Figure 1:
Parhyale hawaiensis.
Shown are adult wild-type Parhyale hawaiensis. The larger animal on top is male, the smaller one below is female.
Parhyale hawaiensis exhibits sexual dimorphism, e.g. in the subchelate gnathopods, which are segmental appendages of T2 and T3.
2.5.1 Ecology and habitat of Parhyale hawaiensis
Parhyale hawaiensis (Figure 1) is a marine amphipod. It is found in tropical oceans around the world, where it aggregates in dense populations in intertidal and shallow water habitats. Parhyale hawaiensis are detritovorous. In their natural ecological environment, they thrive even under rapid changes in temperature, salinity and water quality in general.
Today’s laboratory populations derive from specimen that were isolated from the John G. Shedd Aquarium, Chicago, IL (Browne et al., 2005), where they were considered a pest species in water filter systems. These characteristics make Parhyale well-suited for rearing under laboratory conditions (Rehm et al., 2009b).
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2.5.2 General aspects of Parhyale hawaiensis development
Parhyale hawaiensis develops directly. This means that no free-swimming larva exists;
instead, newly hatched Parhyale exhibit adult morphology with regard to a complete set of segments and fully developed appendages, organs and cuticle (Figure 98). The duration of Parhyale embryogenesis is relatively short (250h at 26°C). Detailed staging information is available: in detail, 30 stages (S1-S30) have been described (Browne et al., 2005). They explain subsequent and partially overlapping developmental processes of early cleavage, gastrulation, germ band formation and segmentation, germ cell migration, appendage development, body morphogenesis, cuticularisation (see also Havemann et al., 2008), mesoderm differentiation, myogenesis and gut development, CNS development and organogenesis.
Specifically, individual stages of Parhyale embryogenesis were defined differently on the base of various features, such as visible early cleavage events (S1-S5), general embryonic morphology (e.g. “soccerball stage” S6 and “rosette stage” S7), emergence of specific morphological traits (e.g. first appearance of head lobes marking the onset of S9, emergence of the dorsal organ at S10 or the proctodeum becoming first visible at S21), developmental and cellular processes (e.g. germ disc condensation during S8, germband row formation at S11 or movement and splitting of the germ cell cluster during S13-S16) and morphogenetic events regarding appendages (e.g. the initiation of limb bud morphogenesis at S15 or the An2 shape change at S22) as well as the gradual folding of the germ band (e.g. the embryo first appearing comma shaped at S18). As a consequence, several stages mark distinct points of time during Parhyale development (e.g. S9, S17 or S27), while others cover a continuous phase of Parhyale embryogenesis (e.g. S12 or S16). In this work, this staging system will be used with the following adjustment: all stages will be treated as temporal phases with one given stage ending at the onset of the next stage (e.g. considering S8, germ disc condensation, ending as soon as the embryo’s head lobes first appear at S9).
2.5.3 The Parhyale hawaiensis body design
Parhyale hawaiensis is a typical representative of the clade amphipoda, in that it exhibits the pivotal synapomorphic character of this group: bidirectional orientation of the
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pereopods, i.e. the appendages of the fourth to eighth thoracic segments (T4-T8) in relation to the body axis, with the T4 and T5 pereopods oriented anterior and the T6-T8 pereopods posterior. With other amphipods, Parhyale also shares sessile compound eyes, large coxal plates and a lateral compression of the body (Figure 1; Browne et al., 2005).
During embryogenesis, reiterated segments are established in anterior-posterior sequence. Except the pre-antennal segment and the asegmental telson, all segments bear paired appendages. Although Parhyale develops directly, naupliar segments emerge earlier than post-naupliar ones. This is reminiscent of Nauplia larvae found in other crustaceans and also likely reflects two different segmentation mechanisms, primary and secondary segmentation (reviewed in Minelli, 2001; Tautz, 2004). From anterior to posterior, the naupliar segments are: preantennal (pAn, carrying labrum and eyes), first antennal (An1), second antennal (An2, corresponding to the intercalary segment in insects, 2.4) and mandibular (Mn). The first antennae are also referred to as the antennules, as compared to the antennae (An2), strictly speaking. During secondary segmentation, the following segments are established (anterior to posterior): first (Mx1) and second maxillary (Mx2, corresponding to the labial segment in insects), followed by eight thoracic segments, the first of which carries the maxillipeds (T1), the second and third carrying subchelate gnathopods (T2, T3) and the remaining carrying pereopods (T4-T8). T2-T8 form the pereon. The appendages of T4 to T8 carry specialised epipods conferring respiratory function, the gills. Posterior to those, six abdominal segments and the telson follow: the anterior three abdominal segments carry pleopods (A1-A3, constituting the pleon), and the posterior three carry uropods (A4-A6, forming the urosome). The telson does not carry paired appendages (Browne et al., 2005).
