' 2005 Wiley-Liss, Inc. genesis 42:124–149 (2005) ARTICLE Stages of Embryonic Development in the Amphipod Crustacean, Parhyale hawaiensis William E. Browne,1 Alivia L. Price,1,2 Matthias Gerberding,2 and Nipam H. Patel2* 1University of Chicago, Department of Molecular Genetics and Cell Biology, Committee on Developmental Biology, Chicago, Illinois 2University of California – Berkeley, Departments of Molecular Cell Biology, Integrative Biology, Center for Integrative Genomics, and HHMI, Berkeley, California Received 1 March 2005; Accepted 12 May 2005 Summary: Studying the relationship between develop- C. elegans. Thus, many critical questions remain with ment and evolution and its role in the generation of bio- regard to how evolution operates over time and how it logical diversity has been reinvigorated by new techni- has come to generate the full range of extant biological ques in genetics and molecular biology. However, diversity. Although we have accumulated significant data exploiting these techniques to examine the evolution of in model species, we are currently left with more ques- development requires that a great deal of detail be known regarding the embryonic development of multiple tions than answers regarding the tempo and mode of evo- species studied in a phylogenetic context. Crustaceans lutionary change due to the vast evolutionary distances are an enormously successful group of arthropods and between the current model systems. This information def- extant species demonstrate a wide diversity of morphol- icit is largely due to the paucity of developmental, genetic, ogies and life histories. One of the most speciose orders and evolutionary data available in nonmodel organisms. within the Crustacea is the Amphipoda. The embryonic The descriptive analyses of gene expression in a wide development of a new crustacean model system, the range of taxa are still relatively few in number. Likewise, amphipod Parhyale hawaiensis, is described in a series our ability to examine the functional basis of observed of discrete stages easily identified by examination of liv- changes in gene expression is restricted to a few species. ing animals and the use of commonly available molecu- lar markers on fixed specimens. Complete embryogene- This problem can be addressed by ‘‘filling in the gaps’’ sis occurs in 250 h at 268C and has been divided into with new model systems more closely related to the cur- 30 stages. This staging data will facilitate comparative rent systems used by researchers. Indeed, the ultimate analyses of embryonic development among crustaceans goal should be to ‘‘cover’’ the Tree of Life with a sufficient in particular, as well as between different arthropod spread of well-studied taxa that the process of evolution groups. In addition, several aspects of Parhyale embry- can be approached and studied with several levels of reso- onic development make this species particularly suit- lution in all major organismal lineages. Ideally, these new able for a broad range of experimental manipulations. taxa should be systems in which embryos are readily genesis 42:124–149, 2005. VC 2005 Wiley-Liss, Inc. obtainable year-round and permit the design and imple- mentation of functional studies with relative ease. Key words: Crustacean; amphipod; arthropod; evolution; development; embryogenesis; segmentation; Engrailed; Several recent studies have examined the evolutionary Distalless relationships among the major groups of arthropods. The data suggest two possible relationships between the Insecta (hexapods) and the Crustacea. One possibility is that the two groups are sister taxa (Boore et al., 1995, INTRODUCTION 1998; Friedrich and Tautz, 1995; Eernisse, 1997; Giribet et al., 2001). The other possibility is a ‘‘Pancrustacea’’ The fields of development, genetics, and evolution have advanced dramatically in the past 25 years. This is in large part due to forward genetics approaches in such Present address for W.E. Browne: Kewalo Marine Lab, University of species as Drosophila melanogaster and Caenorhabdi- Hawaii. tis elegans. The information gathered from this work has Present address for M. Gerberding: Max-Planck-Institut fu¨r Entwicklungs- given us insight into developmental mechanisms and biologie, Tu¨bingen, Germany. their underlying genetic architecture. Only a few addi- * Correspondence to: M. Gerberding, University of California, Berkeley, Department of Integrative Biology, 3060 VLSB # 3140, Berkeley, CA 94720-3140. tional animal taxa (such as Xenopus laevis, Gallus E-mail: [email protected] domestica, Mus musculus, Danio rerio) have come Published online in Wiley InterScience (www.interscience.wiley.com). under the intense scrutiny received by Drosophila and DOI: 10.1002/gene.20145 STAGES OF DEVELOPMENT P. HA