Evolution of regeneration and fission in annelids: insights from engrailed- and orthodenticle-class gene expression

The recent explosion of information on the role of regulatory genes in embryogenesis provides an excellent opportunity to study how these genes participate in post-embryonic developmental processes. We present a detailed comparison of regulatory gene expression during regeneration and asexual reproduction (by fission) in the segmented worm Pristina leidyi (Annelida: Oligochaeta). We isolated three genes from Pristina, one homolog of engrailed and two homologs of orthodenticle, and characterized their expression in different developmental contexts. In situ hybridization studies on worms undergoing normal growth, regeneration and fission demonstrate that in all three processes, Pl-en is expressed primarily in the developing nervous system, and Pl-Otx1 and Pl-Otx2 are expressed primarily in the anterior body wall, foregut and developing nervous system. Our data reveal extensive similarities between expression during regeneration and fission, consistent with the idea that similar developmental processes underlie these two types of development. Thus, we argue that in these annelids fission may have evolved by recruitment of regenerative processes. Furthermore, by comparing our data to existing data from leech embryos, we find evidence that embryonic processes are re-deployed during regeneration and fission.     

Conserved and novel gene expression between regeneration and asexual fission in <Emphasis Type="Italic">Nematostella vectensis</Emphasis>

Due to work in model systems (e.g., flies and mice), the molecular mechanisms of embryogenesis are known in exquisite detail. However, these organisms are incapable of asexual reproduction and possess limited regenerative abilities. Thus, the mechanisms of alternate developmental trajectories and their relation to embryonic mechanisms remain understudied. Because these developmental trajectories are present in a diverse group of animal phyla spanning the metazoan phylogeny, including cnidarians, annelids, and echinoderms, they are likely to have played a major role in animal evolution. The starlet sea anemone Nematostella vectensis, an emerging model system, undergoes larval development, asexual fission, and complete bi-directional regeneration in the field and laboratory. In order to investigate to what extent embryonic patterning mechanisms are utilized during alternate developmental trajectories, we examined expression of developmental regulatory genes during regeneration and fission. When compared to previously reported embryonic expression patterns, we found that all genes displayed some level of expression consistent with embryogenesis. However, five of seven genes investigated also displayed striking differences in gene expression between one or more developmental trajectory. These results demonstrate that alternate developmental trajectories utilize distinct molecular mechanisms upstream of major developmental regulatory genes such as fox, otx, and Hox-like.     

Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and fission evolves in regenerative clades

Regeneration, the ability to replace lost body structures, and agametic asexual reproduction, such as fission and budding, are post‐embryonic developmental capabilities widely distributed yet highly variable across animals. Regeneration capabilities vary dramatically both within and across phyla, but the evolution of regeneration ability has rarely been reconstructed in an explicitly phylogenetic context. Agametic reproduction appears strongly associated with high regenerative abilities, and there are also extensive developmental similarities between these two processes, suggesting that the two are evolutionarily related. However, the directionality leading to this relationship remains unclear: while it has been proposed that regeneration precedes asexual reproduction, the reverse hypothesis has also been put forward. Here, we use phylogenetically explicit methods to reconstruct broad patterns of regeneration evolution and formally test these hypotheses about the evolution of fission in the phylum Annelida (segmented worms). We compiled from the literature a large dataset of information on anterior regeneration, posterior regeneration, and fission abilities for 401 species and mapped this information onto a phylogenetic tree based on recent molecular studies. We used Markovian maximum likelihood and Bayesian MCMC methods to evaluate different models for the evolution of regeneration and fission and to estimate the likelihood of each of these traits being present at each node of the tree. Our results strongly support anterior and posterior regeneration ability being present at the basal node of the annelid tree and being lost 18 and 5 times, respectively, but never regained. By contrast, the ability to fission is reconstructed as being absent at the basal node and being gained at least 19 times, with several possible losses. Models assuming independent evolution of regeneration and fission yield significantly lower likelihoods. Our findings suggest that anterior and posterior regeneration are ancestral for Annelida and are consistent with the hypothesis that regenerative ability is required to evolve fission.     

