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provided by Elsevier - Publisher Connector Developmental Biology 252, 100–118 (2002) doi:10.1006/dbio.2002.0840 Germ Line Development in the Grasshopper Schistocerca gregaria: vasa As a Marker

Chun-che Chang,1 Peter Dearden,2 and Michael Akam3 Laboratory for Development and Evolution, University Museum of Zoology, Department of Zoology, Downing Street, Cambridge CB2 3EJ, United Kingdom

Vasa is a widely conserved marker, both in vertebrates and invertebrates. We identify a vasa orthologue, Sgvasa, and use it to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early , but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most energids as they reach the egg surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after , Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, we suggest that the Vasa-stained cells in the abdominal margin are germ cells, as proposed by Nelson (1934), and not cardioblasts, as has been proposed by others. © 2002 Elsevier Science (USA) Key Words: arthropod; Orthoptera; ; gonad; vasa; embryogenesis; ovary; testis; antibody.

INTRODUCTION cells, where the Bmp4 gene is involved (Lawson et al., 1999; McLaren, 1999). Germ cells, specialized to produce haploid , are Among the holometabolous insects, the presence of dis- specified during embryogenesis in most animals (Dixon, tinct germ plasm and the early segregation of germline 1994; Wei and Mahowald, 1994; Wylie, 1999). In some precursors seems to be widespread, though not ubiquitous animals, these cells are specified by the segregation of an (Eddy, 1975; Nieukoop and Sutasurya, 1981; Zissler, 1992). identifiable germ plasm, a cytoplasmic localization of However, the situation among hemimetabolous insects is electron dense materials in the eggs and early embryos highly variable. There have been few recent studies. In the (Eddy, 1975; Mahowald and Boswell, 1983). Several well- older literature (reviewed in Anderson, 1972; Nieukoop and studied species follow this pattern: Drosophila melano- Sutasurya, 1981), some species are described as possessing gaster (fly), Caenorhabditis elegans (nematode), Danio germ plasm and showing early segregation of germ cells rerio (zebra fish), and Xenopus laevis (frog) (Braat et al., (examples include Collembola, Dermaptera, Homoptera), 1999; Ikenishi, 1998). In other animals, no germ plasm is whereas in other cases, it has been claimed that germ cells identifiable, and germ cells are believed to be specified by arise relatively late in embryogenesis, either during gastru- other mechanisms (Dixon, 1994). For example, mouse lation (Dictyoptera, Heteroptera) or shortly before forma- germ cells are specified by inducing signals from somatic tion of the gonad (Orthoptera). However, these studies rely

1 entirely on cytological criteria to identify germ cells. It Present address: Department of Genetics, University of Leices- remains possible that molecular markers would reveal the ter, University Road, Leicester LE1 7RH, UK. 2 Present address: Biochemistry Department, University of earlier segregation of germ cells, even in those species Otago, 710 Cumberland Road, Box 56, Dunedin, New Zealand. where they have not previously been identified. We have 3 To whom correspondence should be addressed. Fax: ϩ44 1223 reinvestigated germline specification in one such insect, 336679. E-mail: [email protected]. the orthopteran Schistocerca gregaria (a grasshopper, the

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African plague locust). No early segregated germ plasm has regulator in and germ cells (Carrera et al., 2000; been identified in any orthopteran insect (Eddy, 1975; Raz, 2000). During oogenesis in Drosophila, the Vasa pro- Nelson, 1934b). tein is required to promote the localized translation of Previous studies of grasshopper germ cells date from nanos RNA (Gavis et al., 1996) and for the localization of the 19th and early 20th centuries. Neither Wheeler other components of the germ plasm, including gurken (1893), studying Xiphidium ensiferum, nor Roonwal mRNA, which is involved in the establishment of (1937), studying Locusta migratoria, could identify germ polarity (Tinker et al., 1998; Ghabrial and Schupbach, cells until relatively late during embryogenesis (30– 40% 1999). Vasa function is also needed for the posttranslational development), when they appear in the median walls of modification of Oskar protein (Markussen et al., 1995; the coelomic of the abdominal segments. Rongo et al., 1995). However, Nelsen (1934b), studying germ-cell develop- Although vasa is a widely conserved germline marker, its ment in the grasshopper Melanoplus differentialis, products are not always uniquely restricted to the germline. claimed to observe segregation of the germ cells at an For example, in Drosophila, maternal vasa mRNA is earlier stage, at about the time segmentation becomes present uniformly in preblastoderm embryos but is de- apparent in the anterior abdomen (ϳ25% of develop- graded by the time of blastoderm formation (Lasko and ment). Roonwal disputed this claim, identifying the same Ashburner, 1988). vasa RNA is not detected again until the cells as cardioblasts. Neither the description by Roonwal second half of embryogenesis (Lasko and Ashburner, 1988). nor by Nelsen provides unambiguous criteria for identi- Vasa protein, on the other hand, is localized to the posterior fying germ cells. pole of the oocyte, then incorporated into pole cells and For our study, we have used the germline marker vasa to restricted to germ cells throughout all developmental stages reinvestigate the location and origin of germ cells in grass- (Hay et al., 1990; Lasko and Ashburner, 1990). In the silk hopper embryos. Vasa genes were first identified in Dro- moth Bombyx mori, vasa RNA is initially present in all sophila, through that block formation of the cells of the embryonic primordium, and only later becomes germline. In Drosophila, Vasa protein is an essential, ma- restricted to the germline (Nakao, 1999). In zebrafish, it is ternally provided component of the germ plasm and is vasa mRNA that is localized in germ granules, rather than the present in germ cells throughout all developmental stages Vasa protein. Maternally provided Vasa protein is widely (Hay et al., 1988; Lasko and Ashburner, 1988). Drosophila distributed in the embryo and not preferentially localized to vasa encodes an ATP-dependent RNA helicase of the the germline until about cell cycle 13 (Knaut et al., 2000). DEAD-box protein family. Drosophila females homozygous Wolke et al. (2002) recently showed that the enrichment of for null mutations in vasa display a range of defects in vasa RNA and protein in zebrafish primordial germ cells is oogenesis and lay no eggs. Weaker mutations allow oogen- due to preferential stabilization in this issue. esis to proceed, but the eggs produced develop into embryos To detect germ cells in Schistocerca, we have analyzed that lack primordial germ cells (Hay et al., 1988; Lasko and the expression of both vasa RNA and protein. We first show Ashburner, 1988). that vasa is indeed a germline marker in adult gonads and in Vasa homologues have been identified as germline mark- late embryos, where the location of germ cells is known. ers in many species, including other insects (Nakao, 1999), We then examine earlier stages, where the identification of cnidarians (Mochizuki et al., 2001), platyhelminths (Shi- germ cells is unknown or contentious. We have looked in vasa bata et al., 1999), nematodes (Gruidl et al., 1996; Roussell particular for any maternal transmission of RNA or protein that might identify germ plasm or an early segre- and Bennett, 1993), chaetognaths (Carre´ et al., 2002), ascid- gated germline. ians (Fujimura and Takamura, 2000), and vertebrates (Fuji- wara et al., 1994; Kobayashi et al., 2000; Komiya and Tanigawa, 1995; Olsen et al., 1997; Shinomiya et al., 2000; Tsunekawa et al., 2000; Yoon et al., 1997; Yoshizaki et al., MATERIALS AND METHODS 2000). Vasa transcripts or Vasa proteins are localized in germ granules, not only in Drosophila, Caenorhabditis, and zebrafish, whose germline formation is driven by germ Insects plasm (Gruidl et al., 1996; Hay et al., 1988; Olsen et al., 1997; Yoon et al., 1997), but also in mouse germ cells which S. gregaria (Forskål) adults and eggs were obtained from a colony are specified by signal induction (Tanaka et al., 2000; maintained at 27°C in the Department of Zoology, University of Cambridge. Staging of S. gregaria embryos is based on the scheme Toyooka et al., 2000). Thus, vasa is an excellent candidate of Bentley et al. (1979) for embryos from 15 to 100% of develop- for a general marker of germ cells, however they are ment. The staging of younger embryos was estimated as described specified. (Dawes et al., 1994; Dawes, 1996; Dearden and Akam, 2001), and is Why germ cells need vasa is still poorly understood, but given as hours after fertilization at 29–30°C. In our culture of S. it seems likely that Vasa protein acts as a translational gregaria, the 15% stage according to Bentley is reached at approxi-

