Loss of U11 small nuclear RNA in the developing mouse limb results in micromelia.

Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here, we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced owing to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase-to-metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.

Muscle regeneration potential and satellite cell activation profile during recovery following hindlimb immobilization in mice.

Reduced muscle activity leads to muscle atrophy and function loss in patients and animal models. Satellite cells (SCs) are postnatal muscle stem cells that play a pivotal role in skeletal muscle regeneration following injury. The regenerative potential, satellite cell numbers, and markers during recovery following immobilization of the hindlimb for 7 days were explored. In mice exposed to 7 days of hindlimb immobilization, in those exposed to recovery (7 days, splint removal), and in contralateral control muscles, muscle precursor cells were isolated from all hindlimb muscles (fluorescence-activated cell sorting, FACS) and SCs, and muscle regeneration were identified using immunofluorescence (gastrocnemius and soleus) and electron microscopy (EM, gastrocnemius). Expression of ki67, pax7, myoD, and myogenin was quantified (RT-PCR) from SC FACS yields. Body and grip strength were determined. Following 7 day hindlimb immobilization, a decline in SCs (FACS, immunofluorescence) was observed together with an upregulation of SC activation markers and signs of muscle regeneration including fusion to existing myofibers (EM). Recovery following hindlimb immobilization was characterized by a program of muscle regeneration events. Hindlimb immobilization induced a decline in SCs together with an upregulation of markers of SC activation, suggesting that fusion to existing myofibers takes place during unloading. Muscle recovery induced a significant rise in muscle precursor cells and regeneration events along with reduced SC activation expression markers and a concomitant rise in terminal muscle differentiation expression. These are novel findings of potential applicability for the treatment of disuse muscle atrophy, which is commonly associated with severe chronic and acute conditions.

First Record of Fibropapillomatosis in a Green Turtle Chelonia mydas from the Baja California Peninsula.

Fibropapillomatosis (FP) is characterized by multiple fibroepithelial tumors in all parts of the skin and has been reported in sea turtles worldwide. Clinically infected individuals are often emaciated and anemic. In Mexico, however, there are few records of this disease. In this study of green turtles Chelonia mydas in Laguna San Ignacio in Baja California Sur (BCS), we noted one juvenile with multifocal fibropapilloma lesions on the external upper surface of its eyes and hind flippers. Light microscopy revealed hyperkeratosis, epidermal hyperplasia, dermal papillary projections, and fibroblast proliferation. Electron microscopy revealed viral particles. Biopsies of normal skin were done to determine the origin of the turtle through genetic analysis. Its mitochondrial DNA matched that of a haplotype (CMP2) from a Hawaiian green turtle population. Finding FP in a turtle captured in BCS elucidates the need for further monitoring along the west coast of Mexico. Further investigation should include testing tumors to detect and characterize any chelonid herpesviruses and explore any association with FP and other diseases that pose a health risk to other sea turtle species. Received March 26, 2016; accepted August 3, 2016.

Three years of alendronate treatment does not continue to decrease microstructural stresses and strains associated with trabecular microdamage initiation beyond those at 1 year.

UNLABELLED: The effects of a 3-year alendronate treatment on trabecular stresses/strains associated with microdamage initiation were investigated using finite element modeling (FEM). Severely damaged trabeculae in the low-dose treatment group were associated with increased stresses compared with the high-dose treatment group (p = 0.006) and approached significance in the control group (p = 0.02). INTRODUCTION: Alendronate, a commonly prescribed anti-remodeling agent, decreases fracture risk in the vertebrae, hip, and wrist of osteoporotic individuals. However, evaluation of microdamage accumulation in animal and human studies shows increased microdamage density relative to controls. Microstructural von Mises stresses associated with severe and linear damage have been found to decrease after 1 year of alendronate treatment. In the present study, stresses/strains associated with damage were assessed after 3 years of treatment to determine whether they continued to decrease with increased treatment duration. METHODS: Microdamaged trabeculae visualized with fluorescent microscopy were associated with stresses and strains obtained using image-based FEM. Stresses/strains associated with severe, diffuse, and linearly damaged and undamaged trabeculae were compared among groups treated for 3 years with an osteoporotic treatment dose of alendronate, a Paget's disease treatment dose of alendronate, or saline control. Architectural characteristics and mineralization were also analyzed from three-dimensional microcomputed tomography reconstructed images. RESULTS: Severely damaged trabeculae in the osteoporotic treatment dose group were associated with increased stress compared with the Paget's disease treatment dose group (p = 0.006) and approached significance compared to the control group (p = 0.02). Trabecular mineralization in severely damaged trabeculae of the low-dose treatment group was significantly greater compared to severely damaged trabeculae in the high-dose treatment and control group, suggesting that changes at the tissue level may play a role in these findings. CONCLUSIONS: Trabecular level stresses associated with microdamage do not continue to decrease with prolonged alendronate treatment. Changes in mineralization may account for these findings.