WAIENSIS 125 clade in which the insects branch from within the Crus- maturity. Fertilized eggs can be removed from females tacea (Regier and Shultz, 1997; Hwang et al., 2001). In prior to their first cleavage and are sufficiently large to this scenario the insects would represent a terrestrialized perform microinjections (Gerberding et al., 2002) and branch of crustaceans. In either case the crustaceans blastomere isolations (Extavour, 2005) with relative ease. form a key outgroup to the insects and in this context Eggs collected can be hatched individually and the mature will help enormously in our understanding of arthropod animals can subsequently be used in pairwise sister– evolution. The amphipod Parhyale hawaiensis (Dana, brother or mother–son matings to generate inbred lines. 1853) is a crustacean species that is particularly well The length of embryogenesis is relatively short, lasting suited for developmental, genetic, and evolutionary anal- roughly 10 days. Interestingly, the P. hawaiensis body ysis and has the potential of filling an important taxo- plan is established by distinct and invariant cell lineages nomic gap in current comparative studies. very early in embryogenesis (Gerberding et al., 2002). While detailed staging information is available for Dro- sophila (Campos-Ortega and Hartenstein, 1985; Harten- stein, 1993), only relatively simple staging systems exist Parhyale hawaiensis Body Plan for the crustaceans Cherax (Sandeman and Sandeman, The basic body plan of P. hawaiensis follows that of the 1991), and Nebalia (Olesen and Walossek, 2000). typical arthropod in that the ground plan is organized Amphipods (Peracarida; Malacostraca; Crustacea) have around a series of repeating segmental units along the been the subject of study by developmental biologists anterior–posterior (A-P) axis. Several synapomorphic since the late 19th century (e.g., Langenbeck, 1898; characters clearly unite the Amphipoda (Schram, 1986; Weygoldt, 1958). However, detailed descriptive staging Schmitz, 1992). Most recognizable among these charac- information covering the complete course of embryo- ters are the lateral compression of the body, sessile com- genesis in any amphipod is largely lacking. Commonly pound eyes, and the relative orientation of the pereo- referred to as beachhoppers or scuds, amphipods are pods (appendages of thoracic segments T4–T8) to the malacostracan crustaceans and thus closely affiliated body axis (pereopods of T4 and T5 orient anteriorly, per- with more familiar Crustacea such as krill, lobsters, and eopods of T6–T8 orient posteriorly, thus the name for crabs. Within the Crustacea, amphipods rank as one of the group, amphipod) (Fig. 1). Additionally, amphipods the most ecologically successful and speciose extant have large coxal plates (expanded plates attached dor- orders and occur in nearly all known marine, fresh, and sally to the base of thoracic appendages) (Fig. 1). brackish water environments as well as in high humidity Typical of most amphipods, the P. hawaiensis cepha- terrestrial ecosystems (such as tidal zones, coastal flood lon (head) is composed of the anteriormost six seg- plains, and forest leaf litter) (e.g., Barnard and Karaman, ments. The anteriormost preantennal segment bears no 1991; Vinogradov et al., 1996; also Lindeman, 1991; paired appendage. The remaining five segments do pos- Sherbakov et al., 1999; Kamaltynov, 1999; Vainola and sess paired appendages. From anterior to posterior, the Kamaltynov, 1999; Sheader et al., 2000; Poltermann paired appendages of the head are the uniramous first et al., 2000; Gasca and Haddock, 2004). They have pre- antenna (An1), uniramous second antenna (An2), gna- dominately exploited scavenging niches and thus an apt thobasic mandibles (Mn), biramous first maxillae (Mx1), analogy for the group would be ‘‘the flies of the sea.’’ and the biramous second maxillae (Mx2). In addition, This ecological diversity is matched by a high level of the first thoracic segment (T1) is fused to the cephalon. morphological diversity. Several thousand amphipod The T1 appendage pair, the maxillipeds, are triramous, species have been described and the current rate of sev- fused at their base, and extensively modified to assist in eral new species descriptions per year suggests that the feeding (Fig. 1). There is a close arrangement of the gna- upper limit of extant species is far higher than the cur- thal appendages, including the maxillipeds, in a basket rent species count. shape around the mouth to form a highly compact buc- Importantly, several aspects of Parhyale hawaiensis cal mass (Fig. 1b). life history make this particular species amenable to The next seven segments of the thoracic region, the many types of classical and modern laboratory analyses second