© 2002 Elsevier Science (USA). All rights reserved. 102 Chang, Dearden, and Akam

FIG. 1. Cloning, length estimation, and protein sequence of the Schistocerca vasa gene. (A) PCR cloning of a Sgvasa gene with degenerate primers deg-f and deg-r. An ϳ0.40-kb band (arrow) was amplified from Drosophila (Dm), Folsomia candida (Fc, amplification of four potential helicase bands, which are not discussed in this paper), and Schistocerca gregaria (Sg) genomic DNA. Negative control (Ϫctrl): no template DNA. (B) Illustration of Schistocerca vasa gene fragments obtained from various cloning strategies. This scheme shows fragments obtained from PCR cloning (green, 374 bp), cDNA screening [red, ϳ1.9 kb; including the 3Ј-untranslated region (UTR; ϳ0.4 kb)], and 5Ј RACE PCR cloning (blue, 317 bp). In total, 1764 nucleotides of the vasa coding region have been sequenced. Numbers under each bar indicate the sequence position. Sgvasa and other Schistocerca DEAD-box helicase genes have been deposited in GenBank with Accession Nos. AF5100047–AF5100054. (C) Northern blot of adult Schistocerca ovary RNA. The blot was hybridized with radioactive probes PCR-amplified with primers nef3 and ner2 (for positions, see D). A signal from the Schistocerca vasa gene is at ϳ2.2 kb (arrow). Another fainter signal is at ϳ3 kb. This might be a premRNA of the Schistocerca vasa gene. (D) Deduced amino acid sequence of the Sgvasa gene and positions of primers used for cloning the Sgvasa gene and for making expression constructs. RNP1 and ROP1 (blue): primers for 5Ј RACE PCR cloning; nef1 and ner2 (brown): primers for expression constructs; deg-f and deg-r (green): degenerate primers for PCR cloning. nef3 (black) and ner2: primers for making the Northern hybridization probe; Svhf1 and Expr2 (pink): primers for RT-PCR.

© 2002 Elsevier Science (USA). All rights reserved. emLn eeomn nS gregaria S. in Development Line Germ 02Esve cec UA.Alrgt reserved. rights All (USA). Science Elsevier 2002 ©

FIG. 2. Comparison of Schistocerca Vasa protein with other insect Vasa proteins and the Drosophila eIF4a protein. Sequences of Schistocerca Vasa protein (SGV), Bombyx Vasa protein (BV), Drosophila Vasa protein (DMV), and Drosophila (Dm) eIF4a protein were aligned with MacVector 6.0. Sequences conserved among these four DEAD-box helicases are boxed in black, and the eight conserved motifs of the DEAD-box protein family are underlined with black bars. Vasa-specific sequences in this alignment are highlighted with pale green boxes; the ARKF motif is marked with a red margin; GIVGGA and EXEEXW, two insect Vasa-specific motifs, are marked with blue margin. 103 104 Chang, Dearden, and Akam

FIG. 3. Expression of Schistocerca vasa mRNA in adult tissues and eggs during embryogenesis. (A) Adult tissues. Vasa RNA is detected only in the testis and ovary. Total RNA extracted from an equal weight of tissues (30 mg) was used to synthesize the first-strand cDNA. (B) Late oocytes and unlaid eggs. Vasa RNA is detected in late oocytes (2/sample) but not in four individual unlaid eggs (1/sample). Late oocytes (with yellow yolk) were dissected from adult ovarioles; unlaid eggs were dissected from oviducts. (C) Whole-egg RNA. Vasa RNA is detected in eggs 20 h AEL, but no signals were detected in those younger than 12 h AEL. Total RNA was extracted from samples of adult tissues, oocytes, or developing eggs. The RT-PCR was carried out with primers Svh f1 and Expr2, to give an ϳ0.6-kb amplified fragment (arrowhead). Each amplification used cDNA prepared from an equal weight of oocytes or eggs, approximately equivalent to one egg (ϳ30 mg). Schistocerca genomic DNA, RNA from eggs at 50% development, and ovary RNA are templates in the negative control reactions. Schistocerca vasa plasmid DNA is the template in the positive control group.

