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Embryonic development and expression analysis of Distal-less in Caprella scaura (Crustacea, Amphipoda, Caprellidea).

Introduction

The considerable diversity among crustaceans makes them fascinating animals for studying morphological evolution. Several recent studies have provided new insights on how the alteration of genetic mechanisms leads to morphological evolution. For example, studies have revealed that posterior shifts in the expression boundary of the Hox gene Ultrabithorax (Uhx) led to the evolution of maxillipeds, which have morphologically and functionally specialized anterior thoracic appendages for feeding (Averof and Patel, 1997; Abzhanov and Kaufman, 1999, 2000).

The Caprellidea (Crustacea, Amphipoda) may reveal further interesting aspects of morphological evolution because they exhibit unique morphological features that are not observed in other crustaceans. Typically, amphipod crustaceans have seven pereonites (thoracic segments that are not fused to the head) bearing pereopods and six abdominal segments (Fig. 1A). However, in most Caprellidea species, the third and fourth pereopods are absent or strongly reduced, with unclear segments (Fig. 1B). Alternatively, gills develop only in the third and fourth pereonites (the second pereonites in some species; Takeuchi, 1993). Their abdomen also exists in a vestigial form with a remarkably reduced size and no distinct segments (Fig. 1B). Recent molecular phylogenetic studies support the concept that the unique morphology of caprellids was derived from a gammarid-like body plan. This hypothesis is based on the fact that gammarids are closely related to and paraphyletic with caprellids (Ito et al., 2008).

Interestingly, some caprellid families do not exhibit the above-mentioned typical caprellid features. For example, species of the family Caprogammaridae possess segmented abdomens with functional swimming appendages, although their third and fourth pereopods are rudimentary (Takeuchi and Ishimaru, 1991). Another family, Phtisicidae, possesses developed third and fourth pereopods, although the abdomen is vestigial. Our previous molecular phylogenetic analyses of the Caprellidea suggested that the Phtisicidae and Caprogammaridae regained the third and fourth pereopods, and the abdomen, respectively (Ito et al., 2011). Thus, caprellids have a complicated and interesting evolutionary history that does not follow Dollo's law on the irreversibility of evolution (Gould, 1970). In this context, Caprellidea developmental or genetic pathways should be investigated to determine how their pereopods and abdomen degenerated, and more importantly, how they can be re-acquired. Unfortunately, observations of caprellid development are scarce (Pereyaslawzewa, 1888).

[FIGURE 1 OMITTED]

Like other peracarid crustaceans, including the Gammaridea and Isopoda, adult Caprellidea females have a brood pouch on their thoracic segments into which eggs are released. Therefore, it is easy to remove fertilized eggs or embryos from the pouch. In addition, the Caprellidea are direct developers with no planktonic larval stages, as in nauplius or zoea larva. Thus, developmental studies of the Caprellidea should be feasible. Recent molecular develop-mental studies of Gammaridea have been performed, especially on the Gammaridea species Parhyale hawaiensis. Their embryonic development has been described in detail (Browne et al., 2005), and the expression patterns of several genes has been examined (Price and Patel, 2008; Prpic and Telford, 2008). Functional assays of the Hox gene Ubx were performed using RNA interference (Liubicich et al., 2009) or ectopic expression (Pavlopoulos et al., 2009). These technical approaches should help explain the developmental changes that produced the unique body plan of the Caprellidea, since it is derived from the body plan of the Gammaridea (Ito et al., 2008).

In this study, we describe the embryogenesis of a cosmopolitan caprellid species, Caprella scaura Templeton, 1836 (C. scaura typica Mayer, 1890), which exhibits the typical body form of the Caprellidea (the third and fourth pereopods and abdomen are strongly reduced). To investigate the molecular and genetic mechanisms underlying the reduction of pereopods, we examined the expression of Dll, a limb selector gene, in C. scaura embryos.

Materials and Methods

Embryo collection

Adult specimens of Caprella scaura were collected on the dock at Yokohama City (Kanagawa, Japan), transported to the laboratory, and stored in artificial seawater (ASW). When the embryos were released into the brood pouch, they were removed and transferred to a petri dish filled with sterile ASW and incubated at 22[degrees] C.