Improving subchondral bone integrity reduces progression of cartilage damage in experimental osteoarthritis preceded by osteoporosis.

PURPOSE: Impairment of subchondral bone density and quality aggravates cartilage damage in osteoarthritis (OA). Accordingly, we assessed whether improving microstructure and quality at subchondral bone by the bone-forming agent parathyroid hormone (PTH) [1-34] prevent cartilage damage progression in a rabbit model of OA preceded by osteoporosis (OP). METHODS: OP was induced in 20 female rabbits. At week 7, these rabbits underwent knee surgery to induce OA and, at week 12, they started either saline vehicle (n=10) or PTH (n=10) for 10 weeks. Ten healthy animals were used as controls. At week 22, microstructure was assessed by micro-computed tomography and bone remodelling by protein expression of alkaline phosphatase (ALP), metalloproteinase-9 (MMP9), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) at subchondral bone. Cartilage damage was evaluated using Mankin score. RESULTS: PTH reversed the decrease of bone area/tissue area, trabecular thickness, plate thickness, polar moment of inertia, ALP expression and OPG/RANKL ratio, as well as counteracted the increase of fractal dimension and MMP9 expression at subchondral bone of osteoarthritis preceded by osteoporosis (OPOA) rabbits compared to vehicle administration (P<0.05). Likewise, PTH decreased cartilage damage severity in OPOA rabbits. Good correlations were observed between subchondral bone structure or remodelling parameters, and cartilage Mankin score. CONCLUSIONS: Improvement of microstructural and remodelling parameters at subchondral bone by PTH [1-34] contributed to prevent cartilage damage progression in rabbits with early OPOA. These findings support the role of subchondral bone in OA. Further studies are warranted to establish the place of bone-forming agents as potential treatment in OA.

Influence of 14-day hind limb unloading on isolated muscle spindle activity in rats.

During hind limb unloading (HU), the soleus is often in a shortened position and the natural physiological stimulus of muscle spindles is altered, such that muscle spindle activity also changes. Using isolated spindle conditions, the present study investigates the electrophysiological activity and ultrastructure of muscle spindles following HU. Results show that muscle spindle discharges fall into either of two main patterns, single spikes or spike clusters in shortened positions, with a steady frequency of 18-38 spikes/s (mean 29.08 +/- 2.45) in an extended position. Following 14-day HU, afferent discharge activity was significantly altered in soleus muscle spindles. Duration of individual spikes was significantly prolonged, from 0.54 +/- 0.05 ms for control rats to 1.53 +/- 0.25 ms for rats in the HU group. In a shortened position, regular rhythm afferent discharges were obviously depressed, and the majority of muscle spindles became silent, while in an extended position, the discharges remained continuous but with decreased frequency. Results also show that the ultrastructure of muscle spindles experience degenerative changes during HU. Altered muscle spindle afference could possibly modify the activity of motor neurons and further affect the activity of extrafusal fibers.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Scanning electron microscopy of legs of two species of sucking lice (Anoplura: Phthiraptera).