mately 48 h. Fixed Drosophila embryos were kindly provided by F. produced from adult S. gregaria ovaries (Frenk Mora, 1993). 5Ј RACE Roch and R. Barrett. PCR was performed as described by Dawes et al. (1994), using total RNA extracted from ovaries of adult Schistocerca, with two reverse gene-specific primers ROP1 (5Ј-TTTCAGGTTCAGTCTTATTATC- Cloning of Schistocerca Vasa 3Ј) and RNP1 (5Ј-TCTTCCCTTCCACCTCTACCTC-3Ј) (Fig. 1D), Ј PCR cloning was performed from genomic DNA with degenerate and two forward adapter primers Adp (5 -AGAGAACTAGTGTC- Ј Ј primers designed to match the conserved motifs (Fig. 1D): AQTGS- GACGCGG-3 ) and dT17-Adp (5 -AGAGAACTAGTGTCGACGCG- Ј GKT (5Ј-CAGACGGGITCIGGIAA(A/G)AC-3Ј, deg-f primer) and GCCGC(T)17-3 . MLDMGF (5Ј-GA(A/G)AAICCCAT(A/G)TCIA(A/G)CAT-3Ј, deg-r Sequencing reactions were performed with T3/T7 primers and primer). Amplification parameters were: 95°C for 5 min, followed processed on an automated sequencer. Sequences were aligned by by 35 cycles of 94°C for 30 s, 40°C for 30 s, 72°C for 30 s, and finally, using MacVector 6.0 (Oxford Molecular Group) and checked by eye for 72°C for 5 min. PCR products were blunt cloned into plasmid ambiguities, particularly near indels. The molecular weight of Schis- pBluescript II KS (ϩ) (Stratagene). A 370-bp vasa DNA fragment was tocerca Vasa protein was estimated by using the program Statistical used as probe to screen Ͼ4 ϫ 10 5 phage colonies from a cDNA library Analysis of Protein Sequences (http://www.ebi.ac.uk/saps).

© 2002 Elsevier Science (USA). All rights reserved. Germ Line Development in S. gregaria 105

RT-PCR and Northern Blot Analysis because they cross-react with Vasa proteins of germ cells in various arthropods and other species (D. Stern, M. Grbic, C. Extavour, and Total RNA was extracted from adult Schistocerca tissues with an C-c.C., unpublished data). RNeasy Mini Kit (Qiagen) and from eggs with an RNAqueous-Midi Kit (Ambion). First-strand cDNA was synthesized with the gene- specific primer Expr2 (reverse, 5Ј-TCCTGTTCGGCCAATAC- Western Blot and in Situ Analysis Ј GATG-3 ; designed from the HRIGRTGR motif) and AMV reverse Approximately 30 ␮g samples of protein extracted from Schisto- transcriptase (Roche). Reverse transcription used the following condi- cerca adult tissues or eggs were run on 12% acrylamide gels and tions: total RNA was denatured at 65°C for 5 min, the Expr2 primer analyzed by using SuperSignal West Pico Chemiluminescent Sub- was added and annealed at 50°C for 10 min. The reaction was chilled strate (Pierce). Blots were incubated at 4°C overnight with formosa on ice, reverse transcriptase was added, and the reaction was incu- antibodies (diluted 1: 500) or anti-␣-tubulin antibody (Sigma; bated at 42°C for 1 h, then at 70°C for 15 min to stop the reaction. The 1:500). synthesized cDNA was used as template in a PCR with the forward S. gregaria embryos younger than 25% of development were Ј Ј primer Svhf1 (5 -CAGAGGTCGTGTGAACTTCCAG-3 , encoding immersed in fix (3.8% formaldehyde in PBS), pricked about 20 the RGRVNFQ motif) and the reverse primer Expr2. The reaction times through the chorion with a very fine needle (ϳ15 ␮m), conditions were: 95°C for 5 min, followed 40 cycles of 94°C for 30 s, avoiding the most posterior region where the embryonic primor- 55°C for 30 s, 72°C for 30 s, and finally, 72°C for 5 min. 32 dium forms, and left in formaldehyde at 4°C overnight. The next Northern blots were hybridized with P-labeled probe synthe- day, the chorion was peeled off the egg. Embryos older than 30% of Ј sized with the nef3 (forward, 5 -GCTACATTCCCTGAAGA- development were dissected from the yolk in PBS and then fixed at Ј Ј AATTC-3 , encoding the ATFPEEI motif) and ner2 (reverse, 5 - 4°C for 45 min. Ovaries and testes in adult Schistocerca were Ј GAGGCGATCACCATGAATAC-3 , designed from the SIHGDLR dissected in PBS and then fixed for 50 min at room temperature. motif). Probe synthesis and hybridization conditions were as de- Fine forceps were used to remove the ovary/testis sheath after scribed in Dearden et al. (2000). fixation. Antibody staining followed Dawes et al. (1994), except that fixed Production of Fusion Proteins tissues were treated with an Avidin/Biotin Blocking Kit (Vector Laboratories) to block endogenous biotin activity. Signals were A DNA fragment, amplified with primers nef1 and ner2 (Fig. 1D) developed with Vectastain Elite ABC Kit (Vector Laboratory). was cloned by using synthetic BamHI and HindIII sites into pRSET Staining of Drosophila embryos used the standard protocol de- A (Invitrogen), an expression vector encoding a 6-Histidine tag scribed in the Vectastain Kit manual. Formosa antibodies were upstream of the insert. This construct expresses a sequence that diluted 1:100 for all stainings. includes six of the eight conserved DEAD-box protein motifs Probes for in situ hybridization were made following the proto- and the two insect Vasa/AN3-specific motifs (Fig. 2). The same col of Dearden and Akam (2001). Probes were made from the entire methodology was used to produce a pRSET A-Drosophila vasa cloned Sgvasa gene and from two nonoverlapping fragments of this expression construct. Two primers, Drosophila nef1 (forward, sequence. All probes produced identical staining patterns. Images 5Ј-tgtgcccagacgggttctgg-3Ј, encoding the CAQTGSG motif) and in Figs. 4 and 5 were produced from probes made from the Drosophila ner1 (reverse, 5Ј-ATCCCATTGCTCTTCCTCCTC- full-length clone. GAC-3Ј, designed from the VEEEEQWD motif), were used to amplify the Drosophila vasa insert. The template was a pQE31 full-length Drosophila vasa plasmid, a gift from P. Lasko (Lasko RESULTS and Ashburner, 1988; Liang et al., 1994). In addition to the two insect Vasa/AN3-specific motifs, the coding region of the pRSET Isolation of a Vasa Homologue from Schistocerca A-Drosophila vasa construct includes all of the eight conserved DEAD-box motifs. We designed degenerate primers to amplify a fragment of His-tagged fusion proteins were induced according to protocols the vasa gene flanked by motifs that are conserved in the in the QIAexpressionist kit (Qiagen; http://www.qiagen.com/ Drosophila, Xenopus, and mouse vasa sequences (Fujiwara literature). Because the expressed Schistocerca and Drosophila et al., 1994; Hay et al., 1988; Komiya et al., 1994; Lasko and Vasa proteins are highly insoluble, 6 M guanidine hydrochloride Ashburner, 1988). These primers amplify a fragment, ap- was used to extract the target fusion proteins. These were then proximately 400 bp, from both Drosophila and Schistocerca affinity purified with Ni-NTA Superflow resin (Qiagen). genomic DNA (Fig. 1A). The Schistocerca PCR products were cloned and 15 Antibody Production and Purification inserts of the correct size were sequenced. These repre- sented 6 different kinds of DEAD-box genes: putative vasa Affinity purified His-tagged Schistocerca and Drosophila Vasa homologues (2 identical clones); clones similar to the Xe- proteins in homogenized polyacrylamide gel slices were used to nopus AN3 gene (3 very similar clones) (Gururajan et al., immunize two rabbits (Eurogentech; http://www.eurogentec.be/). 1991); and clones similar to the Drosophila RM62 helicase Four injections of Schistocerca protein were followed by a single injection of Drosophila protein (100 ␮g protein/injection). Antisera (10 clones, falling into 4 distinct sequence classes) (Dorer et from the final bleed were affinity purified against the His-tagged al., 1990). Schistocerca Vasa protein by using the protocol of Harlow and Lane We used the PCR cloned Schistocerca vasa fragment as a (1988). We refer to the affinity purified antibodies from the two probe to screen a Schistocerca adult ovary cDNA library rabbits as formosa 1 and formosa 2 (formosa ϭ for most Vasa, (Dearden et al., 2000). Seven positive clones picked for

