Comparison of Developmental Trajectories in the Starlet Sea Anemone Nematostella Vectensis: Embryogenesis, Regeneration, and Two Forms of Asexual Fission
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Invertebrate Biology 126(2): 99–112. r 2007, The Authors Journal compilation r 2007, The American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2007.00081.x Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission Adam M. Reitzel, Patrick M. Burton, Cassandra Krone, and John R. Finnertya Department of Biology, Boston University, Boston, Massachusetts 02215, USA 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 em- bryology 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 de- scribe 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. Additional key words: Nematostella, planula, regeneration, transverse fission Nematostella vectensis STEPHENSON 1935 is a Nematostella vectensis is the only sea anemone spe- euryhaline, eurythermal, burrowing sea anemone cies whose members have been cultured throughout (Anthozoa, Actiniaria) living in coastal estuarine its entire life cycle in the laboratory (Fautin 2002), habitats throughout North America and Southern and recently, it has emerged as an important basal England (Stephenson 1935; Crowell 1946; Hand metazoan system for research in development and 1957). Nematostella vectensis is a member of the fam- genomics (reviewed in Darling et al. 2005). Studies ily Edwardsiidae, a monophyletic clade of burrowing utilizing N. vectensis have led to a better molecular sea anemones with a global distribution (Daly 2002; understanding of anthozoan development while Daly et al. 2002). Like many cnidarians, N. vectensis illuminating critical events in the early evolution of can undergo multiple developmental trajectories, i.e., animals such as the origin of bilateral symmetry the adult polyp may develop via embryogenesis, asex- (Finnerty et al. 2004), anterior–posterior patterning ual fission, or regeneration (Hand & Uhlinger 1992). (Finnerty & Martindale 1999), and triploblasty Unlike the majority of cnidarians, N. vectensis is a (Scholz & Technau 2003; Martindale et al. 2004). relatively well-studied species with a rich literature With completion of its genome sequence and the describing its natural history (Hand & Uhlinger 1994; characterization of 150,000 expressed sequence tags, Sheader et al. 1997), geographic distribution (Hand N. vectensis’s utility as a genomic model system will & Uhlinger 1994 and references therein; Pearson et increase dramatically (Sullivan et al. 2006). al. 2002; Darling et al. 2004), population genetic Despite the increasing prominence of N. vectensis structure (Pearson et al. 2002; Darling et al. 2004), as a laboratory model for development, its complete and conservation (Sheader et al. 1997). developmental repertoire has not yet been thorough- ly explored. The specific developmental sequences of embryogenesis, asexual reproduction, and regenera- tion have not been explicitly compared in one source. a Author for correspondence. Portions of each process have been described in pre- E-mail: [email protected] viously published papers. Frank & Bleakney (1976) 100 Reitzel, Burton, Krone, & Finnerty and Hand & Uhlinger (1992), and later Martin regions (Gierer et al. 1972). However, the Cnidaria (1997), discussed the general features of embryo- have a large diversity of developmental and repro- genesis, the planula larva, and early juvenile mor- ductive mechanisms, making investigations with phology. More detailed observations of early many species across the phylum necessary for a embryogenesis and larval/juvenile development broader, comparative picture (Collins et al. 2005). have been reported in recent papers addressing the Comparisons among additional species would help expression of developmental regulatory genes (e.g., elucidate underlying similarities and differences in Finnerty et al. 2004; Martindale et al. 2004). A num- developmental processes across the Cnidaria. ber of authors (Lindsay 1975; Frank & Bleakney Nematostella vectensis provides an opportunity to 1978; Hand & Uhlinger 1992, 1995) have described elucidate the molecular mechanisms underlying mul- one type of asexual reproduction in N. vectensis that tiple developmental trajectories because members we have termed ‘‘physal pinching’’ (Darling et al. will undergo four distinct trajectories in the labora- 2005). Herein, we describe this process in greater de- tory (embryogenesis, regeneration, asexual reproduc- tail, and we also describe a second, distinct form of tion via physal pinching, and asexual reproduction asexual reproduction that we have termed ‘‘polarity via polarity reversal). Understanding the various reversal.’’ Complete bidirectional regeneration fol- developmental trajectories at the morphological lowing bisection (referred hereafter as just regenera- level is a necessary prerequisite for framing reason- tion) in adults of N. vectensis has been discussed able hypotheses about the underlying molecular largely as an explanation for naturally occurring mechanisms. Previous works with Hydra (Fro¨bius and laboratory-generated ‘‘anomalies’’ (Frank & et al. 2003) and an oligochaete, Pristina leidyi SMITH Bleakney 1978). Adults are frequently collected in 1896 (Bely & Wray 2001), have hypothesized that the the field with multiple oral crowns sharing a common molecular mechanisms underlying alternate develop- physa (Frank & Bleakney 1978). Hand & Uhlinger mental trajectories are likely to be highly similar, as (1995) reported a range of regeneration variants, in- the final phenotype resulting from each process is cluding a four-crowned anemone, by administering identical. However, differences among developmen- specific local injuries to the body wall. tal trajectories in N. vectensis in both temporal se- Members of N. vectensis comprise only one among quence and spatial relationship of particular the thousands of cnidarian species that are capable of developmental events suggest that the underlying developing via alternate developmental trajectories. molecular mechanisms for alternate developmental Understanding the evolution of these trajectories and trajectories may differ in important ways. For exam- the developmental mechanisms will also be strength- ple, embryogenesis proceeds from a single cell, while ened by comparisons with previous work on regeneration and fission occur in fully differentiated development for other species with multiple develop- adults. Likewise, while regeneration is initiated by mental pathways. Within the Cnidaria, a large body external forces, fission is initiated by endogenous sig- of work has investigated alternate developmental tra- nals. Here, we compare and contrast four alternate jectories, especially regeneration and asexual repro- developmental trajectories in N. vectensis at the mor- duction by budding, and to a lesser extent, phological level, with the long-term goal of under- embryogenesis. Most of this work has been carried standing how such developmental flexibility may be out with the hydrozoan Hydra, which has become a encoded in the genome, and how it may evolve. model system for studying asexual reproduction and regeneration (Bode & Bode 1984; Bode 2003). Ex- tensive work manipulating adults with excision and Methods tissue grafts resulted in models for tissue dynamics Animal care and pattern formation in Hydra (Meinhardt 1993). Adults of Hydra that undergo steady-state morpho- Adults of Nematostella vectensis were maintained genesis continually produce cells in the body column in glass or plastic dishes in 33% strength artificial that migrate to the oral and aboral poles, where they seawater (B12%) at room temperature (211–221C). differentiate into region-specific cells. Recent work Adults were fed freshly hatched nauplii (Artemia sp.) has begun characterizing the specific genes and pep- daily, and water was changed weekly. tides involved in cell differentiation and tissue pat- terning during this process (Steele 2002). Individuals Developmental observations of Hydra can regenerate after bisection or even dis- sociation into single cells, resulting in a number of Observations were performed by eye or with abnormal adult phenotypes including multiple oral the aid of a dissecting microscope (model SZX9, Invertebrate Biology vol. 126, no. 2, spring 2007 Reproduction in N. vectensis 101 Olympus, Melville, NY, USA). Images were recorded and similarly observed until regeneration was on a digital camera (Nikon CoolPix 900, Tokyo, complete. Japan) affixed to the dissecting microscope. Scanning electron micrographs were taken for developmental stages between the gastrula and the metamorphosed