Pretarsal, tarsal and tibial structures of the forelegs, midlegs and hindlegs of Pediculus humanus of humans and of Haematopinus apri Goureau, 1866 (Phthiraptera), a parasite of feral hogs, were studied using light microscopy and scanning electron microscopy. Details of the tibial thumb-like process (tl) with the spine of the thumb (spn), tarsal apophysis (ta) and the coupled finger-like process (cfl) can be observed in the leg photomicrograph of both species. A frontal view of the leg in open position shows the articulation of the claw: the structures of an open-closed system, a tooth row (te), rack-system (rs) and two telescopic columns (tc) which are present near the base of the claw in both species. In H. apri, we observed a pad-like structure, the flap-like tibial lobe (fl) on the ventral surface on the tarsus, the euplantulae, with several sensilla basiconica, which is present in each leg.

Versatility of Turing patterns potentiates rapid evolution in tarsal attachment microstructures of stick and leaf insects (Phasmatodea).

In its evolution, the diverse group of stick and leaf insects (Phasmatodea) has undergone a rapid radiation. These insects evolved specialized structures to adhere to different surfaces typical for their specific ecological environments. The cuticle of their tarsal attachment pads (euplantulae) is known to possess a high diversity of attachment microstructures (AMS) which are suggested to reflect ecological specializations of different groups within phasmids. However, the origin of these microstructures and their developmental background remain largely unknown. Here, based on the detailed scanning electron microscopy study of pad surfaces, we present a theoretical approach to mathematically model an outstanding diversity of phasmid AMS using the reaction-diffusion model by Alan Turing. In general, this model explains pattern formation in nature. For the first time, we were able to identify eight principal patterns and simulate the transitions among these. In addition, intermediate transitional patterns were predicted by the model. The ease of transformation suggests a high adaptability of the microstructures that might explain the rapid evolution of pad characters. We additionally discuss the functional morphology of the different microstructures and their assumed advantages in the context of the ecological background of species.

Critical Ischemia Times and the Effect of Novel Preservation Solutions HTK-N and TiProtec on Tissues of a Vascularized Tissue Isograft.

BACKGROUND: We herein investigate critical ischemia times and the effect of novel preservation solutions such as new histidine-tryptophan-ketoglutarate (HTK-N) and TiProtec on the individual tissues of a rat limb isograft. METHODS: Orthotopic hind-limb transplantations were performed in male Lewis rats after 2 hours, 6 hours, or 10 hours of cold ischemia (CI). Limbs were flushed and stored in HTK-N, TiProtec, HTK, or saline solution. Muscle, nerve, vessel, skin, and bone samples were procured on day 10 for histology, immunohistochemistry, confocal and electron microscopy, and quantitative real-time polymerase chain reaction analysis. RESULTS: Histomorphology of the muscle showed a mainly perivascular inflammatory infiltrate, fibrotic degeneration, and neovascularization after 6 hours and 10 hours of CI. However, centrally aligned nuclei observed in muscle fibers suggest for muscle regeneration in these samples. In addition to Wallerian degeneration, nerve injury was significantly aggravated (P = 0.032) after prolonged CI. Proinflammatory and regulatory cytokines were most significantly upregulated after 2-hour CI. Our data suggest no superiority of novel perfusates HTK-N and TiProtec in terms of tissue preservation, compared with HTK and saline. CONCLUSIONS: Limiting CI time for less than 6 hours is the most significant factor to reduce tissue damage in vascularized tissue transplantation. Signs of muscle regeneration give rise that ischemic muscle damage in limb transplantation might be reversible to a certain extent.

Hierarchical bioinspired adhesive surfaces-a review.

The extraordinary adherence and climbing agility of geckos on rough surfaces has been attributed to the multiscale hierarchical structures on their feet. Hundreds of thousands of elastic hairs called setae, each of which split into several spatulae, create a large number of contact points that generate substantial adhesion through van der Waals interactions. The hierarchical architecture provides increased structural compliance on surfaces with roughness features ranging from micrometers to millimeters. We review synthetic adhesion surfaces that mimic the naturally occurring hierarchy with an emphasis on microfabrication strategies, material choice and the adhesive performance achieved.

Anti-inflammatory, anti-nociceptive and antioxidant activities of Balanites aegyptiaca (L.) Delile.