© 2002 Elsevier Science (USA). All rights reserved. 106 Chang, Dearden, and Akam sequencing all contained the same 1.9-kb cDNA sequence. hybridized in parallel give no indication that this signal is 5Ј RACE PCR was used to extend the 5Ј sequence of this an artifact (Fig. 5C). As one of the antisense probes is gene by a further 252 bp. A Northern blot shows that the derived from a region of the transcript that contains no length of the homologous Schistocerca transcript is about conserved motifs, we think it unlikely that it represents 2.2 kb (Fig. 1C), suggesting that the 5Ј end of our RACE hybridization to DEAD-box genes other than those closely product lies close to the start of this transcript (Fig. 1B), but related to vasa. the sequence contains no in-frame start codon. Our Schistocerca vasa clones encode a 1764-bp open ϳ Ј Schistocerca Vasa Protein Is a Germ Cell Marker reading frame followed by 400 bp of the 3 -untranslated in Late Embryonic and Adult Gonads region (Fig. 1B). BLASTP and BLASTN searches show that this open reading frame is most similar to other insect vasa In several species, vasa RNA and protein are initially genes. Figure 2 shows an alignment of the encoded 588- expressed more widely before being localized to the germ- amino-acid sequence with the Vasa proteins of Drosophila line (Ikenishi, 1998; Knaut et al., 2000; Raz, 2000). To (Hay et al., 1988; Lasko and Ashburner, 1988) and Bombyx determine whether the distribution of Vasa protein is (Nakao, 1999), and with Drosophila eIF4-A, a non-Vasa similar to that of the RNA, we generated antibodies against DEAD-box helicase (Dorn et al., 1993). The eight conserved the Schistocerca Vasa protein. These were prepared by motifs found in other animal DEAD-box proteins, and the immunization first with a fragment of Schistocerca Vasa RGG repeat at the N-terminal region, are all found in the and then with a final immunization using Drosophila Vasa, Schistocerca Vasa protein. An ARKF motif, found in Vasa, in the hope that this would specifically boost the response AN3, and PL10 proteins, but not in other DEAD-box to conserved epitopes (see Materials and Methods for fur- helicases, is also found from residues 264 to 267 in the ther details). Schistocerca protein. Two additional motifs are conserved After affinity purification against the Schistocerca Vasa only among the insect Vasa proteins—a GXVGGA motif protein, the antibodies from two rabbits specifically label a (residues 384–389) and an EXEEXW motif at the C-terminal single band of approximately 66 kDa present in Western end (Fig. 2). Based on these conserved sequence features, we blots of total protein extracted from Schistocerca adult conclude that we have cloned a S. gregaria vasa orthologue ovaries and testes (Fig. 6A). This is very close to the size (Sg vasa). predicted for Schistocerca Vasa protein (64.4 kDa for the 588 amino acids cloned). No signal is obtained with similar extracts from whole heads, thoracic muscle, or Malpighian Sg Vasa RNA Is Expressed Specifically in Gonads, tubules. This band is also detected in total protein from but Widely in Embryonic Tissues heart-stage (15%), 27%, and 35% eggs (Fig. 6B). Very weak We used RT-PCR to analyze expression of the Schisto- expression is detected in late oocytes and unlaid eggs (Fig. cerca vasa gene at different stages of embryogenesis and in 6B). the dissected tissues of adults. Schistocerca vasa mRNA is In Drosophila embryos, one of these two antibodies expressed in the ovary and testis, from which the germ cells (formosa 2 antibody) stains specifically the pole cells and originate, but not in a number of other adult tissues (Fig. embryonic germ cells in the gonads (Figs. 7E–7H), although 3A). It is expressed in late oocytes but is not detectable in there is some background staining of other tissues; formosa RNA from unlaid eggs (Fig. 3B) or during very early cleavage 1 antibody has a high background in Drosophila. Moreover, (Ͻ12 h post lay) (Fig. 3C). However, it is detectable again in these two antibodies can specifically identify primordial RNA from eggs at 20 h and in all later stages (Fig. 3C). We germ cells in the pea aphid Acyrthosiphon pisum and the infer that zygotic transcription of Schistocerca vasa begins parasitic wasp Macrocentrus grandii (D. Stern and M. between 12 and 20 h. Grbic, personal communication). We conclude that the In situ hybridization shows that, in oocytes, vasa RNA is antibodies are largely specific for Vasa proteins and do not localized to the cytoplasm of germline cells in the distal cross-react with most widely distributed DEAD-box pro- part of the germarium and of young oocytes in the vitel- teins. We used one of these antibodies (formosa 1) for most larium. Signal intensity decreases as the oocytes grow (Figs. experiments reported here, but similar results were ob- 4A and 4B). In gonads of embryos slightly older than 70% of tained with both. development, a vasa signal is specifically detected in germ In the ovary of adult Schistocerca, this antibody stains cells (Figs. 4C and 4D). the cytoplasm of germline cells in the germarium and the Schistocerca vasa RNA is widely expressed in younger cytoplasm of oocytes in the vitellarium (Figs. 7A and 7B). embryos. Probes from the full-length Sgvasa cDNA clone The staining of the oocytes decreases as they mature (Fig. and from two nonoverlapping fragments were prepared and 7B), in very much the same pattern as seen for vasa RNA hybridized in situ to RNAs that are widely distributed (Fig. 4A). The somatic cells (terminal filament and follicle within the embryo (Fig. 5; and data not shown). Slightly cells) are unstained (Fig. 7A). higher signals are seen in the mesoderm, particularly of the In the testis of adult Schistocerca, the antibody stains abdominal segments (Fig. 5). Sense strand control probes intensely the cytoplasm of germ cells at the apex of the