Observation of embryo genesis

The embryos in the petri dish were viewed under a microscope, and brightfield images were collected at regular intervals (every 1 to 3 h in the early phase and 6 to 12 h in the middle-to-last phase of embryogenesis). For fluorescent microscopic observation, some embryos were fixed in 4% paraformaldehyde, 0.1 mol 1-1 3-(N-morpholino)propanesulfonic acid (pH 7.5), and 0.4 mol 1-1 NaCl, and stored in phosphate-buffered saline (PBS) with 0.1% of sodium azide at 4[degrees]C.

A nucleic acid-specific fluorescent dye, YOY0-1, was used for the observation of fluorescence. Before staining, the embryos (which were preserved in PBS with 0.1% sodium azide) were washed twice in PBS and treated with 1 mg/ml RNase at 37 [degrees]C for 1 h to remove all cytoplasmic RNA. The embryos were then placed in 1 Amoi r' YOY0-1 at room temperature for 1 h in the dark and washed three times with PBS. The stained embryos were observed by confocal laser scanning microscopy (Zeiss LSM510). Three-dimensional images of the embryos were reconstructed from Z-series images using a Zeiss LSM Image Browser, ver. 4.0 (Carl Zeiss).

Cloning and molecular phylogenetic analysis of CsDII

To clone the partial fragment of CsDll (Caprella scaura Distal-less), we designed the following primer set for PCR based on the homeodomain sequence of Artemia Dll (Panganiban and Rubenstein, 2002): 5'-CCGAATTCAARCCNMGNACNATHTA-3' and 5' -CCGGATCCRTTYTG-RAACCADATYTT-3'. A longer cDNA fragment of CsDll was obtained by rapid amplification of cDNA ends (RACE) PCR using a BD SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. The following primers were used: first 3' RACE PCR, 5'-CCGATTTACTCCAGTCTGCAGCTACAGCAGC-3'; second (nested) 3' RACE PCR, 5'-TCGCAGCTAAACTAGGCCTAACGCAAACACAGG-3'. The amplified cDNA fragments were purified using a Sephaglas Bandprep kit (Amersham) after electrophoresis in a 1% agarose gel, and then subcloned using the pGEM-T Easy Vector system (Promega).

The isolated gene fragment (GenBank Acc. No. AB649425) was confirmed as Dll by molecular phylogenetic analyses using DII/Dlx gene sequences from C. swum, P. hawaiensis (Gamrnariclea), Drosophila tnelanogayter (Insecta), Tetranychus urticae (Chelicerata), and mice. The NK homeobox gene tinman and Abdominal-B of D. melanogaster were selected as outgroup genes. The homeobox regions of these genes (52 amino acids) were used for phylogenetic analysis.

Phylogenetic trees were constructed by the maximum likelihood (ML) method with PhyML 3.0 (Guindon and Gascuel, 2003). ML analyses were performed following the DCMut + G +F model selected by the Akaike information criterion (AIC) in Modelgenerator ver. 0.85 (Keane et al., 2006). SPR branch swapping was applied to all ML analyses; 1000 bootstrap pseudoreplicates were performed to evaluate the confidence for each node.

In situ hybridization

Generally, the in situ hybridization protocol in this study was as described previously (Rehm et al., 2009). For in situ hybridization, living embryos were dissected to remove the inner and outer membranes and stretch out the embryo using tungsten needles in an ASW/formaldehyde fixative solution (9 parts ASW and 1 part 37% formaldehyde). The embryos were kept in the fixative for about 20 min, dehydrated with a graded methanol series, and then preserved in 100% methanol at --30 [degrees]C.

