structure. The X. tropicalis Cx43 gene displays the typical two exon-one intron connexin configuration, where the first exon is non-coding. The predicted amino acid sequence of the XtCx43 protein is highly homologous to that of X. laevis, chicken and mammals. Expression of XtCx43 cDNA in N2A cells results in gap-junction plaque formation. Promoter activity of a 3.5 kb upstream region of the X. tropicalis Cx43 gene, including exon 1, mimics endogenous timing of expression after injection of reporter constructs in X. laevis embryos.     

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.     

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae)

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianus Ahl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensis Perret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.     

Parallel microtubules and other conserved elements of dorsal axial specification in the direct developing frog, <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

Specification of the dorsal axis in commonly studied frogs, such as Xenopus laevis and Rana pipiens, depends on a microtubule-mediated movement of cytoplasm in the fertilized egg. The Puerto Rican tree frog, Eleutherodactylus coqui, has an egg that is twenty times the volume of that of X. laevis, raising the question as to whether the mechanism of dorsal axial specification is conserved in these large eggs. Fertilized eggs of E. coqui develop a transient array of parallel microtubules, similar to other frogs, but proportionately larger. The array persists after first cleavage, longer than in other frogs, and is gone by the third cleavage. Correlated with the longer life of the parallel microtubules, both 2- and 8-cell E. coqui embryos remain sensitive to gravity-mediated axial specification, a sensitivity lost in X. laevis before the 2-cell stage. Activation of the Wnt/beta-catenin pathway by injected Xwnt8 RNA causes axial formation as in X. laevis. The results indicate that elements of dorsal axial specification are conserved in E. coqui, but they occur later compared to in X. laevis.     

Expression profile of Xenopus banded hedgehog, a homolog of mouse Indian hedgehog, is related to the late development of endochondral ossification in Xenopus laevis

Late development of endochondral ossification occurs at the boundary between the growth cartilage and bone marrow during the formation of long bones in Xenopus laevis. Since the Indian hedgehog (Ihh) is involved in endochondral ossification in mouse, we investigated the expression of Xenopus banded hedgehog (X-bhh), which is a homolog of mouse Ihh. RT-PCR analysis demonstrated that the X-bhh mRNA was detected from an early stage of limb formation to formation of femurs in mature frogs, and it was associated with the expression of Xenopus-ptc1 (X-ptc1), Xenopus-gli1 (X-gli1), Xenopus-type II collagen (X-col II), Xenopus-runx2 (X-runx2), and Xenopus-osteocalcin (X-ocn) mRNAs. In situ hybridization revealed that chondrogenic cells observed at early limb development expressed X-bhh and X-gli1. At later stages of limb development, chondrocytes, located slightly away from the boundary between the cartilage and bone marrow, expressed the X-bhh, X-ptc1, and X-gli1 mRNAs; however, the mesenchymal cells at the boundary failed to express these mRNAs. The X-bhh, X-ptc1, and X-gli1 mRNAs as well as those of X-runx2 and X-ocn were expressed by the mesenchymal cells in the periosteal region at the tip of the cortical bone, indicating an intimate relationship between X-bhh expression and bone formation in this region. Considered collectively, the present study suggests that X-bhh evolutionally acquired the function to induce osteogenesis; however, the expression profile of X-bhh in epiphysis is closely related to the late development of endochondral ossification in X. laevis.     

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.