Asexual Reproduction and Segmental Regeneration, but not Morphallaxis, are Inhibited by Boric Acid in Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae)

Body fragmentation, in some animal groups, is a mechanism for survival and asexual reproduction. Lumbriculus variegatus (Müller, 1774), an aquatic oligochaete worm, is capable of regenerating into complete individuals from small body fragments following injury and reproduces primarily by asexual reproduction. Few studies have determined the cellular mechanisms that underlie fragmentation, either regenerative or asexual. We utilized boric acid treatment, which blocks regeneration of segments in amputated fragments and blocks architomic fission during asexual reproduction, to investigate mechanistic relationships and differences between these two modes of development. Neural morphallaxis, involving changes in sensory fields and giant fiber conduction, was detected in amputated fragments in the absence of segmental regeneration. Furthermore, neural morphallactic changes occurred as a result of developmental mechanisms of asexual reproduction, even when architomy was prevented. These results show that fragmentation in L. variegatus, during injury or asexual reproduction, employs developmental and morphallactic processes that can be mechanistically dissociated by boric acid exposure. In regeneration following injury, compensatory morphallaxis occurred in response to fragmentation. In contrast, anticipatory morphallaxis was induced in preparation for fragmentation during asexual reproduction. Thus, various forms of regeneration in this lumbriculid worm can be activated independently and in different developmental contexts.     

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.     

Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission

Abstract. The starlet sea anemone, Nematostella vectensis , is a small burrowing estuarine animal, native to the Atlantic coast of North America. In recent years, this anemone has emerged as a model system in cnidarian developmental biology. Molecular studies of embryology and larval development in N. vectensis have provided important insights into the evolution of key metazoan traits. However, the adult body plan of N. vectensis may arise via four distinct developmental trajectories: (1) embryogenesis following sexual reproduction, (2) asexual reproduction via physal pinching, (3) asexual reproduction via polarity reversal, and (4) regeneration following bisection through the body column. Here, we compare the ontogenetic sequences underlying alternate developmental trajectories. Additionally, we describe the predictable generation of anomalous phenotypes that can occur following localized injuries to the body column. These studies suggest testable hypotheses on the molecular mechanisms underlying alternate developmental trajectories, and they provoke new questions about the evolution of novel developmental trajectories and their initiation via environmental cues.     

Molecular signaling in feather morphogenesis

The development and regeneration of feathers have gained much attention recently because of progress in the following areas. First, pattern formation. The exquisite spatial arrangement provides a simple model for decoding the rules of morphogenesis. Second, stem cell biology. In every molting, a few stem cells have to rebuild the entire epithelial organ, providing much to learn on how to regenerate an organ physiologically. Third, evolution and development ('Evo-Devo'). The discovery of feathered dinosaur fossils in China prompted enthusiastic inquiries about the origin and evolution of feathers. Progress has been made in elucidating feather morphogenesis in five successive phases: macro-patterning, micro-patterning, intra-bud morphogenesis, follicle morphogenesis and regenerative cycling.     

Modes of Developmental Outgrowth and Shaping of a Craniofacial Bone in Zebrafish

The morphologies of individual bones are crucial for their functions within the skeleton, and vary markedly during evolution. Recent studies have begun to reveal the detailed molecular genetic pathways that underlie skeletal morphogenesis. On the other hand, understanding of the process of morphogenesis itself has not kept pace with the molecular work. We examined, through an extended period of development in zebrafish, how a prominent craniofacial bone, the opercle (Op), attains its adult morphology. Using high-resolution confocal imaging of the vitally stained Op in live larvae, we show that the bone initially appears as a simple linear spicule, or spur, with a characteristic position and orientation, and lined by osteoblasts that we visualize by transgenic labeling. The Op then undergoes a stereotyped sequence of shape transitions, most notably during the larval period occurring through three weeks postfertilization. New shapes arise, and the bone grows in size, as a consequence of anisotropic addition of new mineralized bone matrix along specific regions of the pre-existing bone surfaces. We find that two modes of matrix addition, spurs and veils, are primarily associated with change in shape, whereas a third mode, incremental banding, largely accounts for growth in size. Furthermore, morphometric analyses show that shape development and growth follow different trajectories, suggesting separate control of bone shape and size. New osteoblast arrangements are associated with new patterns of matrix outgrowth, and we propose that fine developmental regulation of osteoblast position is a critical determinant of the spatiotemporal pattern of morphogenesis.     

Conservation of epigenetic regulation, ORC binding and developmental timing of DNA replication origins in the genus Drosophila

There is much interest in how DNA replication origins are regulated so that the genome is completely duplicated each cell division cycle and in how the division of cells is spatially and temporally integrated with development. In the Drosophila melanogaster ovary, the cell cycle of somatic follicle cells is modified at precise times in oogenesis. Follicle cells first proliferate via a canonical mitotic division cycle and then enter an endocycle, resulting in their polyploidization. They subsequently enter a specialized amplification phase during which only a few, select origins repeatedly initiate DNA replication, resulting in gene copy number increases at several loci important for eggshell synthesis. Here we investigate the importance of these modified cell cycles for oogenesis by determining whether they have been conserved in evolution. We find that their developmental timing has been strictly conserved among Drosophila species that have been separate for approximately 40 million years of evolution and provide evidence that additional gene loci may be amplified in some species. Further, we find that the acetylation of nucleosomes and Orc2 protein binding at active amplification origins is conserved. Conservation of DNA subsequences within amplification origins from the 12 recently sequenced Drosophila species genomes implicates members of a Myb protein complex in recruiting acetylases to the origin. Our findings suggest that conserved developmental mechanisms integrate egg chamber morphogenesis with cell cycle modifications and the epigenetic regulation of origins.     

