M. D. Candia Carnevali

Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study

The myosin composition of lateral muscle in Poecilia reticulata from birth to adult was studied by ATPase histochemistry and immunostaining with myosin isoform-specific antibodies. At birth the muscle consists of two layers containing developmental isoforms of myosin. In deep layer fibres the developmental myosin is replaced by the adult fast-white isoform soon after birth. In the epaxial and hypaxial monolayer fibres the myosin composition present at birth (J1) is replaced within 3 days by another (J2). In some fibres, this J2 composition is retained in the adult, but in others it is slowly replaced by the adult slow-red muscle isoform. Close to the lateral line, all monolayer fibres are already in transition between the J2 myosin and the adult slow-red form at birth, and rapidly complete the transition to slow-red form. These fibres, together with others generated de novo in an underlying hyperplastic zone, form the red muscle layer of the adult. The pink muscle develops during the first month after birth, and by 31 days it consists of an outer, middle and inner layer. A few middle layer fibres are already present at birth, while the outer layer fibres first appear 3 days after birth. The thin inner layer is probably a transitional form between the middle pink and adult white types, and appears at about 31 days. A morphometric analysis showed that growth of the white muscle occurs principally by hypertrophy. Even at the magnification level of the electron microscope, no satellite cells or myoblasts which could give rise to new fibres were found in the white muscle, except in the far epaxial and hypaxial regions and only in the first 10 days. A zone of hyperplastic growth was also found lying just under the superficial monolayer close to the lateral line, and this presumably contributes fibres to the red and pink muscle layers.

Pattern of bromodeoxyuridine incorporation in the advanced stages of arm regeneration in the feather star Antedon mediterranea

Abstract.The overall process of arm regeneration in the crinoid Antedon mediterranea is a typical epimorphic process (blastemal regeneration). This can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase. The crucial problem of the identification of cell lineages responsible for both repair and regenerative processes has been approached by monitoring cell proliferation during the advanced regenerative phase using light-microscopic and ultrastructural immunocytochemical methods to detect the incorporation of the thymidine analogue bromodeoxyuridine (BrdU) into regenerating tissues. Various treatment protocols and BrdU incubation times have been employed and provided information not only on the sources, sites of proliferation, and recruitment times of the new cells, but also on the cell lineage involved and subsequent fate (differentiation and/or migration) of the labelled cells. Our results are consistent with the following conclusions: (1) The arm regeneration process is due to a massive intervention of active proliferating cells identifiable as migratory, morphologically undifferentiated cells (amoebocytes and coelomocytes). (2) The preferential proliferation sites of these cells are the terminal blastema, the coelomic epithelium, and the brachial nerve of both the regenerating arm and the stump, even far from the amputation. (3) The two main cell components contributing to the regenerate have different origins: the blastemal cells and all the cell lineages derived from the amoebocytes; the coelomic cells from the migratory coelomocytes, in their turn derived from proliferation of the coelomic epithelium. (4) The blastemal regeneration of Antedon is due to a combined recruitment of pluripotent elements, implying the intervention of presumptive stem cells (amoebocytes) and the transdifferentiation/dedifferentiation of differentiated elements of the coelomic epithelium.

MUSCLE GROWTH IN RESPONSE TO CHANGING DEMANDS OF FUNCTIONS IN THE TELEOST SPARUS AURATA (L.) DURING DEVELOPMENT FROM HATCHING TO JUVENILE

Abstract Growth of laterarl muscle in the teleost fish Sparus aurata (L.) was examined from hatching to juvenile by a basic morphofunctional approach that takes into account structural and ecophysiological aspects and combines in vivo observations and LM and TEM microscopic analysis. As shown in most teleost fishes, muscle growth proceeds by a double mechanism of hyperplasia and hypertrophy that contribute differentially to the overall development of the lateral muscle, giving rise in each myomere to a typical pattern of structurally and functionally different fibre types (slow-red and fast-white fibres, plus pink intermediate fibres) in a nerve-dependent process. During larval life the muscle growth takes place mainly due to hyperplastic growth at the level of specific proliferative zones of the myomeres, from which slow, pink and white muscle fibres are derived. In those species that reach a large adult size a new typical hyperplastic process disseminated throughout the fast white muscle layer takes place during post-larval life. In contrast, hypertrophic growth occurs in all stages, but is the dominant mechanism of muscle growth only in juvenile and adult. The suitable recruitment of the different fibre types enables the fish to optimize its performances according to specific functional and metabolic requirements related to the swimming behaviour and hydrodynamic regimes. The different mechanisms of growth are here analysed in their detailed structural and ultrastructural aspects in order to interpret their adaptive significance in the light of the fish life cycle, with particular reference to locomotion and feeding behaviour.

