Integumentary structure and its relationship to wiping behaviour in the common Indian tree frog, Polypedates maculatus

Skin of the Indian tree frog, Polypedates maculatus (Rhacophoridae), was studied in the context of self-wiping behaviour which functions to expel and distribute cutaneous secretions recently shown to retard evaporative water loss. The secretions contain both mucus and lipids and are derived from a common gland considered to be homologous with characteristic anuran mucous glands. The glands are bipotent and secrete both mucus and lipoid products which are evidently mixed within the glandular lumen. Another type of gland resembling characteristic anuran serous (or granular) glands is found in dorsal but not ventral skin, whereas the lipid-secreting mucous glands are found in skin associated with all body surfaces. There is no distinct, lipid-secreting gland present in the skin of this species other than the mucous glands. These histochemical data complement the earlier finding that resistance to evaporative water loss in this species is relatively small compared with phyllomedusine 'waterproof frogs which also exhibit wiping behaviour associated with secretion of lipids. Thus, wiping behaviour may have evolved in association with mucous secretions before dominant lipoid secretions resulted from strong selection for water conservation.     

Abnormal Limb Regeneration without Tumor Production in Adult Newts Directed by Carcinogens, 20-Methylcholanthrene and Benzo (α) pyrene

The effects of potent carcinogens, 20-methylcholanthrene (MC) and benzo(α)pyrene (BP), on limb regeneration were studied in adult newts. A microcrystal of these carcinogens was administered directly to the blastema of forelmibs on day 7 after amputation. The formation of the regeneration cone was delayed and the cone was shifted in abnormal polarity depending upon the site of micro-crystal administration. These carcinogens affected morphogenesis of skeletal structures of regenerating limbs. Subregeneration and superregeneration of either or both carpals and digits, absence of either or both ulna and radius, and accessory limb formation have been recorded as abnormalities caused by these carcinogens. Non-carcinogenic benzocompounds did not show such effects as those of MC and BP. The regeneration blastema of the limb appears to be resistant to carcinogenic effects of the carcinogens used since tumor formation has never been observed in our study so far.     

Anatomical Network Comparison of Human Upper and Lower,Newborn and Adult,and Normal and Abnormal Limbs,with Notes on Development,Pathology and Limb Serial Homology vs. Homoplasy

How do the various anatomical parts (modules) of the animal body evolve into very different integrated forms (integration) yet still function properly without decreasing the individual’s survival? This long-standing question remains unanswered for multiple reasons, including lack of consensus about conceptual definitions and approaches, as well as a reasonable bias toward the study of hard tissues over soft tissues. A major difficulty concerns the non-trivial technical hurdles of addressing this problem, specifically the lack of quantitative tools to quantify and compare variation across multiple disparate anatomical parts and tissue types. In this paper we apply for the first time a powerful new quantitative tool, Anatomical Network Analysis (AnNA), to examine and compare in detail the musculoskeletal modularity and integration of normal and abnormal human upper and lower limbs. In contrast to other morphological methods, the strength of AnNA is that it allows efficient and direct empirical comparisons among body parts with even vastly different architectures (e.g. upper and lower limbs) and diverse or complex tissue composition (e.g. bones, cartilages and muscles), by quantifying the spatial organization of these parts—their topological patterns relative to each other—using tools borrowed from network theory. Our results reveal similarities between the skeletal networks of the normal newborn/adult upper limb vs. lower limb, with exception to the shoulder vs. pelvis. However, when muscles are included, the overall musculoskeletal network organization of the upper limb is strikingly different from that of the lower limb, particularly that of the more proximal structures of each limb. Importantly, the obtained data provide further evidence to be added to the vast amount of paleontological, gross anatomical, developmental, molecular and embryological data recently obtained that contradicts the long-standing dogma that the upper and lower limbs are serial homologues. In addition, the AnNA of the limbs of a trisomy 18 human fetus strongly supports Pere Alberch''s ill-named "logic of monsters" hypothesis, and contradicts the commonly accepted idea that birth defects often lead to lower integration (i.e. more parcellation) of anatomical structures.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.     

Limb Regeneration in the Fiddler Crab, Uca pugilator: Histological, Physiological and Molecular Considerations

This paper summarizes our recent work on the histological, physiologicaland molecular aspects of limb regeneration in the fiddler crabUca pugilator and new information is presented on mitotic activityin the blastema during the first days of blastemal organization.We also report for the first time the localization of vertebrategrowth factor immunoreactivity (FGF 2 and FGF 4) in the regeneratingblastema. In the first part of this paper we review recent histologicalfindings concerning the physical events that accompany autotomyof limbs and propose a new function for the autotomy membrane—thetethering of the regenerating pedal nerve to the walls of thecoxa. In the second part of the paper we review our recent findingson the identification and characterization of the Uca ecdysteroidreceptor (UpEcR, and its potential dimer partner, the retinoid-X-receptor,UpRXR). Using Uca-specific antibody probes raised in our lab,we have been able to identify specific cells in the early blastemathat express receptor proteins. The regenerating limb of thefiddler crab is responsive to both steroids and retinoids andmRNA for steroid and retinoid receptors are expressed in theregenerating limb buds during all stages of regeneration. TheDNA and deduced amino acid sequences of the ecdysteroid receptoris very similar to the sequences of insect EcRs, while the retinoidreceptor is similar to insect protein (ultraspiracle) in theDNA-binding domain, but closer to vertebrate RXRs in the ligandbinding domain     