The nature of the gnathocephalic and the T1 appendages reflects the feeding mode of Parhyale: food is transported from posterior to anterior, passing all feeding appendages until it reaches the stomodeum. In adult animals, the segments pAn through T1 form the cephalon or head, with the respective segmental appendages concentrated as the morphologically distinct buccal mass (Browne et al., 2005).
Parhyale exhibits sexual dimorphism. Male adults have specialised gnathopods (T3) that are used for mating (2.5.4). The endites of females’ T2-T5 appendages constitute the brood pouch, into which the fertilised eggs are deposited (Browne et al., 2005).
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2.5.4 Parhyale hawaiensis mating behaviour
Adult Parhyale reach sexual maturity ca. 6 weeks after hatching. Mature adults reproduce throughout the year, roughly every 2-3 weeks (Browne et al., 2005). In detail, sexually mature male and female Parhyale hawaiensis enter premating amplexus with the larger male grasping the female with its gnathopods (sexually dimorphic T3 appendages). A mating pair remains in this position over several days, until the female moults. At this moment, the male deposits its sperm into the female’s paired oviducts and subsequently releases her. The female sheds her eggs through the oviducts into a ventral marsupium, or brood pouch, fertilizing them in the process (see also Figure 2). After that, the female’s cuticle hardens. The brood pouch is composed of the specialized appendage endites of T2-T5.
Embryos of one batch usually develop synchronously. Their numbers may range from 1 up to 25 (Browne et al., 2005).
Figure 2: Ovigerous female Parhyale hawaiensis (top) and shed cuticle (bottom). Until hatching, Parhyale eggs develop in the mother’s brood pouch.
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2.5.5 Early developmental processes of Parhyale hawaiensis
In Parhyale, the intial cleavages of the zygote are total. Two slightly inequal cleavages and a subsequent distinctly inequal cleavage give rise to four macromeres and four micromeres, each of which exhibit an unambiguous morphology. As shown by cell lineage experiments, each of these eight cells gives rise to specific progeny having invariant cell fates (Gerberding et al., 2002). Importantly, this shows that at this early time of development, anterior-posterior, dorso-ventral and bilateral polarity has been established in Parhyale. This mode of early cleavage is specific to amphipods and is believed to have evolved convergently to the mode of holoblastic cleavage found in lophotrochozoans and annelids. Apart from amphipods, several other malacostracan taxa exhibit holoblastic cleavages during the intial phase of embryogenesis as well (e.g., Biffis et al., 2009; Hertzler et al., 1994). These cases are also believed to resemble secondary cleavage evolution (Scholtz and Dohle, 1996). In contrast, Drosophila and Tribolium embryos undergo intial syncytial cleavages that result in an early syncytial blastoderm (e.g., Lecuit, 2004; Schröder et al., 2008).
2.5.5.1 Parhyale hawaiensis development from S1 through S7
As mentioned above, the implementation of axial polarity is preceded by the specification of cell lineage founders within the S4 Parhyale embryo. At this stage, the Parhyale embryo is composed of four macromeres and four micromeres. From each of these cells, specific germ layers or germ layer fractions encompassing tissues with 'polarised' positional identity derive (Gerberding et al., 2002). In detail, three ectodermal precursors will subsequently contribute to the developing germ band, one to the left embryonic hemisphere (El), one to the right (Er) and the remaining precursor to the posterior part of the embryo (Ep).
