Yann Bassaglia

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Abstract Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3–15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.

Fast and slow rat muscles degenerate and regenerate differently after whole crush injury.

SummaryThe whole-crush injured rat skeletal muscle was used as a model to explore the regenerating potentialities of fast and slow muscles. Laminin was chosen to follow changes in basal lamina and desmin to visualize new muscular elements; they were revealed by immunofluorescence on cryostat sections of either fast (extensor digitorum longus) or slow (soleus) regenerating muscle. Soleus myolysis was rapid, extensive and heterogeneous. Basal laminae were nearly destroyed. In contrast, extensor digitorum longus maintained its basal lamina framework during myolysis. Soleus reconstruction began early, following the pattern of remaining basal laminae as closely as possible, but regeneration stagnated from day 16 and the regenerated muscle was fibrotic. In extensor digitorum longus, reconstruction progressed slower than in soleus, but regularly from the periphery toward the centre of the muscle. The regenerated extensor digitorum longus showed a quasi-normal structure from day 16. At the end of the process, the elimination of old basal lamina was completed in extensor digitorum longus, but was not achieved in soleus. We propose that the old basal lamina should help the initiation of reconstruction. This new model also underlines the importance of the turnover of basal laminae in muscular regeneration, and will be useful to understand the background of the different regenerative response of both muscles.

Somatic muscle development in Sepia officinalis (cephalopoda - mollusca): a new role for NK4.

Cephalopods are emerging as new developmental models. These lophotrochozoans exhibit numerous morphological peculiarities among molluscs, not only regarding their nervous system but also regarding their circulatory system, which is closed and includes three hearts. However, the molecular control of cardiac myogenesis in lophotrochozoans is largely unknown. In other groups, cardiac development depends on numerous different genes, among them NK4 seems to have a well‐conserved function throughout evolution. In this study, we assessed the expression pattern of SoNK4, the Sepia officinalis NK4 homologue, during Sepia officinalis development by whole‐mount in situ hybridization. SoNK4 expression begins before morphogenesis, is not restricted to prospective cardiac muscles but above all concerns mesodermal structures potentially rich in muscles such as arms and mantle. These results suggest an important role of SoNK4 in locomotory (somatic) muscles development of Sepia officinalis, and thus a new role for NK4. Developmental Dynamics 237:1944–1951, 2008.

Reflectin genes and development of iridophore patterns in Sepia officinalis embryos (Mollusca, Cephalopoda)

Background: In the cuttlefish Sepia officinalis, iridescence is known to play a role in patterning and communication. In iridophores, iridosomes are composed of reflectins, a protein family, which show great diversity in all cephalopod species. Iridosomes are established before hatching, but very little is known about how these cells are established, their distribution in embryos, or the contribution of each reflectin gene to iridosome structures. Results: Six reflectin genes are expressed during the development of iridosomes in Sepia officinalis. We show that they are expressed in numerous parts of the body before hatching. Evidence of the colocalization of two different genes of reflectin was found. Curiously, reflectin mRNA expression was no longer detectable at the time of hatchling, while reflectin proteins were present and gave rise to visible iridescence. Conclusion: These data suggest that several different forms of reflectins are simultaneously used to produce iridescence in S. officinalis and that mRNA production and translation are decoupled in time during iridosome development. Developmental Dynamics 242:550–561, 2013.

The Pax gene family: Highlights from cephalopods

Pax genes play important roles in Metazoan development. Their evolution has been extensively studied but Lophotrochozoa are usually omitted. We addressed the question of Pax paralog diversity in Lophotrochozoa by a thorough review of available databases. The existence of six Pax families (Pax1/9, Pax2/5/8, Pax3/7, Pax4/6, Paxβ, PoxNeuro) was confirmed and the lophotrochozoan Paxβ subfamily was further characterized. Contrary to the pattern reported in chordates, the Pax2/5/8 family is devoid of homeodomain in Lophotrochozoa. Expression patterns of the three main pax classes (pax2/5/8, pax3/7, pax4/6) during Sepia officinalis development showed that Pax roles taken as ancestral and common in metazoans are modified in S. officinalis, most likely due to either the morphological specificities of cephalopods or to their direct development. Some expected expression patterns were missing (e.g. pax6 in the developing retina), and some expressions in unexpected tissues have been found (e.g. pax2/5/8 in dermal tissue and in gills). This study underlines the diversity and functional plasticity of Pax genes and illustrates the difficulty of using probable gene homology as strict indicator of homology between biological structures.

Relationship Between Plasminogen Activators and Regeneration Capacities of Rat Skeletal Muscles

Mammalian skeletal muscle regeneration may be divided in two major parts. The first is a degenerative period leading to muscle atrophy as a result of the catabolic conditions due to tissue breakdown, cell death, and subsequent presence of large amounts of lysosomal and non lysosomal proteinases.1,2 The second step is characterized by tissue remodelling resulting in a relatively complete regeneration.3–8 This step was described by Bischoff 9 as dependent upon the capacity of neutral proteinases to digest the basement membrane. It involves changes in the amount and distribution of various components of the basement membrane10 such as fibronectin2, laminin and type IV collagen.12,13 The participation of the plasminogen activator (PA) system in these modifications has been described.14 In addition, a large amount of information links tissue remodelling to the activity of serine proteinases such as PAs.15

NEUROGENESIS IN CEPHALOPODS: "ECO-EVO-DEVO" APPROACH IN THE CUTTLEFISH SEPIA OFFICINALIS (MOLLUSCA-CEPHALOPODA)

Cephalopods are new evolutionary and ecological models. By their phylogenetic position (Lophotrochozoa, Mollusca), they provide a missing master piece in the whole puzzle of neurodevelopment studies. Their derived and specific nervous system but also their convergence with vertebrates offer abundant materials to question the evolution and development of the nervous system of Metazoa (evo-devo studies). In addition, their various adaptions to different modes of life open new fields of investigation of developmental plasticity according to ecological context (eco-evo-devo approach). In this paper, we review the recent works on cephalopod nervous developmental investigations. We show how cephalopods, and especially Sepia officinalis, an animal of economical interest, can be used as suitable models to extend our knowledge on cephalopod ecology and on nervous system evolution among molluscs.

Eye Development in Sepia officinalis Embryo: What the Uncommon Gene Expression Profiles Tell Us about Eye Evolution

In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including pax, six, eya, and dac playing also key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.

Could FaRP-Like Peptides Participate in Regulation of Hyperosmotic Stress Responses in Plants?

The ability to respond to hyperosmotic stress is one of the numerous conserved cellular processes that most of the organisms have to face during their life. In metazoans, some peptides belonging to the FMRFamide-like peptide (FLP) family were shown to participate in osmoregulation via regulation of ion channels; this is, a well-known response to hyperosmotic stress in plants. Thus, we explored whether FLPs exist and regulate osmotic stress in plants. First, we demonstrated the response of Arabidopsis thaliana cultured cells to a metazoan FLP (FLRF). We found that A. thaliana express genes that display typical FLP repeated sequences, which end in RF and are surrounded by K or R, which is typical of cleavage sites and suggests bioactivity; however, the terminal G, allowing an amidation process in metazoan, seems to be replaced by W. Using synthetic peptides, we showed that amidation appears unnecessary to bioactivity in A. thaliana, and we provide evidence that these putative FLPs could be involved in physiological processes related to hyperosmotic stress responses in plants, urging further studies on this topic.