Modularity of gene-regulatory networks revealed in sea-star development

Evidence that conserved developmental gene-regulatory networks can change as a unit during deutersostome evolution emerges from a study published in BMC Biology. This shows that genes consistently expressed in anterior brain patterning in hemichordates and chordates are expressed in a similar spatial pattern in another deuterostome, an asteroid echinoderm (sea star), but in a completely different developmental context (the animal-vegetal axis). This observation has implications for hypotheses on the type of development present in the deuterostome common ancestor.     

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.     

Myogenesis during holothurian intestinal regeneration

Echinoderms are well known as being able to regenerate body parts and thus provide excellent models for studying regenerative processes in adult organisms. We are interested in intestinal regeneration in the sea cucumber, Holothuria glaberrima, and focus here on the regeneration of intestinal muscle components. We have used immunohistochemical techniques to describe the formation of the intestinal muscle layers. Myoblasts are first observed within the regenerating structure, adjacent to the coelomic epithelia. Within a few days, these cells acquire muscle markers and form a single cell layer that underlies the epithelia. Animals injected with BrdU at various regeneration stages have been subsequently analyzed for the presence of muscle differentiation markers. BrdU-labeled muscle nuclei are observed in myocytes of 3-week regenerates, showing that these cells originate from proliferating precursors. The peak in muscle precursor proliferation appears to occur during the second week of regeneration. Therefore, new muscle cells in the regenerating intestine originate from precursors that have undergone cell division. Our results suggest that the precursor cells arise from the coelomic epithelia. We also provide a comparative view of muscle regeneration in an echinoderm, a topic of interest in view of the many recent studies of muscle regeneration in vertebrate species. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from NIH-RCMI (RRO-3641-01) and the University of Puerto Rico     

Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.     

The Retinal Homeobox (Rx) gene is necessary for retinal regeneration

The Retinal Homeobox (Rx) gene is essential for vertebrate eye development. Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in a variety of species. Here we show that Rx function is also necessary during retinal regeneration. We performed a thorough characterization of retinal regeneration after partial retinal resection in pre-metamorphic Xenopus laevis. We show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx and other RPC marker genes. We used an shRNA-based approach to specifically silence Rx expression in vivo in tadpoles. We found that loss of Rx function results in impaired retinal regeneration, including defects in the cells that repopulate the wound and the RPE at the wound site. We show that the regeneration defects can be rescued by provision of exogenous Rx. These results demonstrate for the first time that Rx, in addition to being essential during retinal development, also functions during retinal regeneration.     

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.     

Amputation induces stem cell mobilization to sites of injury during planarian regeneration

How adult stem cell populations are recruited for tissue renewal and repair is a fundamental question of biology. Mobilization of stem cells out of their niches followed by correct migration and differentiation at a site of tissue turnover or injury are important requirements for proper tissue maintenance and regeneration. However, we understand little about the mechanisms that control this process, possibly because the best studied vertebrate adult stem cell systems are not readily amenable to in vivo observation. Furthermore, few clear examples of the recruitment of fully potent stem cells, compared with limited progenitors, are known. Here, we show that planarian stem cells directionally migrate to amputation sites during regeneration. We also show that during tissue homeostasis they are stationary. Our study not only uncovers the existence of specific recruitment mechanisms elicited by amputation, but also sets the stage for the systematic characterization of evolutionarily conserved stem cell regulatory processes likely to inform stem cell function and dysfunction in higher organisms, including humans.     

Serum amyloid A protein in an echinoderm: its primary structure and expression during intestinal regeneration in the sea cucumber Holothuria glaberrima

Serum amyloid A (SAA) proteins comprise a family of highly conserved apolipoproteins found in all mammals thus far investigated, and also in ducks and salmonid fishes. However, no invertebrate SAA homologues have been detected to date. Here we report the characterization of the first SAA homologue in a nonvertebrate deuterostome, the echinoderm Holothuria glaberrima. A 971-base-pair cDNA was obtained from a regenerating intestine cDNA library. The clone contains a 369-nucleotide open reading frame corresponding to a 122-amino-acid protein exhibiting a high degree of homology to members of the SAA superfamily. Sequence alignments of the holothuroid and vertebrate SAA proteins make evident a remarkable degree of conservation, even between phylogenetically disparate groups. Northern blots and immunohistochemistry show that SAA expression increases during regeneration of the holothuroid digestive tract as compared with normal nonregenerating tissue, and that the SAA protein is expressed by cells of the coelomic epithelium of the regenerating intestine. While SAA expression during the initial wound healing stage of regeneration is minimal, it increases during subsequent stages, peaking at day 15 of regeneration, concomitantly with lumen formation and the organization of the muscular layers of the regenerating digestive tract. Although in vertebrates SAA proteins may be part of a well-conserved anti-inflammatory mechanism, their exact biological function remains obscure. Our results suggest the possibility that SAA proteins, although structurally conserved, may possess enough functional diversity to participate in processes other than anti-inflammatory responses.     