© 2002 Elsevier Science (USA). All rights reserved. Germ Line Development in S. gregaria 107

FIG. 4. Expression of Schistocerca vasa mRNA in adult ovarioles and in embryonic gonads. (A) A Schistocerca adult ovariole follicle hybridized with anti-sense Sgvasa mRNA probes. Intensity of in situ signals decreases in the growing oocytes and finally becomes almost undetectable. (B) Magnification of the inset in (A). Tf, terminal filament; G, germarium; V, vitellarium. In the germarium, in situ signals (red arrowhead) are only found in the germline stem cells that are developing into oocytes. In the vitellarium, signals are concentrated in the cytoplasm of oocytes (red arrow). Follicle cells (green arrow) are devoid of staining. Oocyte nuclei, black arrow. (C) An embryonic gonad hybridized with anti-sense Sgvasa mRNA probes. This gonad, dissected from a Schistocerca embryo slightly after 70% of development, extended from abdominal segments 3–6 at the dorsal side of the abdomen (shown in D). In situ signals (arrow) were identified in the cytoplasm of embryonic germ cells. Anterior is on the upper left; the view is dorsal. Scale bars, 100 ␮m. The reiterated staining in the lateral margins of the segments visible in (D) is probably artefactual binding to developing tracheae.

© 2002 Elsevier Science (USA). All rights reserved. 108 Chang, Dearden, and Akam

testicular follicle (Fig. 7C). More distal cells stain much more weakly (Fig. 7D). Again, the somatic cells of the follicular sheath are unstained (Fig. 7C). Ovary and testis can be distinguished in Schistocerca embryos at 95% development. At this stage, the nymph is virtually fully formed, with pigmented cuticle, eyes, and bristles. The gonads lie along the midline of the abdomen, dorsal to the gut and ventral to the heart, embedded in a thin sheet of fat body (Roonwal, 1937) (Figs. 8A and 8D). The germ cells are conspicuously large and are becoming organized into follicles, the shape and distribution of which distinguish the ovary from the testis (Nelsen, 1934a) (Figs. 8C and 8E). Our anti-Vasa antibodies stain an asymmetric crescent of cytoplasm in the germ cells of both ovaries and testes (Figs. 8C and 8E). In 70% embryos, Vasa-positive cells form two rows of large cells, one on either side of the midline, embedded within the sheet of visceral mesoderm that will form the fat body and gonad. They extend from approximately abdomi- nal segment 3 (A3) to A6. Staining is cytoplasmic and often asymmetrically localized, much as in the gonads of older embryos (Fig. 8F). No sign of the follicular organization of the gonad is yet apparent, but because of the clear similari- ties between these staining cells and those of the gonads in later embryos, we infer that they are the primordial germ cells.

Vasa Protein Is Expressed in Most Cleavage Energids, but Becomes Localized to the Serosa and a Subset of Marginal Embryonic Cells after Gastrulation The early development of the Schistocerca gonad is not well described, and the time of segregation of the germ cells is contentious (Nelsen, 1934a; Roonwal, 1937). Knowing that Vasa marks the germline at late embryonic stages, we attempted to trace the origins of these cells by staining younger embryos with our anti-Vasa antibodies. However, given that vasa RNA appears to be widely expressed in early embryos (Fig. 5), we cannot assume that Vasa protein will be expressed exclusively in the germ cell lineage. Newly laid eggs contain no detectable localized Vasa protein (Fig. 9A), though a trace of Vasa protein is detected on Western blots of unlaid eggs (Fig. 6B). We suspect that some Vasa protein is uniformly distributed in the thin layer of superficial cytoplasm that surrounds the egg or is buried in islands of cytoplasm deep within the yolk. We know that these deep islands are not efficiently stained in whole- mount preparations. However, when cleavage energids FIG. 5. Expression of vasa mRNA in Schistocerca embryos. (A, B) (nuclei with surrounding cytoplasm) emerge at the sur- Schistocerca embryos at 25 and 30% of development hybridized with anti-sense Sgvasa mRNA probes. The whole embryo stains. Signals are enriched in the segmental mesoderm (green arrowhead). (C) 30% embryo hybridized with corresponding sense probe as control. No signal is detected. (D) Magnification of the A2–A5 region from the 30% embryo shown in (B). Only faint signals are and 30% panel). Anterior is up; focal plane is on the dorsal side. detected at the dorsal margin of the germ band (red arrow), the Scale bars, 100 ␮m. M1, mandibular segment; T1, first thoracic expected location of potential germ cells. (Compare Figs. 10F, 11 segment; A1, A2, and A5, abdominal segments.