The compass depressors ofParacentrotus lividus (Echinodermata, Echinoida): ultrastructural and mechanical aspects of their variable tensility and contractility

SummaryThe compass depressors are bands of soft tissue which connect the compass ossicles of the echinoid lantern to the inner edge of the test. They are essentially ligaments with on one side a thin layer of muscle cells. The ligamentous component consists mainly of a parallel array of collagen fibrils with interspersed 12 nm microfibrils. The most notable cellular constituents are granule-containing cell bodies and their processes which resemble the juxtaligamental cells that have been found in all echinoderm mutable collagenous tissues and which may control the tensility of these tissues. The muscle cells occupy about 8% of the total cross-sectional area of the compass depressor and are located in a richly innervated pseudostratified myoepithelium. When subjected to constant low loads in creep tests the compass depressor stretches to a fixed length beyond which there is no further extension. The length at this creep limit coincides with the maximum length to which the compass depressor is stretched by natural movements of the intact lantern. Stress-strain tests show that treatment with 1 mM acetylcholine or 100 mM K+ ions can increase reversibly the stiffness of the compass depressor to an extent that cannot be due to contraction of the myoepithelium, suggesting that the mechanical properties of the ligament are under physiological control. Tension-length data on the myoepithelium suggest that it generates a maximum active tension when the compass depressor is stretched to the creep limit. The implications of these results for the function of the compass depressors are discussed.

Localization of calcitonin gene-related peptide mRNA in developing olfactory axons

Abstract During development of the olfactory pathway, calcitonin gene-related peptide (CGRP) expression is regulated both temporally and spatially. We had previous evidence that between E13 and E19 CGRP mRNA was present at the level of olfactory axons but the resolution of light-microscope in situ hybridization did not permit the axons to be distinguished from the closely apposed ensheathing cells. In this study, the localization of CGRP mRNA was studied at early developmental stages (E13–15) through in situ hybridization at the transmission electron-microscope (TEM) level. CGRP transcripts were observed exclusively in axons and not in ensheathing cells. The distribution of transcripts in the axons suggests that they are associated with intermediate filaments rather than microtubules. In addition, a careful ultrastructural analysis provided evidence that polysomes and membrane-bound ribosomes are present in such axons, suggesting that the peptide could be synthesized locally.

The mechanically adaptive connective tissue of echinoderms: Its potential for bio-innovation in applied technology and ecology

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

Regenerative Response and Endocrine Disrupters in Crinoid Echinoderms: An Old Experimental Model, a New Ecotoxicological Test

The regenerative phenomena that reproduce developmental processes in adult organisms and are regulated by endocrine and neurohumoral mechanisms can provide new sensitive tests for monitoring the effects of exposure to anthropogenic chemicals such as endocrine disrupter (ED) contaminants. These pollutants in fact can be bioaccumulated by the organisms, causing dysfunctions in steroid hormone production/metabolism and activities and inducing dramatic effects on reproductive competence, development and growth in many animals, man included. Current research is exploring the effects of exposure to different classes of compounds well known for their ED activity, such as polychlorinated biphenyls (PCBs), nonylphenols and organotins, on regenerative potential of echinoderms, a relatively unexplored and promising applied approach which offers the unique chance to study physiological developmental processes in adult animals. The selected test species is the crinoid Antedon mediterranea, which represents a valuable experimental model for investigation into the regenerative process from the macroscopic to the molecular level. The present study employs an integrated approach which combines exposure experiments, chemical analysis and biological analysis utilizing classical methods of light (LM) and electron (TEM and SEM) microscopy and immunocytochemistry. The experiments were carried out on experimentally induced arm regenerations in controlled conditions with exposure concentrations comparable to those of moderately polluted coastal zones in order to reproduce common conditions of exposure to environmental contaminants. The results of the exposure tests were analysed in terms of effects at the whole organism, at the tissue and cellular level, and possible sites of action of EDs. Our results show that prolonged exposure to these compounds significantly affects the regenerative mechanisms by inducing appreciable anomalies in terms of regeneration times, overall growth, general morphology and histological and cellular pattern. A concentration/effect relationship could be found for all the substances. Interestingly, contrasting results in terms of inhibition or acceleration of regeneration phenomenon were obtained for the different chemicals.