Temporal and spatial distribution patterns of echinoderm larvae in La Parguera, Puerto Rico

This study describes temporal and spatial abundance patterns of echinoderm larvae in La Parguera, Puerto Rico. For the temporal study, larvae were sampled by a series of monthly tows taken with a 64 microm mesh net between the new and full moon from April 2005 to July 2006, September 2006 and August 2007. In order to measure spatial variation of echinoderm larval abundances, oblique tows were taken with 64 and 202 microm mesh nets at seven different sites within the shelf, at the shelf-edge, and at a nearby oceanic stations during August 2007. Overall, Echinoidea (sea urchin) exhibited the highest abundance with a total of 11 921 larvae, representing 52.5% of the total collection. Ophiuroidea (brittle star) ranked second in abundance with 45.6% of the total larvae. Holothuroidea (sea cucumber) and Asteroidea larvae (sea star) accounted for less than 2% of the total echinoderm larval collection. Early larval stages (2-8 day old) of Diadema antillarum represented 20% of the total Echinoidea larvae. There was no marked seasonal trend of echinoderm larval abundance; Echinoidea and Ophiuroidea larvae were present in all monthly samples indicating that reproduction occurs year-round. Peak abundances of later-stage Echinoidea larvae were observed during January, July and October and of later-stage Ophiuroidea larvae during June, August and October. The observed peaks of later-stage larval abundances may be indicative of higher recruitment activity during these months. There was a significant difference of echinoderm larval abundance between spatial stations, with higher abundances collected at the shelf-edge. Later-stage (approximately 24 day old) D. antillarum larvae were mostly collected at shelf-edge and oceanic locations. In addition, the 64 microm mesh net was more efficient for collection of echinoderm larvae than the 202 microm mesh net.     

Phylogenetic analysis of the Wnt gene family and discovery of an arthropod wnt-10 orthologue

Wnt genes encode a conserved family of secreted signaling proteins that play many roles in arthropod and vertebrate development. We have investigated both the phylogenetic history and molecular evolution of this gene family. We have identified a novel Wnt gene in a diversity of arthropods that it is likely an orthologue of the vertebrate Wnt-10 group. Wnt-10 is one of only two cases in which orthology between protostome and deuterostome genes could be consistently assigned based on our analyses. Despite difficulties in assessing orthologies, all of our trees suggest that the most recent common ancestor of protostomes and deuterostomes possessed more than the five Wnt genes known from either arthropods or nematodes. This suggests that Wnt gene loss has occurred during protostome evolution. In addition, we examined the rate of amino acid evolution in the two arthropod/deuterostome orthology groups we identified. We found little rate variation across taxa, with the exception that Drosophila Wnt-1 is evolving more rapidly than all vertebrate and most arthropod orthologues.     

Bioelectricity and epimorphic regeneration

All cells have electric potentials across their membranes, but is there really compelling evidence to think that such potentials are used as instructional cues in developmental biology? Numerous reports indicate that, in fact, steady, weak bioelectric fields are observed throughout biology and function during diverse biological processes, including development. Bioelectric fields, generated upon amputation, are also likely to play a key role during vertebrate regeneration by providing the instructive cues needed to direct migrating cells to form a wound epithelium, a structure unique to regenerating animals. However, mechanistic insight is still sorely lacking in the field. What are the genes required for bioelectric‐dependent cell migration during regeneration? The power of genetics combined with the use of zebrafish offers the best opportunity for unbiased identification of the molecular players in bioelectricity. BioEssays 29:1133–1137, 2007. © 2007 Wiley Periodicals, Inc.     

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.     

Metaphylogeny of 82 gene families sheds a new light on chordate evolution

Achieving a better comprehension of the evolution of species has always been an important matter for evolutionary biologists. The deuterostome phylogeny has been described for many years, and three phyla are distinguishable: Echinodermata (including sea stars, sea urchins, etc...), Hemichordata (including acorn worms and pterobranchs), and Chordata (including urochordates, cephalochordates and extant vertebrates). Inside the Chordata phylum, the position of vertebrate species is quite unanimously accepted. Nonetheless, the position of urochordates in regard with vertebrates is still the subject of debate, and has even been suggested by some authors to be a separate phylum from cephalochordates and vertebrates. It was also the case for agnathans species -myxines and hagfish- for which phylogenetic evidence was recently given for a controversial monophyly. This raises the following question: which one of the cephalochordata or urochordata is the sister group of vertebrates and what are their relationships? In the present work, we analyzed 82 protein families presenting homologs between urochordata and other deuterostomes and focused on two points: 1) testing accurately the position of urochordata and cephalochordata phyla in regard with vertebrates as well as chordates monophyly, 2) performing an estimation of the rate of gene loss in the Ciona intestinalis genome. We showed that the urochordate phyla is the vertebrate sister group and that gene loss played a major role in structuring the urochordate genome.     

Using zebrafish as the model organism to understand organ regeneration

The limited regenerative capacity of several organs, such as central nervous system(CNS), heart and limb in mammals makes related major diseases quite difficult to recover. Therefore, dissection of the cellular and molecular mechanisms underlying organ regeneration is of great scientific and clinical interests. Tremendous progression has already been made after extensive investigations using several model organisms for decades. Unfortunately, distance to the final achievement of the goal still remains. Recently, zebrafish became a popular model organism for the deep understanding of regeneration based on its powerful regenerative capacity, in particular the organs that are limitedly regenerated in mammals. Additionally, zebrafish are endowed with other advantages good for the study of organ regeneration. This review summarizes the recent progress in the study of zebrafish organ regeneration, in particular regeneration of fin, heart, CNS, and liver as the representatives. We also discuss reasons of the reduced regenerative capacity in higher vertebrate, the roles of inflammation during regeneration, and the difference between organogenesis and regeneration.     

Unusual gene order and organization of the sea urchin hox cluster

While the highly consistent gene order and axial colinear patterns of expression seem to be a feature of vertebrate hox gene clusters, this pattern may be less well conserved across the rest of the bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is 5'-Hox1, 2, 3, 11/13c, 11/13b, 11/13a, 9/10, 8, 7, 6, 5-3'.) The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.     

Kohamaic acid A,a novel sesterterpenic acid,inhibits activities of DNA polymerases from deuterostomes

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.