As a consequence, the establishment of anterior-posterior polarity starts conceptually within the S4 embryo due to the specification of the Ep macromere. This polarity is maintained during subsequent cleavages leading to the 'soccerball stage' (S6) and to gastrulation, as derivatives of the three ectodermal founders retain the relative position to each other within the embryo (Gerberding et al., 2002). During early gastrulation ('rosette stage', S7) mesodermal and germ line cells cluster at the anterior of the condensing germ disc, indicating
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the anterior-posterior polarity of the embryo and marking the site where head structures will be formed (Browne et al., 2005; Gerberding et al., 2002).
2.5.5.2 Parhyale hawaiensis germ disc condensation during S8
As a result of the completion of gastrulation, the germ disc aggregates at the anterior ventral region of the egg during S8 (Browne et al., 2005). It covers slightly more than a third of the embryo's surface and is characterised by evenly distributed, large cells that contain coin-shaped nuclei. Around the periphery of the germ disc, single cells of similar morphology are scattered. The anterior-posterior polarity of the germ disc is reflected by its relative position within the embryo, with the posterior end expanding farther ventrally than the anterior end does dorsally (Browne et al., 2005).
2.5.5.3 Emergence of head lobes in Parhyale hawaiensis at S9
As embryogenesis proceeds to stage 9, the germ disc elongates along the anterior-posterior axis and acquires a distinct tripartite morphology that comprises two emerging head lobes and the post-naupliar, posterior region from which trunk segmentation originates. The head lobes represent the developing left and right head hemispheres that encompass the prospective pre-antennal region of the embryo as well as the first and second antennal segments. The site of the future mandibular segment, which is considered the most posterior naupliar segment in crustaceans (reviewed in Minelli, 2001), shows no hemispheric division and represents the base from which both head lobes expand towards anterior. The emergence of the extra-embryonic dorsal organ, composed of cells found anterior of the developing embryonic head, marks the onset of stage 10 of Parhyale embryogenesis (Browne et al., 2005).
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2.5.6 Technical repertoire of the genetic system Parhyale hawaiensis
Several characteristics make Parhyale hawaiensis amenable to embryological and molecular techniques (Rehm et al., 2009b). Batches of synchronously developing embryos can be obtained from ovigerous females shortly after fertilisation, i.e. before they undergo first cleavages. They can be incubated outside the brood pouch until they reach a desired age.
In addition, the embryos are relatively large (size) and therefore accessible for injection-based techniques (Rehm et al., 2009d). The fact that from S6, the pigmented yolk fraction of an embryo is separated from the opaque germ rudiment enables fast and reliable staging of an individual embryo (Browne et al., 2005).
Specifically developed and adjusted histological techniques (whole mount in situ hybridisation and antibody staining) allow for visualisation of transcript and protein expression (MM; e.g. Browne et al., 2006; Prpic and Telford, 2008; Rehm et al., 2009a; for a technical overview, see Rehm et al., 2009c). pMinos-based stable transgenesis can be applied to Parhyale. Parhyale-specific genetic drivers, in particular an hsp70b-derived heat shock element as well as a muscle-specific enhancer element, have been isolated and can be used for transgenesis of expression constructs (Pavlopoulos and Averof, 2005; Pavlopoulos et al., 2009). Parhyale embryogenesis starts with a series of total and unequal cleavages. These lead to four micromeres and four macromeres, each of which found invariant cell lineages (Gerberding et al., 2002). This particular feature can be used for clonal analyses (e.g., Extavour, 2005; Price et al., 2010). Adult Parhyale are only weakly pigmented and therefore transparent, enabling easy detection of fluorescent dyes and markers (Browne et al., 2005;
Pavlopoulos and Averof, 2005). The amphipod body plan of Parhyale (2.5.3) opens various possibilities to study morphological phenotypes.
However, although attempts to apply siRNA and morpholinos for performing loss-of-function experiments have provided preliminary results (Liubicich et al., 2009; Ozhan-Kizil et al., 2009) and injection of capped mRNA has been successful in causing ectopic expression in Parhyale (Pavlopoulos et al., 2009), functional techniques had not been available at the beginning of this work. Still, the development of versatile, robust functional techniques is far from being completed.
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