The anti-inflammatory and anti-nociceptive activities of methanol (ME), butanol (BE) extracts and of two new saponins isolated from Balanites aegyptiaca bark were evaluated. The study was carried out in vivo and in vitro. The samples, extracts and pure substances, were intra-gastrically administered to animals. Two different animal models, the carrageenin-induced edema, in the rat, and acetic acid-induced writhing test in mice, were adopted. Moreover, the antioxidant power of extracts, fractions and individual constituents from Balanites aegyptiaca has been evaluated in vitro, using a method based on the Briggs-Rauscher (BR) oscillating reaction. Results obtained demonstrate that both ME or BE have a significant effect at the highest dose on the number of abdominal writhes induced by acetic acid, with a 38 and 54% inhibition respectively, but no significant difference was observed for extracts at the lowest dose and for the pure compounds compared with control animals. The same extracts exhibit a significant reduction on the rat paw edema. The inhibition produced by ME is about the same (28+/-3% lowest dose, 32+/-3% highest dose) after administration. A more evident effect is obtained by BE (41+/-3% and 68+/-6% respectively) and single saponins B1 and B2 (62+/-5% and 59+/-6% respectively) after oral administration. The antioxidant activity obtained seems to be in good accordance with the pharmacological results. The histological sections of rat paw confirm the antiflogistic activity of the plant extracts.

Tissue patterning in the developing mouse limb.

The developing mouse fore- and hindlimbs begin as bumps on the flank of the embryo and grow out to form miniature models of the adult limb during a five day period from E9.5 to E14.5. In this paper I show a series of embryos taken at half-day intervals during limb development and outline the timetable of patterning for each of the component tissues of a limb: epidermis, connective tissues, muscle, nerves and blood vessels. Scanning electron micrographs, supplemented by histological sections, are presented to define a set of standard stages for the description of mouse limb development. I discuss my observations of the mouse limb in the light of current theories of vertebrate limb development, which are based on classic manipulation experiments in the chick as well as more recent molecular data from the mouse system. The limb skeletal pattern in a mouse is laid down in a proximodistal direction, as it is in a bird: the E11.5 forelimb reveals the first signs of a humerus and by E14.5 even the most distal phalanges of the hand are formed. At this late stage ossification sleeves are seen around the proximal limb elements as the cartilage template begins to be converted to a bony skeleton. Myogenic cells stainable with the MF20 antibody against early muscle myosin heavy chain are first seen in the mouse forelimb at E11.5, which is also when the first nerve fascicles begin to enter the limb. From E11.5 to E14.5 both muscle and nerve patterns mature to give distinct muscles at all proximodistal levels of the limb, each muscle with its own nerve branch, and a cutaneous nerve plexus that extends to the fingertips. The developing skin of the mouse limb matures from a bi-layered epidermis overlying an avascular, but otherwise nondescript, prospective dermal layer of mesenchyme at E9.5, to a 4- or 5-layered epidermis with early hair placodes and the first signs of a distinct dermal layer at E14.5. Notable differences between mouse and chick limb development lie in the relatively late formation of the apical ectodermal ridge in the mouse and its unexpectedly close relations with blood vessels, in the absence of anterior and posterior necrotic zones and, possibly, in a late migration of myogenic cells into the mouse limb bud.

SEM search for sound production and sound perception organs in a variety of Drosophila species.

The discovery of new song types in Hawaiian Drosophila species has raised a question of alternative sound production and sound perception mechanisms in Drosophila. For example D. disjuncta males, which produce song with a carrier frequency of 5,000-6,000 Hz, do not vibrate their wings but only tremble them in a very small amplitude while singing. In our SEM search we found at the wing base of the flies of this species a special structure which could play a part in song production. Our search for auditory sense organs in Drosophila species revealed that the aristae on fly antennae are structurally not as simple as has been assumed. In Hawaiian D. planitibia subgroup species, the fly aristae are more regular than in other Drosophila species and they are also covered with small hairs. We did not find any sign of tympanic hearing organs in any of the studied species, but we discovered on fly legs and wings sensilla which could play a part in sound/vibration perception. Our SEM survey provides a good starting point in the search for alternative pathways for sound production and perception via transmission electron microscopy combined with behavioral studies and electrophysiological recordings in Hawaiian Drosophila species.