Regeneration of Limbs and Sensory Organs in Ixodid Ticks (Acari,Ixodoidea, Ixodidae,and Argasidae)

We present a review of our own and literature data on reparative regeneration in ixodoid ticks (chelicerate arthropods). Ticks have a high potential for reparative regeneration and a close relationship between regeneration and development determined by similar hormonal regulatory mechanisms. These mechanisms depend on ecdysteroid hormones, which participate in the initiation of both processes, and juvenile hormones, which direct these processes either to the maintenance of larval features or to the development of nymphal and adult features. We present a detailed analysis of the regeneration of Haller's sensory organs in ixodid ticks and propose an hypothesis about the role of juvenile hormones in the modification of morphogenetic processes in this group. Furthermore, we present data on the effects of insect juvenile hormone analogs (methoprene and fenoxycarb) on the regeneration of Haller's organ, which support this hypothesis. Studies on reparative regeneration in arthropods provide a broader view of the problem of repair morphogenesis in animals.     

Limb Regeneration in the Fiddler Crab, Uca pugilator: Hormonal and Growth Factor Control

This paper summarizes recent work on various aspects of hormonalcontrol of regeneration in the crustacean, Uca pugilator. Hormonalcontrol in this crab is effected by means of the crustacteansteroid hormones, the ecdysteroids. New evidence is presentedsupporting a role for the retinoid hormones, all-trans retinoicacid and 9-cis retinoic acid, in the control of regenerationin these animals. The possible role of fibroblast growth factorsin organization of the limb blastema is explored and the similaritiesbetween vertebrate and invertebrate control of regenerationare discussed.     

Regeneration of Limbs in Males of the Tanner Crabs Chionoecetes bairdi and Ch. Opilio

The frequency of the loss of pereiopods and the features of their regeneration were studied in males of the tanner crabs Chionoecetes bairdi and Ch. opilio, sampled in the Sea of Okhotsk in 2001. It was established that after the loss of an appendage a complete recovering of body symmetry in both crab species occurs in four moltings, during which the length of the regenerating pereiopod averages respectively 39–41, 70.5, 89.5, and 96–97% of the norm. The patterns of limb regeneration were compared between tanner crabs and representatives of fam. Lithodidae.     

Solution Structure and Phylogenetics of Prod1, a Member of the Three-Finger Protein Superfamily Implicated in Salamander Limb Regeneration

Background

Following the amputation of a limb, newts and salamanders have the capability to regenerate the lost tissues via a complex process that takes place at the site of injury. Initially these cells undergo dedifferentiation to a state competent to regenerate the missing limb structures. Crucially, dedifferentiated cells have memory of their level of origin along the proximodistal (PD) axis of the limb, a property known as positional identity. Notophthalmus viridescens Prod1 is a cell-surface molecule of the three-finger protein (TFP) superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules.

Methodology/Principal Findings

With the aim of identifying potential orthologs of Prod1, we have solved its 3D structure and compared it to other known TFPs using phylogenetic techniques. The analysis shows that TFP 3D structures group in different categories according to function. Prod1 clusters with other cell surface protein TFP domains including the complement regulator CD59 and the C-terminal domain of urokinase-type plasminogen activator. To infer orthology, a structure-based multiple sequence alignment of representative TFP family members was built and analyzed by phylogenetic methods. Prod1 has been proposed to be the salamander CD59 but our analysis fails to support this association. Prod1 is not a good match for any of the TFP families present in mammals and this result was further supported by the identification of the putative orthologs of both CD59 and N. viridescens Prod1 in sequence data for the salamander Ambystoma tigrinum.

Conclusions/Significance

The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be correlated with the absence of the Prod1 gene.     