© 2002 Elsevier Science (USA). All rights reserved. Germ Line Development in S. gregaria 109

FIG. 6. Expression of Schistocerca Vasa protein in adult tissues, unlaid eggs, and eggs during embryogenesis. (A) Western blot of adult Schistocerca tissue proteins probed with formosa 1 antibody. Schistocerca Vasa protein (ϳ66 kDa) is specifically detected in the testis and ovary (arrowhead). Total protein in each lane is about 30 ␮g. The lower panel shows a parallel blot probed with mouse anti-␣-tubulin antibody. ␣-Tubulin (55 kDa, arrow) is detected in every tissue except thoracic muscle. (Protein stains (not shown) confirm that, despite the absence of tubulin staining, the thoracic muscle extract is undegraded, though dominated by a few abundant muscle proteins.) (B) Western blot of total egg protein extracts probed with formosa 1 antibody. Schistocerca Vasa protein (ϳ66 kDa) is clearly identified in eggs at heart stage (15%), 27%, and 35% of development (arrowhead). A very faint signal was detected in late oocytes and unlaid eggs (hollow arrowhead). Adult ovary protein was used as the positive control. Total protein in each lane is about 8 ␮g. Except for dissected late oocytes and adult ovaries, in whole-egg extracts, the Vasa protein band is distorted by the bulk of yolk protein running at about 80 kDa.

face of the egg, these do not stain (Figs. 9A and B), 9F). Because RT-PCR first detects vasa transcripts in eggs suggesting that the energids do not bring Vasa protein to around 20 h, we suspect that this Vasa protein is being the surface with them. synthesized from newly transcribed RNA in these cells. Vasa protein appears in these superficial energids a few Between 15 and 30 h, the remainder of the egg cortex is hours after they reach the egg surface, and probably at populated by energids that have migrated forward, deep about the same time that these energids form closed cells within the egg, before moving to the surface and rapidly (Ho et al., 1997). Stained energids are first visible near the forming cells (Ho et al., 1997). At any one time, the most posterior pole, at about 15 h after egg laying (AEL) (Fig. anterior of these surface energids are Vasa-negative, but

© 2002 Elsevier Science (USA). All rights reserved. FIG. 7. Schistocerca adult ovaries and testes, and Drosophila embryos, stained with formosa antibodies. (A, B) Schistocerca ovariole stained with formosa 1 antibody. Tf, terminal filament; G, germarium; V, vitellarium. (A) is a magnification of the inset in (B). Germline stem cells in the germarium are weakly stained (arrowhead). Early developing oocytes are more strongly stained (red arrow). Intensity of staining decreases as oocytes mature. Follicle cells (green arrow) are devoid of staining. Black arrows, oocyte nuclei. (C, D) Schistocerca testicular follicles stained with formosa 1 antibody. (C) is the magnification of the inset in (D). Staining is present in the cytoplasm of germline stem cells at the apex of the follicle (arrowhead). Follicle cells (green arrow) are devoid of staining. (E–H) Drosophila embryos stained with formosa 2 antibody. (E, F) Stage 5 embryo; (G, H) Stage 12 embryo. (E) and (G) are enlargements of the germ cells in (F) and (H), respectively. Anterior is to the left. Scale bars, 100 ␮m (B, D, F, H,); 10 ␮m (A, C). FIG. 8. Schistocerca embryonic gonads at 95 and 70% of development stained with formosa 2 antibody. (A, C) Gonad in a 95% embryo. The pattern of developing follicles identifies this as a developing ovary in a female (Nelson, 1934a). (B) Schematic drawing of the inset in (A), indicating the relative positions of embryonic germ cells (GC) and other cells that will from the somatic parts of the ovariole (SC, somatic cells; B, boundary of primordial ovarioles; ML, midline of gonad). (C) Magnification of the inset in (A). The cytoplasm of germ cells is specifically stained (red arrow), often as a crescent around the nucleus. Nuclei of germ cells (black arrow) and somatic cells (green arrow) are unstained. (D, E) Gonad embedded in fat body in a 95% embryo. The pattern of developing follicles identifies this as a developing testis in a male (Nelson, 1934a). (E) is a magnification of the inset in (D). The cytoplasm of primordial germ cells is stained primarily toward the center of each follicle (red arrow). Somatic cells (green arrow) and fat body (blue arrow) are unstained. (F) Gonad dissected from a Schistocerca embryo at 70% of development. The large germ cells are embedded within the dorsal sheet of visceral mesoderm. This is located ventral to the heart and dorsal to the gut (see diagram in G) and extends from abdominal segments 3 to 6. Staining is localized to the cytoplasm of germ cells, often concentrated at one side of the cell (red arrow). Developing fat body cells are indicated with blue arrows. Somatic cells of the gonad are not obvious at this stage. Dorsal is uppermost. Scale bars, 100 ␮m (A, D, F); 10 ␮m (C, E). All of these gonads are stained with formosa 2 antibody. Gonads stained with formosa 1 antibody show similar results (not shown). 112 Chang, Dearden, and Akam

© 2002 Elsevier Science (USA). All rights reserved. Germ Line Development in S. gregaria 113 behind these, all superficial cells accumulate Vasa protein and may represent the presence of low levels of Vasa (Figs. 9C–9E). Thus, it seems that the expression of Vasa protein, correlating with the RNA in situ pattern (Fig. 5), protein initiates de novo in each cell shortly after it reaches and/or of some other closely related DEAD box protein, the egg surface; it is not the proliferation of the first which is expressed widely at this stage of development. Vasa-positive energids at the posterior pole that populate As the lateral margins of the embryo fold dorsally, from the egg surface (Fig. 9F). Once they reach the surface, some 25% development onwards, these lateral staining cells are of the Vasa-positive cells migrate back toward the posterior brought toward the midline. In 35% embryos, Vasa-positive pole, where they aggregate to form the embryonic primor- cells are identified slightly disassociated from the outer dium (Ho et al., 1997). layer of the germ band, close to the dorsolateral walls of The majority of energids throughout the egg stain with abdominal segments A2 to A9 (Figs. 10H and 10I). At 40 and anti-Vasa antibody, including both those that will form the 45%, they occupy a similar position, but somewhat deeper embryo and those that will form the serosa (an extraembry- in the body cavity (data not shown), at the position where onic membrane that rapidly becomes polyploid and secretes Roonwal and Nelson both identified germ cells. We believe a cuticle around the egg) (Figs. 9G–9J). However, a sparse that these cells become the primordial germ cells, because population of nuclei that remain just below the surface they already display the asymmetric localization of Vasa remain unstained (Fig. 9J). These are probably the nuclei of protein characteristic of the primordial germ cells in 70% syncytial energids that remain connected to the yolk (Ho et embryos (Fig. 10I cf. Fig. 8F). We have not been able to al., 1997). follow the further development of the gonads between 45 Serosal cells continue to stain strongly as they differen- and 70% in whole mounts, because secretion of the first tiate, but most of the cells that aggregate to form the embryonic cuticle begins at about 45%, but Roonwal and embryonic primordium rapidly cease to stain; by 48 h Nelson were able to follow these stages of gonad develop- (corresponding to the 15% stage of development of Bentley), ment in sectioned material (Nelsen, 1934b; Roonwal, 1937). the embryonic primordium appears to be virtually un- stained when incubated with antibody in situ,onthe surface of the egg (Figs. 9I and 9J). However, if the embryo is DISCUSSION dissected free from the serosa and yolk before antibody treatment, cells around the internal margin of the embryo In newly laid eggs of Drosophila, C. elegans, and ze- stain preferentially (Fig. 9K). brafish, vasa RNA and/or protein are localized in the germ Between 15 and 20% of development, the marginal cells granules, components of the germ plasm that will later be of the embryo continue to stain preferentially with the recruited into germ cells (Eddy, 1975; Saffman and Lasko, anti-Vasa antibody (Figs. 9M and 10A and 10C). From 25 to 1999). Vasa is subsequently expressed in the germline 30% of development, these strongly Vasa-positive cells are throughout much of development. In these and other spe- restricted to the abdominal margins, though lower levels of cies, vasa has proven to be an excellent germline marker. staining are seen throughout the embryo (Figs. 10D–10G). We have cloned an orthologue of vasa from Schistocerca, This generalized staining is antibody-specific (most other and find that Vasa protein is expressed specifically in antibodies give little background staining in 30% embryos) germline cells of late embryos and adults. It provides a