The Aristotle's lantern of the sea-urchin Stylocidaris affinis (Echinoida, Cidaridae): functional morphology of the musculo-skeletal system

SummaryThe Aristotles lantern, or masticatory apparatus, of regular sea-urchins is a complex musculo-skeletal system which is thought to have contributed significantly to the evolutionary success of these animals. This paper gives an account of the antomical relationships and functional morphology of both skeletal and soft tissue components in the lantern and related structures of the sea-urchin Stylocidaris affinis (Cidaridae), and compares these features with their equivalent in the previously described lantern of the sea-urchin Paracentrotus lividus (Echinidae, Camarodonta). There are major differences in the skeletons of these lanterns which involve mostly the arrangement and morphology of elements participating in movement, i.e. joints and articular surfaces, and which highlight the generally heavier and less mobile nature of the lantern in the Cidaridae. There are remarkably few differences, however, in the microstructure of the skeletal stereom. Significant dissimilarities were found in the anatomical arrangement of muscles and ligamentous structures and in their macro- and microstructure. The implications of these morphological features for the functioning of the lantern of the Cidaridae are discussed in the context of an integrated model of lantern biomechanics.

Changes in ubiquitin conjugates and Hsp72 levels during arm regeneration in echinoderms.

Abstract: All organisms show a common defensive mechanism that results in the expression of conserved heat shock proteins (Hsps). These proteins function in a wide range of stressful conditions. We have monitored their levels in species of regenerating echinoderms with different mechanisms of regeneration and from different geographical locations. The effect of an artificial higher temperature on expression of Hsps was also studied. Two stress proteins (Hsp72 and ubiquitin) that are important in processes such as development and protein degradation were investigated. Using Western blot analysis and immunocytochemistry, we found significant changes in the level (Hsp72) and pattern of conjugation (ubiquitin) that corresponded with the repair phase (early regenerative stages) and with the later growth and regeneration of new tissues. Animals from the intertidal environment showed a distinctly sustained expression pattern of Hsp72 compared with benthic animals which suggests a functionally adaptative and dynamic stress response program.

Correlations between the biochemistry and mechanical states of a sea-urchin ligament: a mutable collagenous structure.

Mutable collagenous tissues (MCTs) of echinoderms can be regarded as intelligent and dynamic biomaterials, due to their ability to reversibly change their mechanical properties in a short physiological time span. This mutability phenomenon is nervously mediated and involves secreted factors of the specialized ‘juxtaligamental’ cells, which, when released into the extracellular matrix (ECM), change the cohesive forces between collagen fibrils. MCTs exist in nature in several forms, including some associated with echinoderm autotomy mechanisms. Since the molecular mechanism of mutability is still incompletely understood, the aim of this work was to provide a detailed biochemical analysis of a typical mutable collagenous structure and to identify possible correlations between its biochemistry and mechanical states. A better understanding of the mutability phenomena is likely to provide a unique opportunity to develop new concepts that can be applied in the design of dynamic biomaterial for tissue regeneration, leading to new strategies in regenerative medicine. The MCT model used was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was analyzed in different mechanical states, mimicking the mutability phenomenon. Spectroscopic techniques, namely Fourier transform infrared (FT-IR) and confocal Raman microscopy, were used to identify the specific molecular components that contribute to the CDL biochemical microenvironment and to investigate the possibility that remodelling/synthesis of new ECM components occurs during the mutability phenomenon by analogy with events during pregnancy in the uterine cervix of mammals (which also consists mainly of mechanically adaptable connective tissues). The results demonstrate that CDL ECM includes collagen with biochemical similarities to mammalian type I collagen, as well as sulphated glycosaminoglycans (GAGs). CDL mutability seems to involve a molecular rearrangement of the ECM, without synthesis of new ECM components. Although there were no significant biochemical differences between CDLs in the various mechanical states were observed. However, subtle adjustments in tissue hydration seemed to occur, particularly during stiffening.