Rudimentary, “functionless” first metapodials of Canis latrans: Variation and association in length with longer,functional metapodials

A tenet of evolutionary theory is that phenotypic variation of a trait is inversely related to the intensity of stabilizing selection pressure. Among homologous bones, such as metapodials, a rudimentary, “nonfunctional” bone is expected to be more variable in length than nonrudimentary bones. This study compares variation and association in length among metapodials using 277 adult skeletons of Canis latrans. Canis latrans has a short, “functionless” first metacarpal (mc1) and “rudimentary, vestigial” first metatarsal (mt1). Results show that among the 10 metapodials, mt1 has the highest variation in length; other metapodials do not differ significantly from one another in their variation. Correlation coefficients for length of mc1 and mt1 with their ipsilateral metapodials 2-5 are significantly lower than coefficients for all other ipsilateral pairs. The correlation coefficient between left and right mt1 is significantly the lowest among all bilateral pairs of metapodials. Results are interpreted as follows. Mt1's high variation and low association in length are the outcome of less intense stabilizing selection pressure compared with other metapodials. The nonsignificant difference for variation in length between mc1 and metapodials 2-5 may be that mc1 is functional for development of a pollical dewclaw that helps restrain small prey.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

Embryogenesis of the glowworm Rhagophthalmus ohbai Wittmer (Insecta: Coleoptera, Rhagophthalmidae), with emphasis on the germ rudiment formation

The early embryonic development and features of the developing embryo of the glowworm Rhagophthalmus ohbai are described chiefly by light microscopy, with emphasis on the germ rudiment formation and its phylogenetic implication. The egg period is 30-34 days at about 23 degrees C. The newly laid egg is a short ellipsoid, 1.09 by 0.78 mm in size, and the size increases to 1.15 by 0.95 mm by 17 days after oviposition. Cleavage is of the typical superficial type. The germ disk is formed by cell aggregation of the embryonic area at the anterior end of the egg. The central part of the germ disk then sinks into the yolk and the spherical germ rudiment is formed by fusion of the amnioserosal folds extended from all margins of the germ disk. The inner region of the germ rudiment soon becomes slender and develops into the short embryo, whereas the outer region facing the anterior end is extended to form the thin amnion. The embryo then rapidly elongates, the elongation being accompanied by embryo segmentation and formation of appendages. The submerged condition of the embryo persists until about 17 days after oviposition (about 1 day before embryonic revolution) and thereafter the embryo becomes superficial in position. The presence of the following embryonic characters in R. ohbai supports the molecular data placing it within the Lampyridae: 1) formation of a spherical germ rudiment near the anterior end of the egg, and 2) the submerged condition of the developing embryo persists until shortly before revolution.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Pattern formation in a pentameral animal: induction of early adult rudiment development in sea urchins

We investigated adult rudiment induction in the direct-developing sea urchin Heliocidaris erythrogramma microsurgically. After removal of the archenteron (which includes presumptive coelomic mesoderm as well as presumptive endoderm) from late gastrulae, larval ectoderm develops properly but obvious rudiments (tube feet, nervous system, and adult skeleton) fail to form, indicating that coelomic mesoderm, endoderm, or both are required for induction of adult development. Recombination of ectoderm and archenteron rescues development. Implanted endoderm alone or left coelom alone each regenerate the full complement of archenteron derivatives; thus, they are uninformative as to the relative inductive potential of the two regions. However, in isolated ectoderm, more limited regeneration gives rise to larvae containing no archenteron derivatives at all, endoderm only, or both endoderm and left coelom. Adult nervous system begins to develop only in the latter, indicating that left coelom is required for the inductive signal. Isolated ectoderm develops a vestibule (the precursor of adult ectoderm) and correctly regulates vestibular expression of the ectodermal territory marker HeET-1, indicating that the early phase of vestibule development occurs autonomously; only later development requires the inductive signal. Another ectodermal marker, HeARS, is regulated properly in the larval ectoderm region, but not in the vestibule. HeARS regulation thus represents an early response to the inducing signal. We compare HeARS expression in H. erythrogramma with that in indirect developers and discuss its implications for modularity in the evolution of developmental mode.     

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.     

The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis

Summary The crystallins of normal and a^p mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (a^p/a^p; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.     

Podoplanin is expressed by a sub-population of human foetal rib and knee joint rudiment chondrocytes

The aim of this study was to determine if podoplanin was expressed by rudiment chondrocytes in human foetal cartilages. Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development.     

Ion flow regulates left–right asymmetry in sea urchin development

The degree of conservation among phyla of early mechanisms that pattern the left–right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H⁺/K⁺-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H⁺/K⁺-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H⁺ and/or K⁺, and also of Ca²⁺, functions in the establishment of LR asymmetry in sea urchin development.Electronic Supplementary Material Supplementary material is available for this article at