THE CONTROL OF SEXUAL MORPHOGENESIS IN THE ASCOMYCOTINA

(1) A series of factors controls sexual morphogenesis in the Ascomycotina, a process involving the formation of novel structures such as ascocarps (fruit bodies) and asci (sacs containing spores) during sexual reproduction. (2) Environmental and genetic factors must be correct before Ascomycetes may sexually reproduce. Compatibility in many heterothallic species is under polygenic control, with the mating type loci and also other genetic factors determining the productivity of sexual crosses. (3) Classical genetic studies have shown that sexual morphogenesis involves the expression of a series of developmentally regulated genes, and this has been confirmed by recent molecular studies which have demonstrated changes in patterns of mRNA and protein synthesis during ascocarp formation. (4) Hyphal differentiation leading to the formation of mature fruit bodies occurs in response to a series of signals, which include various physical and chemical factors. (5) Chemical sex factors have been identified which are believed to have important regulatory or nutritional roles in sexual morphogenesis. These include the following. (a) Diffusible sex hormones which may regulate developmental switching between asexual and sexual modes of reproduction, including (i) pheromones involved with the induction of gametangia and gamete attraction, and (ii) sex morphogens involved with triggering particular stages of fruit body formation. (b) Sexual growth substances which are required as nutrients, and may be precursors for the production of sex hormones, or metabolites used in the synthesis of novel sexual structures. Most of these sex factors are lipids. (6) Certain sex morphogens and sexual growth substances have been shown to exhibit activity in a variety of fungal species, suggesting that fungi of related phylogenetic descent may utilize similar metabolites or signalling factors during sexual reproduction. (7) Phenoloxidase enzymes may catalyse hyphal aggregation in developing fruit bodies. (8) Initial stages of ascocarp development may occur independently of the events of the sexual cycle. However, a link(s) with the functional ascogenous hyphae is needed for the formation of morphologically mature ascocarps. (9) Suitable environmental conditions are sufficient to trigger sexual morphogenesis in homothallic Ascomycetes. However, an extra level of control is present in heterothallic species, with a compatible partner required to complete sexual reproduction. This may be partly because novel regulatory products, formed by the combined action of the mating type loci of different partners, are required for further ascocarp development. (10) Further research is required to identify more fungal chemical sex factors and to determine the role of environmental stress in controlling sexual morphogenesis, and how this may be related to temporal patterns in the expression of mating type genes.     

Morphological characterization of the asexual reproduction in the acorn worm Balanoglossus simodensis

The acorn worm Balanoglossus simodensis reproduces asexually by fragmentation and subsequent regeneration from the body fragments. We examined the morphogenesis of its asexual reproduction. At first, we collected asexually reproducing specimens and observed their morphogenesis. Then, we succeeded in inducing the asexual reproduction artificially by cutting the worm at the end of the genital region. The process of morphogenesis is completely the same between naturally collected and artificially induced specimens. The stages during morphogenesis were established on the basis of the external features of the asexually reproducing fragments. The internal features of the fragments were also examined at each stage. In a separate phase of the study, the capacity for regeneration of some body parts was also examined by dividing intact worms into about 10 fragments. Although the capacity for regeneration varied among the different body parts, some fragments regenerated into complete individuals in 1 month. The process of regeneration was the same as that in the asexually produced fragments.     

DEVELOPMENTAL QUANTITATIVE GENETICS AND THE EVOLUTION OF ONTOGENIES

A model is presented which permits integration of developmental information into genetic discussions about evolutionary change in morphology. Development of a trait is described in terms of an ontogenetic trajectory whose properties are defined by a small number of parameters. Some evolutionary aspects of development are examined from the perspective of this quantitative genetic model. Particular attention is given to the developmental origin of pleiotropic effects, developmental constraints, heterochrony, and the growth and morphogenesis of complex morphologies. The role of genetic maternal effects in mammalian development is briefly examined, particularly as it relates to selection on developmental traits.     