An ultrastructural study of the perichondrium in cartilages of the chick embryo.

The ultrastructure of perichondrial tissue of cartilage rudiments (metatarsus, tibiotarsus and sternum) of the chick embryo at various stages of development (H.H. stages 28-45) was investigated by transmission electron microscopy. Previous microscopic and submicroscopic data were generally confirmed, but new findings indicated: (a) the existence of a temporary syncytial state of perichondroblasts during the earliest developmental stages, (b) the existence of a perichondrial cambial layer of stem cells, (c) involvement of perichondroblasts in the appositional growth of cartilage. Electron microscopy revealed clear temporal relations between cell differentiation, perichondrial growth and the structure and production of perichondrial ECM. In addition, the boundaries between cartilage and perichondrial tissue were demonstrated unambiguously. Perichondrial structure varied specifically with each cartilage segment; in particular the perichondrium in long bone rudiments (where ossification starts early) contrasted with that in the permanent cartilage medial process of the sternum.

Regeneration of tactile lamellar corpuscles of the rat after postnatal freeze injury.

Tactile lamellar corpuscles were studied after freeze injury of rat toe pads under normal conditions and following permanent denervation in 1- to 65-day-old animals. In the innervated skin, digital corpuscles redifferentiated in all age groups examined during development and maturation. Characteristic of the reinnervated skin was a great diversity in the shape and size of newly formed corpuscles. Small corpuscles with only 1-3 lamellae around their terminals and well-developed corpuscles of about normal size with up to 15 lamellae were sometimes found within the same sample of skin. The regenerated corpuscles were reduced in number; they reappeared in only 50% of dermal papillae in the toe pads after freeze injury in 7-week-old rats, compared with approximately 100% of dermal papillae that contained lamellar corpuscles in normal toe-pad skin. In denervated toes, occasional corpuscular lamellar structures appeared first after freeze injury applied to 34-day-old rats. In the toe pads denervated and injured by freezing in 42- and 49-day-old rats, lamellar structures redifferentiated in about 10% of the papillae, and in 23.5% after freeze injury applied to 2-month-old rats. Unsatisfactory preservation of basal laminae at the former sites of the corpuscles and in the acellular peripheral nerve stumps, and/or insufficient migration of Schwann cells, may be responsible for the absence or abortive regeneration of lamellar structures in denervated skin of food pads after freeze injury in young rats.

Sequences of intrafusal fiber formation are muscle-dependent in rat hindlimbs.

A rat muscle spindle typically contains four intrafusal fibers-one nuclear bag2, one nuclear bag1 and two nuclear chain fibers. We compared the sequence of formation of the three intrafusal fiber types among the tibialis anterior (TA), soleus (SOL) and medial gastrocnemius (MG) muscles using immunocytochemistry of spindle-specific myosin heavy chain isoforms. Spindles of the TA began to differentiate earlier and acquired the full complement of intrafusal fibers sooner than spindles of the SOL or MG muscles. At the onset of spindle assembly, the intrafusal myotubes expressed myosin heavy chains similar to those expressed by extrafusal myotubes. The first intrafusal myotube then differentiated into the bag2 fiber regardless of the muscle. However, the fate of the second-forming intrafusal myotube varied among the muscles studied. It usually differentiated into a chain fiber in the TA, into a bag1 fiber in the SOL, and into either a bag1 or a chain in the MG. The fate of the third-forming intrafusal myotube was reciprocal to that of the second; i.e. in those spindles in which the bag1 fiber was second to form, a chain was third, and vice versa. The fourth and last intrafusal myotube gave rise to a chain fiber. The inter- and intramuscular variability in the fate of intrafusal myotubes of the second and third generation argues against the existence of a program intrinsic to the myotubes that would mandate their differentiation along specific paths. Rather, an extrinsic regulatory factor, probably associated with the primary afferent neuron, may govern differentiation of pluripotential myotubes into particular types of intrafusal fiber. The fate of the intrafusal myotubes might then depend on the timing of the regulatory effect of afferents relative to the stage of development of the intrafusal bundle.

Carrageenan-induced arthritis in the rat.

