Fundamental aspects of arm repair phase in two echinoderm models

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored.Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools.Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models.Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.     

De Novo Transcriptome Analysis of Two Seahorse Species (Hippocampus erectus and H. mohnikei) and the Development of Molecular Markers for Population Genetics

Seahorse conservation has been performed utilizing various strategies for many decades, and the deeper understanding of genomic information is necessary to more efficiently protect the germplasm resources of seahorse species. However, little genetic information about seahorses currently exists in the public databases. In this study, high-throughput RNA sequencing for two seahorse species, Hippocampus erectus and H. mohnikei, was carried out, and de novo assembly generated 37,506 unigenes for H. erectus and 36,113 unigenes for H. mohnikei. Among them, 17,338 (46.23%) unigenes for H. erectus and 17,900 (49.57%) for H. mohnikei were successfully annotated based on the information available from the public databases. Through comparing the unigenes of two seahorse species, 7,802 candidate orthologous genes were identified and 5,268 genes among them could be annotated. In addition, gene ontology analysis of two species was similarly performed on biological processes, cellular components, and molecular functions. Twenty-four and twenty-one unigenes in H. erectus and H. mohnikei were annotated in the biosynthesis of unsaturated fatty acids pathways, and both seahorses lacked the Δ12 and Δ15 desaturases. Total of 8,992 and 9,116 SSR loci were obtained from H. erectus and H. mohnikei unigenes, respectively. Dozens of SSR were developed and then applied to assess the population genetic diversity, as well as cross-amplified in a related species, H. trimaculatus. The H_O and H_E values of the tested populations for H. erectus, H. mohnikei, and H. trimaculatus were medium. These resources would facilitate the conservation of the species through a better understanding of the genomics and comparative genome analysis within the Hippocampus genus.     

Homeobox Genes Expressed During Echinoderm Arm Regeneration

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.     

MicroRNA-Like Small RNAs Prediction in the Development of Antrodia cinnamomea

Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.     

Color Vision Variation as Evidenced by Hybrid L/M Opsin Genes in Wild Populations of Trichromatic Alouatta New World Monkeys

Platyrrhine (New World) monkeys possess highly polymorphic color vision owing to allelic variation of the single-locus L/M opsin gene on the X chromosome. Most species consist of female trichromats and female and male dichromats. Howlers (genus Alouatta) are an exception; they are considered to be routinely trichromatic with L and M opsin genes juxtaposed on the X chromosome, as seen in catarrhine primates (Old World monkeys, apes, and humans). Yet it is not known whether trichromacy is invariable in howlers. We examined L/M opsin variation in wild howler populations in Costa Rica and Nicaragua (Alouatta palliata) and Belize (A. pigra), using fecal DNA. We surveyed exon 5 sequences (containing the diagnostic 277th and 285th residues for λ_max) for 8 and 18 X chromosomes from Alouatta palliata and A. pigra, respectively. The wavelengths of maximal absorption (λ_max) of the reconstituted L and M opsin photopigments were 564 nm and 532 nm, respectively, in both species. We found one M–L hybrid sequence with a recombinant 277/285 haplotype in Alouatta palliata and two L–M hybrid sequences in A. pigra. The λ_max values of the reconstituted hybrid photopigments were in the range of 546~554 nm, which should result in trichromat phenotypes comparable to those found in other New World monkey species. Our finding of color vision variation due to high frequencies of L/M hybrid opsin genes in howlers challenges the current view that howlers are routine and uniform trichromats. These results deepen our understanding of the evolutionary significance of color vision polymorphisms and routine trichromacy and emphasize the need for further assessment of opsin gene variation as well as behavioral differences among subtypes of trichromacy.     

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.     

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells^1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca²⁺ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca²⁺ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans^5-7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca²⁺-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca²⁺ changes and response to light^8-12 as well as the role of inner segment Ca²⁺ stores in Ca²⁺ homeostasis^13,14. Fluorescent dyes can also be used for measuring Mg²⁺ concentration¹⁵, pH, and as tracers of aqueous and membrane compartments¹⁶. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells^17-19.Download video file.^{(70M, mov)}