Comparative anatomy and histology of developmental and parasitic stages in the life cycle of the lined sea anemone Edwardsiella lineata

The evolution of parasitism is often accompanied by profound changes to the developmental program. However, relatively few studies have directly examined the developmental evolution of parasitic species from free-living ancestors. The lined sea anemone Edwardsiella lineata is a relatively recently evolved parasite for which closely related free-living outgroups are known, including the starlet sea anemone Nematostella vectensis. The larva of E. lineata parasitizes the ctenophore Mnemiopsis leidyi, and, once embedded in its host, the anemone assumes a novel vermiform body plan. That we might begin to understand how the developmental program of this species has been transformed during the evolution of parasitism, we characterized the gross anatomy, histology, and cnidom of the parasitic stage, post-parasitic larval stage, and adult stage of the E. lineata life cycle. The distinct parasitic stage of the life cycle differs from the post-parasitic larva with respect to overall shape, external ciliation, cnida frequency, and tissue architecture. The parasitic stage and planula both contain holotrichs, a type of cnida not previously reported in Edwardsiidae. The internal morphology of the post-parasitic planula is extremely similar to the adult morphology, with a complete set of mesenterial tissue and musculature despite this stage having little external differentiation. Finally, we observed 2 previously undocumented aspects of asexual reproduction in E. lineata: (1) the parasitic stage undergoes transverse fission via physal pinching, the first report of asexual reproduction in a pre-adult stage in the Edwardsiidae; and (2) the juvenile polyp undergoes transverse fission via polarity reversal, the first time this form of fission has been reported in E. lineata.     

Expression profiles during honeybee caste determination

Background  

Depending on their larval environment, female honeybees develop into either queens or workers. As in other polyphenisms, this developmental switch depends not on genomic differences between queens and workers but on the differential expression of entire suites of genes involved with larval fate. As such, this and other polyphenic systems can provide a novel tool for understanding how genomes and environmental conditions interact to produce different developmental trajectories. Here we use gene-expression profiles during honeybee caste determination to present the first genomic view of polyphenic development.     

Constructing cranial ontogenetic trajectories: A comparison of growth,development, and chronological age proxies using a known‐age sample of Macaca mulatta

Recent morphometric research has generated opposing conclusions regarding the ontogenetic trajectories of catarrhine crania, possibly due to the ontogenetic proxies used to calculate them. Therefore, we used three surrogates: size, molar eruption, and chronological age to generate trajectories in a known‐age sample to produce ontogenetic trajectories and determine the similarities and differences between them. Forty‐three landmarks from an ontogenetic series of 160 Macaca mulatta crania, with associated ages at death, were used to produce ontogenetic trajectories of cranial shape change. These were computed by sex through multivariate regression of Procrustes aligned coordinates against three surrogates for ontogeny: natural log of centroid size (growth), molar eruption stage (development), and chronological age. These trajectories were compared by calculating the angles between them. Each trajectory was also used to produce simulated adults from juveniles, which were then compared with each other and actual adults. The different trajectories are nearly parallel as each of the surrogates track similar aspects of ontogenetic cranial shape change, but chronological age was the most divergent. Simulated adults produced using the developmental stage trajectories were most similar to actual adults. When simulated adults were produced from opposite sex trajectories, they resembled the sex from which the trajectory was produced, not the sex of the juvenile specimen. We discuss properties of the trajectories produced from each of the surrogates, the possible reasons for previously opposing conclusions, how these properties can inform future investigations, and how our investigation bears on analyses of heterochrony.     

The power of regeneration and the stem-cell kingdom: freshwater planarians (Platyhelminthes)

The great powers of regeneration shown by freshwater planarians, capable of regenerating a complete organism from any tiny body fragment, have attracted the interest of scientists throughout history. In 1814, Dalyell concluded that planarians could "almost be called immortal under the edge of the knife". Equally impressive is the developmental plasticity of these platyhelminthes, including continuous growth and fission (asexual reproduction) in well-fed organisms, and shrinkage (degrowth) during prolonged starvation. The source of their morphological plasticity and regenerative capability is a stable population of totipotent stem cells--"neoblasts"; this is the only cell type in the adult that has mitotic activity and differentiates into all cell types. This cellular feature is unique to planarians in the Bilateria clade. Over the last fifteen years, molecular studies have begun to reveal the role of developmental genes in regeneration, although it would be premature to propose a molecular model for planarian regeneration. Genomic and proteomic data are essential in answering some of the fundamental questions concerning this remarkable morphological plasticity. Such information should also pave the way to understanding the genetic pathways associated with metazoan somatic stem-cell regulation and pattern formation.