Digoxigenin (DIG)-labeled RNA probes were synthesized from the cDNA using a DIG RNA labeling kit (Roche). After rehydration and three washes in PBT (PBS + 0.1% Tween-20), the embryos were post-fixed in PBT/formaldehyde fixative solution (9 parts PBT and 1 part 37% formaldehyde) for 30 min and washed in 500 ill of hybridization buffer (50% formamide, 5X saline-sodium citrate I SSC], 0.25% Triton X-100, 1 % SDS, and 100 p.g/m1 yeast RNA). After being placed in the hybridization buffer and incubated at 65 [degrees]C for 30 min, the embryos were hybridized with DIG-labeled probes in 500 ill of hybridization buffer (final concentration about I rig/g1) at 65 "C for 20 h. After hybridization, excess probe was removed by washing the embryos once with hybridization buffer at 65 [degrees]C for 30 min, four times with 2x SSC at 65 [degrees]C for 30 min, twice with 2 X SSC at room temperature for 10 min, once with 2x SSC and PBT (1: I) solution for 20 min, and three times with PBT for 20 min. Next, the embryos were incubated in PBT containing bovine serum albumin (BSA) for 30 min at room temperature. Subsequently, the embryos were incubated with alkaline phosphatase-conjugated anti-DIG antibodies at 4 [degrees]C overnight. The embryos were then washed three times with PBT for 30 min and transferred to alkaline phosphatase reaction buffer (0.1 mol r' Tris [pH 9.51, 0.1 mol 1-1 NaC1, and 0.05 mol r' MgCl2). Finally, the embryos were placed in Nitro blue tetrazolium/5-bromo-4- chloro-3-indoly1 phosphate (NBT/BCIP) solution (Roche) to visualize positive immunoreactions.

Results

Life cycle of Caprefla scaura

Since Caprellidea are direct developers, juveniles hatch with a form almost identical to that of adults and then molt at regular intervals. It was previously reported that females develop their brood pouch (Fig. 2A) and sexually mature during the seventh and ninth instar (about 20-40 days after the first instar) in C. scaura (Sakaguchi, 1990). While males grow faster than females, they also mature after the seventh instar (about 9 days after the first instar) (Sakaguchi, 1990). Mature females molt and lay eggs in their brood pouch 5-27 days (12.3 days on average) after the previous molting (Sakaguchi, 1990). Although the number of eggs laid depends on the size or nutritional condition of the female, we observed that sufficiently mature females released approximately 50 eggs during one oviposition. The average lifespan in captivity is about 60 days for males and 100 days for females (Sakaguchi, 1990).

Staging of embryonic development in Caprella scaura

Prior to egg deposition into the brood pouch, mature oocytes or eggs were seen in the thoracic segments of adult females (Fig. 3A). According to a previous report (Lim and Alexander, 1986), the mating of C. scaura commences with female molting, followed by copulation, which is accomplished by rapid pumping of the male copulatory pulp. The eggs were released to the brood pouch about 1.5 h after molting. We observed this mating behavior; however, we could not tell at which time fertilization occurred. We observed further development after removing the eggs from the brood pouch and transferring them to a petri dish. We will now describe the developmental stages relative to the time that one-cell-stage eggs were transferred to the petri dish.

From the one-cell stage to hatching, embryonic development took about 100 h at 22 [degrees]C. The following events occurred during embryonic development in C. scaura: (1) transition from holoblastic cleavage to superficial cleavage, (2) germdisc condensation, (3) germband formation and elongation, (4) germband flexure and limb bud formation, (5) limb bud elongation and segmentation, and (6) dorsal closure and reversal of cephalic direction.

Fertilized eggs possess a gray-brown yolk, which appears to be evenly distributed (Fig. 2B). The lengths of the long and short axes were approximately 220 and 190 p,m, respectively.

[FIGURE 2 OMITTED]

Early cleavage was holoblastic (Fig. 2C, D). The first cleavage occurred about 1 h after transfer; the cleavage furrow was perpendicular to the long axis and produced two cells slightly unequal in size (Fig. 2C). The second cleavage was observed 2 h after transfer. The second cleavage furrow was parallel to the long axis. As in gammarids, the second cleavage occurred unequally and generated four cells: one large cell, one small cell, and two equal-sized cells (Fig. 2D) (Gerberding and Scholtz, 1999; Gerberding et al., 2002; Browne et al., 2005).

After several cleavages, the yolk gradually segregated toward the interior while the nucleated cells remained on the embryo surface. The transition from holoblastic to superficial cleavage was observed in stage 5 of gammarid embryo- genesis (Scholtz and Wolff, 2002; Browne et al., 2005). About 6 h after transfer, the clear nucleated cells covered the gray-brown yolk (Fig. 2E). This stage corresponds to stage 6 in Parhyale hawaiensis, when whitish nucleated cells are evenly distributed around the egg (Browne et al., 2005).

Nucleated cells aggregated at the anterior pole of the egg to form the embryonic germdisc in 9-h-old embryos (Fig. 2F, G). At a later stage, the germdisc began to elongate in the posterior direction and form the germband with a grid-like arrangement of cells (32 h after transfer; Fig. 2H, I). Similar developmental processes were observed in P. hawaiensis. Germdisc condensation corresponds roughly to stages 7-10 and germband elongation to stages 11-16 in P. hawaiensis (Browne et al., 2005).