FIG. 9. Schistocerca eggs and embryos from 0 to 17% of development stained with formosa 1 antibody. (A) Eggs from 0 to 15% of development, showing the extent of energid migration and Vasa staining. Eggs staged by hours after egg laying (h AEL); 48 h AEL ϭ 15% stage of development. Posterior (P) is to the left. Black arrowheads indicate the most anterior point at which cleavage energids have appeared at the surface of the egg; red arrowheads under each egg indicate the anterior limit of Vasa-stained cells/energids. Staining is first detected in energids at the posterior pole of the 15 h AEL egg (small red arrow; the posterior end of this egg has been detached and rotated to show the posterior pole; also see F). At 36 h AEL, both stained and unstained energids/cells are present at the anterior pole of the egg. (B–E) Higher magnification of eggs at 12–18 h AEL. Posterior is up. The nuclei of energids that do not stain for Vasa are visible by DAPI fluorescence (B, C, E; blue spots indicated with green arrows). Fluorescence is quenched in stained energids (red arrows). (C) 15h AEL. The few Vasa-positive energids lie close to the posterior pole. (D) 18 h AEL. Most energids are Vasa-positive (red arrow). (E) Enlargement of the box in (D), showing that the most anterior energids remain Vasa-negative. (F–H) Polar views of eggs and forming embryos. (F) Stained energids (red arrow) at the posterior pole in the 15 h AEL embryo. (G, H) Vasa-positive cells in the forming embryonic primordium (36 h AEL) and at gastrulation (42 h AEL). Note gastrulation furrow in (H). (I, J) Embryonic primordium in the 15%/48 h AEL egg. Lateral view. Staining in the embryo is now much weaker. Many cells of the embryonic primordium are unstained. Outside the embryo, there is a population of large heavily stained cells (red arrow, probably the forming serosa, which is just beginning to fold over the embryo) interspersed with a population of unstained nuclei which may be yolk syncytial nuclei (green arrow). (K–M) Embryos slightly older than those in (I, J), dissected free from the surface of the yolk before staining, and viewed from the inner (dorsal) surface. (K, M) Stained with formosa 1 antibody; (L) Control without primary antibody. The antibody stains the margin of the dissected embryo (red arrows), in a region that is unstained in whole mounts. Note that staining is absent in regions where the serosa and yolk remain attached (open arrowheads); Vasa-positive cells in the serosa are marked with a red arrowhead in (M). Anterior is toward the top. Scale bars: 1 mm (A); 100 ␮m(B–I, M); 50 ␮m(J–L).

