Does the egg capsule protect against chronic UV-B radiation? A study based on encapsulated and decapsulated embryos of cuttlefish Sepia officinalis.

Although the egg capsule plays a crucial role in the embryonic development of cephalopods, its ability to protect embryos from Ultraviolet (UV) radiation is unknown. Our study evaluated the photoprotection mechanisms of S. officinalis to UV-B radiation and estimated the ability of the black capsule to act as a physical shield against it. Embryos with and without capsule and juveniles were exposed to four experimental UVB conditions for 55 days. The effects of different UVB doses were evaluated in terms of morphological abnormalities and differences in gene expression between each group. We observed that the development might be severely impaired in embryos exposed to UVB without capsule protection, and these effects were time- and UVB-dose-dependent. In addition, we found variations in gene expression levels (light-sensitive, stress response and DNA repair) in different tissues as a function of UVB doses. We suggest a relationship between morphological abnormalities and the limit of molecular regulation. These results suggest that the quantitative differences in expression are essential for defining the survivability of the embryo face to UVB. Thus, we demonstrated that the egg capsule could ensure successful embryonic development of the cuttlefish S. officinalis even at high doses of UVB.

Diversity of Light Sensing Molecules and Their Expression During the Embryogenesis of the Cuttlefish (Sepia officinalis).

Eyes morphologies may differ but those differences are not reflected at the molecular level. Indeed, the ability to perceive light is thought to come from the same conserved gene families: opsins and cryptochromes. Even though cuttlefish (Cephalopoda) are known for their visually guided behaviors, there is a lack of data about the different opsins and cryptochromes orthologs represented in the genome and their expressions. Here we studied the evolutionary history of opsins, cryptochromes but also visual arrestins in molluscs with an emphasis on cephalopods. We identified 6 opsins, 2 cryptochromes and 1 visual arrestin in Sepia officinalis and we showed these families undergo several duplication events in Mollusca: one duplication in the arrestin family and two in the opsin family. In cuttlefish, we studied the temporal expression of these genes in the eyes of embryos from stage 23 to hatching and their expression in two extraocular tissues, skin and central nervous system (CNS = brain + optic lobes). We showed in embryos that some of these genes (Sof_CRY6, Sof_reti-1, Sof_reti-2, Sof_r-opsin1 and Sof_v-arr) are expressed in the eyes and not in the skin or CNS. By looking at a juvenile and an adult S. officinalis, it seems that some of these genes (Sof_r-opsin1 and Sof_reti1) are used for light detection in these extraocular tissues but that they set-up later in development than in the eyes. We also showed that their expression (except for Sof_CRY6) undergoes an increase in the eyes from stage 25 to 28 thus confirming their role in the ability of the cuttlefish embryos to perceive light through the egg capsule. This study raises the question of the role of Sof_CRY6 in the developing eyes in cuttlefish embryos and the role and localization of xenopsins and r-opsin2. Consequently, the diversity of molecular actors involved in light detection both in the eyes and extraocular tissues is higher than previously known. These results open the way for studying new molecules such as those of the signal transduction cascade.

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.

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, Paxß, PoxNeuro) was confirmed and the lophotrochozoan Paxß 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.

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.

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.

ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca ­ Cephalopoda).

New molecular resources regarding the so-called "non-standard models" in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family,we point out for both families a striking molecular diversity in S. officinalis.

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.

Muscle satellite cells and endothelial cells: close neighbors and privileged partners.

Genetically engineered mice (Myf5nLacZ/+, Myf5GFP-P/+) allowing direct muscle satellite cell (SC) visualization indicate that, in addition to being located beneath myofiber basal laminae, SCs are strikingly close to capillaries. After GFP(+) bone marrow transplantation, blood-borne cells occupying SC niches previously depleted by irradiation were similarly detected near vessels, thereby corroborating the anatomical stability of juxtavascular SC niches. Bromodeoxyuridine pulse-chase experiments also localize quiescent and less quiescent SCs near vessels. SCs, and to a lesser extent myonuclei, were nonrandomly associated with capillaries in humans. Significantly, they were correlated with capillarization of myofibers, regardless to their type, in normal muscle. They also varied in paradigmatic physiological and pathological situations associated with variations of capillary density, including amyopathic dermatomyositis, a unique condition in which muscle capillary loss occurs without myofiber damage, and in athletes in whom capillaries increase in number. Endothelial cell (EC) cultures specifically enhanced SC growth, through IGF-1, HGF, bFGF, PDGF-BB, and VEGF, and, accordingly, cycling SCs remained mainly juxtavascular. Conversely, differentiating myogenic cells were both proangiogenic in vitro and spatiotemporally associated with neoangiogenesis in muscular dystrophy. Thus, SCs are largely juxtavascular and reciprocally interact with ECs during differentiation to support angio-myogenesis.

Proteasomes are tightly associated to myofibrils in mature skeletal muscle.

Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of alpha1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils.

Angiogenin distribution in human term placenta, and expression by cultured trophoblastic cells.

Human angiogenin is a 14-kDa secreted protein with angiogenic and ribonucleolytic activities. Angiogenin is associated with tumour development but is also present in normal biological fluids and tissues. To further address the physiological role of angiogenin, we studied its expression in situ and in vitro, using the human term placenta as a model of physiological angiogenesis. Angiogenin was immunodetected by light and transmission electron microscopy, and its cellular distribution was established by double immunolabelling with cell markers including von Willebrand factor, platelet/endothelial cell adhesion molecule-1 (PECAM-1), CD34, Tie-2, vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Angiogenin immunoreactivity was detected in villous and extravillous trophoblasts, the trophoblast basement membrane, the endothelial basal lamina, foetal blood vessels, foetal and maternal red blood cells, and amnionic cells. Its expression was confirmed by in situ hybridisation with a digoxygenin-labelled cDNA probe and reverse transcriptase-polymerase chain reaction amplification. Villous cytotrophoblasts, isolated and differentiated in vitro into a functional syncytiotrophoblast, expressed and secreted angiogenin. Given its known biological activities in vitro and its observed pattern of expression, these data suggest that, in human placenta, angiogenin has a role not only in angiogenesis but also in vascular and tissue homeostasis, maternal immune tolerance of the foetus, and host defences.