At about 50 h after transfer, the germband started to fold inward toward the interior of the egg at the posterior-ventral side (Fig. 3A, B). The germband flexure was observed clearly under brightfield microscopy (Fig. 3A). This stage corresponds to stage 17 of P. hawaiensis development.

At about 53 h after transfer, folding of the germband and the formation of the limb buds were obvious (Fig. 3C, D). Although clear stripes of engrailed expression were observed in the abdomens of P. hawaiensis specimens during a comparable stage (stage 19; Browne et al., 2005), no posterior structure was confirmed in C. scaura. At this stage, limb buds appeared in all pereonites, including the third and fourth (Fig. 3D). However, in 74-h-old embryos, further elongation and segmentation of the limb buds occurred in all but the third and fourth pereonites. Instead, oval projections appeared in the third and fourth pereonites (Fig. 3E--G). Pereopods developed in the gammarids at a comparable stage to P. hawaiensis (stages 22 and 23; Browne et al., 2005). Each gill developed as a side branch on the coxa, the most proximal limb segment. The oval projection of the caprellid third and fourth pereonites projects from more proximal segments, which may correspond to the coxa.

At about 90 h after transfer (Fig. 3H, I), the extra-embryonic area disappeared after dorsal closure, and the stomodeum direction was changed from upward to downward relative to the egg (Fig. 31). At this stage, the third and fourth pereopods were still composed of two segments, likely coxa in the proximal and gill in the distal (Fig. 31). Considering that coxae of the caprellid third and fourth pereopods are degenerative, they may be absorbed after hatching. The embryos hatched at about 100 h after transfer. Thus, the embryonic period is much shorter than in P. hawaiensis, which takes about 250 h to hatch (Browne et al., 2005).

The Caprella scaura homolog of Dll and its expression

To evaluate the molecular mechanism of pereopod reduction, we isolated a caprellid homolog of Distal-less. The amino acid sequences of the CsDll homeodomain were found to be almost identical to Dll from other arthropods (Fig. 4A). Molecular phylogenetic analysis supported a close relationship between CsDll and Dll from other organisms (Fig. 4B).

The expression of CsDll was examined in embryos in which appendage buds had elongated (corresponding to the stage shown in Fig. 3E--G). Due to the difficulty of removing the extracellular membranes, we did not examine CsDll expression at earlier stages. We detected strong CsDll expression in elongating antennae or pereopods (Fig. 4C, D). However, we did not detect any expression in the third and fourth pereonites or in the oval projections on these segments (Fig. 4C, D).

Discussion

Embryonic development of the Amphipoda has been well studied in Gammaridea (Wolff and Scholtz, 2002; Browne et al., 2005). During embryogenesis, the early cleavages are holoblastic, after which nucleated cells emerge at the surface of the embryo. After the germdisc elongates to form the germband, it begins to fold from the posterior ventral side, after which limb bud elongation occurs. The present study demonstrated that the developmental events found in the embryogenesis of the Gammaridea are highly conserved in Caprella scaura. However, in C. scaura embryos, pronounced abdominal segments were not observed throughout their development. In addition, although limb bud formation occurred in all thoracic segments just after the germband flexure, limb bud elongation and segmentation did not occur in the third and fourth pereonites.

The pereopods of the Amphipoda are uniramous and can be divided into two main parts: basal (coxopodite) and distal (telopodite). Some pereopods bear gills and coxal plates on their coxopodite. The time course of pereopod development was examined for Parhyale hawaiensis (Browne et al., 2005) and Orchestia cavimana (Ungerer and Wolff, 2005). In pereopods, precursors of gills and coxal plates develop as dorsal projections from the coxae, while the segmented coxopodite and telopodite give rise to the main limb branch (Browne et al., 2005; Ungerer and Wolff, 2005). This study found that, in the third and fourth thoracic pereopods of C. scaura, oval projections developed on the proximal segment. Therefore, the oval projections are likely primordial gills, and the proximal segment corresponds to the coxa. Considering the absence of coxae in adult third and fourth pereonites, the coxae may be absorbed after hatching.