From Clinging to Digging: The Postembryonic Skeletal Ontogeny of the Indian Purple Frog,Nasikabatrachus sahyadrensis (Anura: Nasikabatrachidae)

The Indian Purple frog, Nasikabatrachus sahyadrensis, occupies a basal phylogenetic position among neobatrachian anurans and has a very unusual life history. Tadpoles have a large ventral oral sucker, which they use to cling to rocks in torrents, whereas metamorphs possess adaptations for life underground. The developmental changes that underlie these shifts in habits and habitats, and especially the internal remodeling of the cranial and postcranial skeleton, are unknown. Using a nearly complete metamorphic series from free-living larva to metamorph, we describe the postembryonic skeletal ontogeny of this ancient and unique monotypic lineage. The torrent-dwelling larva possesses a dorsoventrally flattened body and a head with tiny dorsal eyes, robust lower and upper jaw cartilages, well-developed trabecular horns, and a definable gap between the trabecular horns and the tip of the snout. Unlike tadpoles of many other frogs, those of Nasikabatrachus retain larval mouthparts into late metamorphic stages. This unusual feature enables the larvae to maintain their clinging habit until near the end of metamorphosis. The subsequent ontogenetic shift from clinging to digging is correlated with rapid morphological changes and behavioral modifications. Metamorphs are equipped with a shortened tibiafibula and ossified prehallical elements, which likely facilitate initial digging using the hind limbs. Subsequently, the frogs may shift to headfirst burrowing by using the wedge-shaped skull, anteriorly positioned pectoral girdle, well-developed humeral crests and spatula-shaped forelimbs. The transition from an aquatic life in torrents to a terrestrial life underground entails dramatic changes in skeletal morphology and function that represent an extreme in metamorphic remodeling. Our analysis enhances the scope for detailed comparative studies across anurans, a group renowned for the diversity of its life history strategies.     

Tail Regeneration in Normal,Blinded and Pinealectomized Gekkonid Lizards,Hemidactylus flaviviridis,Exposed to Four Different Light Conditions During Three Seasons (Temperatures)

Tail regeneration was followed for 60 days in 1470 normal (NL), blinded (BL) and pinealectomized (PX) gekkonid lizards, Hemidactylus flaviviridis, fed on cockroaches ad libitum and exposed to continuous light of high intensity (2500 lux), continuous light of low intensity (638 lux), 12 h of light (high intensity) and 12 h of darkness and continuous darkness in summer (March – May; cage temperature 30°C) monsoon (August – October; cage temperature 26°C) and winter (November – January; cage temperature 17°C) seasons. A comparative assessment of the new growth (regenerate) showed that the initiation of regeneration, the daily growth, the total length of tail replaced at the end of regeneration and the total percentage replacement of the lost (autotomized) tails were all enhanced by continuous light and depressed by continuous darkness. Furthermore, there was no significant difference between any of the parameters in NL and BL Hemidactylus; however, PX lizards generally showed retardation in the regeneration process. Seasonal differences in tail regeneration were noted—the best regenerative performance was obtained during the summer months and the worst during the winter months, with the regenerative performance during the monsoon season in between. Thermal and photoperiodic influences on tail regeneration in lizards are discussed.     

Magnetic Resonance Imaging Allows the Evaluation of Tissue Damage and Regeneration in a Mouse Model of Critical Limb Ischemia

Magnetic resonance imaging (MRI) provides non-invasive, repetitive measures in the same individual, allowing the study of a physio-pathological event over time. In this study, we tested the performance of 7 Tesla multi-parametric MRI to monitor the dynamic changes of mouse skeletal muscle injury and regeneration upon acute ischemia induced by femoral artery dissection. T2-mapping (T2 relaxation time), diffusion-tensor imaging (Fractional Anisotropy) and perfusion by Dynamic Contrast-Enhanced MRI (K-trans) were measured and imaging results were correlated with histological morphometric analysis in both Gastrocnemius and Tibialis anterior muscles. We found that tissue damage positively correlated with T2-relaxation time, while myofiber regeneration and capillary density positively correlated with Fractional Anisotropy. Interestingly, K-trans positively correlated with capillary density. Accordingly, repeated MRI measurements between day 1 and day 28 after surgery in ischemic muscles showed that: 1) T2-relaxation time rapidly increased upon ischemia and then gradually declined, returning almost to basal level in the last phases of the regeneration process; 2) Fractional Anisotropy dropped upon ischemic damage induction and then recovered along with muscle regeneration and neoangiogenesis; 3) K-trans reached a minimum upon ischemia, then progressively recovered. Overall, Gastrocnemius and Tibialis anterior muscles displayed similar patterns of MRI parameters dynamic, with more marked responses and less variability in Tibialis anterior. We conclude that MRI provides quantitative information about both tissue damage after ischemia and the subsequent vascular and muscle regeneration, accounting for the differences between subjects and, within the same individual, between different muscles.     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Eugregarines reduce susceptibility of the hide beetle, Dermestes maculatus, to apicomplexan pathogens and retard larval development

Eugregarines are abundant in a great diversity of invertebrates, and yet their relationships with their hosts are subject to controversy and confusion. We tested the effect of the eugregarine, Pyxinia crystalligera, on growth, development, and susceptibility to two Apicomplexa pathogens of the hide beetle, D. maculatus. Heavy infection with eugregarines provided partial protection from two pathogenic members of Apicomplexa, M. trogodermae and A. tribolii. Infection with P. crystalligera caused lower weight in beetle larvae, but did not significantly retard pupation or adult emergence. A. tribolii infection of Lepidoptera and M. trogodermae infection of D. maculatus are reported for the first time.