This study documents a model of carrageenan-induced chronic inflammatory arthritis in the rat, using quantitative histomorphometric assessment. Ten Sprague-Dawley female rats were randomly assigned to one of two groups. Arthritis was induced in the right tibiofemoral joint by 7 intra-articular injections of 0.02 mL of 1% carrageenan in the arthritic group over 24 days. The control (normal) group was injected with 0.02 mL of saline in the right tibiofemoral joint. Sagittal sections of the right knee joint (distal femur and proximal tibia) were assessed by histomorphometry using the LECO 2001 image analysis system. Articular cartilage thickness, epiphyseal plate thickness, subchondral bone plate thickness, trabecular bone volume and thickness of the synovial lining cell layer were measured. Differences between normal and arthritic groups were statistically significant for articular cartilage thickness of the femur, epiphyseal plate thickness of both the femur and tibia, subchondral bone plate thickness of the tibia and the thickness of the synovial lining cell layer. These findings demonstrate that carrageenan-induced arthritic changes are similar to other, established models of arthritis in the rat.

Fine structural aspects of the anterior necrotic zone in the leg bud of the chick embryo.

A fine structural study was undertaken on the area called the anterior necrotic zone of leg buds of the chick embryo at stages 22-29. Physiologically dying cells, which were distributed in the mesenchymal area, were characterized by increased electron density of the cytoplasm, appearance of electron lucent vacuoles and condensed chromatin pattern. Many of the dying cells seemed to be fragmented into several pieces of cell debris during the necrotic process. Two kinds of cells were observed to take up the necrotic cells; one type, large cells 15 microns or more in size and irregular in shape, which had lysosomes, extended microvillus-like cytoplasmic projections and pseudopods, took up pieces of necrotic cells by phagocytosis and stored them in acid phosphate-negative and -positive vacuoles. The other type was several microns in diameter with numerous free ribosomes and stored a small number of vacuoles containing cell debris. At stage 29, profiles of necrotic cells lying free in the mesenchymal area, and cells containing one or two vacuoles filled with cell debris decreased in number as well as large cells containing numerous vacuoles, whose contents seemed to be degraded. When pieces of the anterior portion of chick leg buds at stage 24 were organ-cultured for 3 days after glutaraldehyde-fixed mouse red blood cells were injected, the red blood cells were taken up into cells in the mesenchymal area. Formation of large cells containing numerous vacuoles stuffed with red blood cells was rare. These findings may imply that most necrotic cells in the anterior necrotic zone are disposed of by macrophages and that some are endocytosed by mesenchymal cells.

Ultrastructural comparison between regenerating and developing hindlimbs of Xenopus laevis tadpoles.

Hindlimbs of Xenopus laevis tadpoles at stages 50 to 55 of embryonic development were amputated in order to study the fine structure associated with ontogenetic decline in regenerative ability of this anuran. Regenerating hindlimbs were compared with their contralateral developing limb so as to determine the similarities and differences in ectodermal-mesenchymal ultrastructural relationships in these systems. Prior to stage 53, mesenchymal cells in regenerating limbs, as well as mesenchyme cells in developing limbs appear undifferentiated; the cells are not visibly different. However, at stage 54 muscle and cartilage differentiation in the developing foot is distinct. Furthermore, in all larvae with regenerates at stages 50 to 52 the basal lamina is not evident subjacent to the apical tip of the epithelium which covers the amputation surface. A distinct basal lamina is present, however, beneath the epidermis in all embryonic limbs examined, including the apical tip, as well as in regenerates of stages 53 to 55 tadpoles. Also, a greater apical accumulation of extracellular matrix (ECM) and organized collagen is observed among the mesenchymal cells in regenerates of stages 53 to 55 tadpoles, and among the mesenchyme cells in developing limbs of stages 50 to 55, compared with pre-stage 53 regenerates. In cases in which an embryonic limb bud is composed of relatively undifferentiated cells (stages 50 to 52), events following amputation result in the complete regeneration of a limb. However, when amputated embryonic limbs contain differentiating tissues, (e.g., muscle and cartilage) only selective tissues undergo regeneration resulting in malformed (heteromorphic) regenerates.(ABSTRACT TRUNCATED AT 250 WORDS)