To examine the molecular aspects of the degeneration of the third and fourth pereopods, we examined Dll expression, which is involved in the differentiation of the distal part of appendages (Scholtz et al., 1998). Dll was not expressed in the third/fourth pereopods or the gill primordia (Fig. 4). In the case of the caprellid thoracic limb, reduction of the third/fourth pereopods might be caused by the suppression of Dll. However, we were unable to examine the expression of Dll at earlier stages due to technical difficulty in removing the extracellular membranes. Thus, Dll may be ex-pressed in the third and fourth pereopod buds of the Caprellidea during early development.

Several reports have demonstrated that arthropod degeneration of the distal part of appendages is accompanied by the repression of Dll expression. For example, this occurs in the first thoracic appendage of Daphinia manga (water flea), which lacks a prominent ventral branch typically carried by the second to fourth appendages, and this loss correlates with a lack of Dll expression (Shiga et al., 2002). Another example is the crustacean mandible, whose third head appendage functions as the masticatory apparatus. In the Crustacea, the telopodite of the mandible is strongly reduced in size or completely missing, and only the coxopodite can function in mastication (Manton, 1964, 1977). It was previously shown that Dll is expressed in the early mandibular bud, but not in the later embryonic stages of a gammarid species, which does not have the mandibular telopodite (Scholtz et al., 1998). This indicates that Dll plays multiple roles in arthropod appendage development; early expression may be required for initiation of the proximal-distal axis, while late Dll expression is necessary for the maintenance and differentiation of the distal fate (Scholtz et al., 1998). Dll expression was detected before the limb bud was morphologically visible in amphipod pereonites (Hejnol and Scholtz, 2004; Browne et al., 2005), indicating that the function of Dll in early limb development is conserved among amphipod pereopods. Therefore, it is likely that at earlier stages Dll is expressed in the third and fourth pereopod buds of the Caprellidea (Fig. 3C, D). We conclude that Caprellidea lost Dll expression during the late phase of development in third and fourth pereopod, and that this evolutionary loss is linked with the degeneration of third and fourth pereopods.

On the basis of this hypothesis, we were interested in the genetic mechanisms mediating the suppression of Dll in the Caprellidea lineage. To address this hypothesis, the evaluation of Hox gene expression patterns may be informative since modifications to Hox gene expression patterns or functions have been linked to changes in the morphologies and functions of appendages. For example, Dll of D. manga is directly repressed by Anntenapedia (Shiga et al., 2002). In the gammarid P. hawaiensis, ectopic expression of Ubx leads to the transformation of maxillipeds to pereopods (Pavlopoulos et al., 2009), while the inhibition of Ubx results in the opposite effect (Liubicich et al., 2009).

Another interesting aspect of the evolution of the body plan of the Caprellidea is that full development of the third and fourth pereopods and the abdomen may have been regained in some families. An ancestral state reconstruction, based on a molecular phylogenetic analysis of the Caprellidea, suggested that both the family Caprogammaridae (possessing developed abdomens) and family Phtisicidae (exhibiting developed third and fourth pereopods) re-acquired their abdomen and pereopods, respectively (Ito et al., 2011). There are several cases in which the re-acquisition of complicated characteristics are indicated from the phylogenetic framework (Whiting et al., 2003; Kohlsdorf and Wagner, 2006; Collin et al., 2007; Collin and Miglietta, 2008); however, additional genetic or developmental studies are required.

In this study, we provided a brief description of Caprellidea embryogenesis, focusing on the reduction of pereo:. pods. This will serve as the basis for additional molecular analyses of the developmental process. In addition, the developmental biology of the gammarid P. hawaiensis has been actively studied in recent years. It is possible to compare the gene network regulating pereopod and abdomen formation between the Gammaridea and Caprellidea. This may reveal the genetic mechanisms underlying the body plan evolution of the Caprellidea and how pereopods and an abdomen can be restored after they are lost.

Acknowledgments

This work is supported by a research grant from the Research Institute of Marine Invertebrates and by Sasagawa Scientific Research Grant from the Japanese Science Society to AI.

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Received 26 June 2011; accepted 9 August 2011.

* To whom correspondence should be addressed. E-mail: hwada[AT]biol.tsukuba.ac.jp

ATSUSHI ITO (1), MASAKAZU N. AOKI (2), KENSUKE YAHATA (1), AND HIROSHI WADA (1)*

(1.) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan: and

(2.) Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan
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