© 2002 Elsevier Science (USA). All rights reserved. 114 Chang, Dearden, and Akam valuable marker for following germline development from begin to accumulate detectable Vasa protein (Fig. 9C). We 25% embryogenesis onwards. However, its expression is think it likely that this is zygotically synthesized protein, not specific to the germline during early embryogenesis. though it is possible that some uniformly distributed ma- Vasa RNA is not maternally transmitted, and there is no ternal protein at the cortex concentrates in energids as they evidence that vasa protein is maternally transmitted as part reach the surface. of a localized germ cell determinant. Subsequently, Sgvasa appears to be widely transcribed throughout the first 30% of embryogenesis. Our antibody Oogenesis suggests that Vasa protein accumulates to high levels in extraembryonic cells, and in all early embryonic cells, but Schistocerca vasa RNA and protein are both expressed at that protein levels in the embryo fall shortly after gastrula- high levels during the early stages of oogenesis, with levels tion in all but a small population of cells around the falling rapidly as the oocyte matures. RNA and protein margins of the embryo. By 25% development, strongly expression match closely. This pattern is very similar to staining cells persist only around the margin of the abdo- that seen in the ovary of Drosophila, except that in Dro- men. sophila, vasa is transcribed from nurse cell nuclei, whereas This protein pattern is not obviously mirrored by the in Schistocerca, there are no nurse cells, and expression is results of RNA in situ hybridization, which suggest that presumably from the oocyte nucleus itself. In Drosophila, vasa RNA is more widely distributed, and not particularly vasa function is needed for germline development during concentrated in these marginal cells. However, Western early stages of oogenesis (Lasko and Ashburner, 1988). This blots of protein extracted from the same stage (Fig. 6) give role may be conserved in Schistocerca. no indication that the antibody is cross-reacting with some One minor discrepancy between RNA and protein pat- other DEAD-box protein that is abundant only at these terns is apparent in the most apical cells in the germarium, stages, so it is likely that either translational control or which stain for protein, but not detectably for RNA. This differences in protein stability account for the specific may simply reflect the different sensitivity of the two accumulation of Vasa protein in the marginal cells. Post- techniques, but it is also possible that some other cross- transcriptional control of Vasa accumulation has been reacting DEAD box protein is expressed in these germ line documented in zebrafish (Wolke et al., 2002). stem cells. In later embryonic stages, both Sgvasa RNA and protein In Drosophila, Vasa has a distinct role during later stages specifically accumulate in the presumptive germ cells of of oogenesis. Some fraction of the Vasa protein present the embryonic gonads, providing a good marker for the during oogenesis persists into the fertilized egg, where it organization of follicles in both ovaries and testes. In adults, localizes to the polar granules as one component of the RT-PCR and Western blots again confirm the gonad- germline determinant. This process depends on the prior specific accumulation that is seen by antibody staining. localization of oskar RNA and a number of other factors (Lasko, 1999). Vasa is required for the translation of oskar, The Specification of Germ Cells in Schistocerca gurken, and nanos RNA, and probably also of other genes in the germline (Markussen et al., 1995; Rongo et al., 1997). In Previous studies of the germline in Orthoptera identified the absence of this localized Vasa protein, no pole cells or germ cells by cytological criteria (Nelsen, 1934b, in Mela- germline form in the resultant embryo. noplus differentialis; Roonwal, 1937, in Locusta migrato- We can detect no vasa RNA in mature Schistocerca eggs. ria). At about 40% development, Roonwal described them We can detect a trace of cross-reacting protein in these eggs as cells “larger and paler . . . than those of the surrounding on Western blots, but have no evidence that this is localized mesoderm,” lying “in the median walls of the dorsal pouch to any particular part of the egg. It is not localized at the egg of the coelomic cavities of the second to the fifth abdominal cortex, but current techniques do not allow us to stain segments.” The description, location, and subsequent be- efficiently the cytoplasm that is buried deep within these havior of these cells suggest that they were describing the large yolky eggs. However, no Vasa protein appears to be population of Vasa-positive cells that we identify. Accord- carried to the surface by cleavage energids as they bring this ing to their descriptions, these cells migrate with the dorsal cytoplasm to the surface of the egg. We therefore think it mesoderm and are brought near to the dorsal midline of the unlikely that Vasa serves as a maternally transmitted embryo during dorsal closure (Fig. 11). We have not fol- germline determinant. lowed gonad development between 45 and 70% embryogen- esis, but our observations before 45% and later of the forming gonad are consistent with their descriptions. Embryogenesis Nelson thought that he could identify germ cells earlier RT-PCR shows that transcription of Schistocerca vasa than this, as large cells with weakly staining chromatin initiates very early in embryogenesis, at a stage where there located at the dorsolateral margins of the germ band during are only scattered cleavage energids (12–20 h AEL). Shortly early stages of abdomen segmentation (corresponding to after reaching the egg cortex (from 15 h AEL), these energids about 25% development in Schistocerca). From here, he

© 2002 Elsevier Science (USA). All rights reserved. Germ Line Development in S. gregaria 115

FIG. 10. Schistocerca embryos from 20 to 35% of development stained with formosa 1 antibody. Developmental stages: (A–C) 20%; (D, E) 25%; (F, G) 30%; (H, I) 35% (flattened embryo). (C), (E), (G), and (I) are enlargements of the insets in (A), (D), (F), and (H), respectively. All embryos are dorsal views, with anterior up. (A, C) 20% embryo. The antibody preferentially stains the margin of the embryo (arrows). (B) Without primary antibody, no staining is detected. (D–I) 25% to 35% of development. Strong staining at the dorsolateral margin of the abdomen (D, E) resolves into a population of individual large cells with stained cytoplasm (F–I). We identify these Vasa-positive cells as primordial germ cells (arrows). These are carried toward the midline as the embryo folds dorsally (F) and then appear to migrate internally (H). Low levels of background staining are present in most other tissues. This appears dense in the thickened folds of the head and limb buds (arrowheads). Scale bars, 100 ␮m (A, B, D, F, H); 10 ␮m (C, E, G, I).

believed that the cells migrated to the dorsal mesoderm. do these cells originate, and when are they specified as Roonwal disputed this identification, asserting that these germline cells? We do not have firm answers to either of cells were cardioblasts, and that germ cells arose de novo in these questions, but the specific accumulation of Vasa the mesoderm. However, Nelson’s description of an earlier protein suggests that the marginal cells may be specified as segregation is consistent with the Vasa staining cells that a distinct cell population as early as the late heart-stage we observe at the margins of the germ band. We think it embryo, around the time that the embryo with its amnion likely that these marginal cells become the future germline. is beginning to detach from the serosa. The primordial germ Assuming this to be the case, two questions arise: where cells may originate either by the segregation of early speci-

© 2002 Elsevier Science (USA). All rights reserved. 116 Chang, Dearden, and Akam

FIG. 11. Diagram summarizing Vasa expression in the Schistocerca embryo, as revealed by formosa antibodies. From 20% development onwards, strongly Vasa-positive cells can be followed from the outer margin of the germ band into the forming dorsal mesoderm (30–35%, red), and thence into the gonads (70%). In earlier stages, all cells stain with our antibodies, though preferential staining of the outer margin of the germ bands arises at or soon after gastrulation. Diagrams below embryos from 25 to 70% indicate the position of Vasa-positive cells (red) in cross-section at the level of A2 (scissors).

fied energids, or by inductive signaling that takes place Johnston’s Labs for protein chemistry expertise; Paul Lasko for during or shortly after the formation of the embryonic Drosophila vasa cDNA; John Rodford for the diagrams; Chuck primordium. There is no shortage of possible sources for Cook, Marion Rozowski, and Cassandra Extavour for comments on such signals; for example, we know that dpp (an insect the manuscript. This work was supported by the Wellcome Trust, Bmp4 homologue) is expressed in a ring surrounding the and by a scholarship to C-c.C. from the Ministry of Education and the Academia Sinica, Taiwan, ROC. margin of the embryo at or shortly before the time that we see localization of Vasa protein accumulation (Dearden and Akam, 2001). REFERENCES Distinguishing between these two models for germ line specification remains an important objective, because if Anderson, D. T. (1972). The development of hemimetabolous signal induction proves to be the case, this would refute the insects. In “Developmental Systems: Insects,” Vol. 1 (S. J. commonly held assumption that the Drosophila germ Counce and C. H. Waddington, Eds.), pp. 96–165. Academic plasm-driven mode is universal among insects. At present, Press, London. the best we can say is that we have no evidence for the Bentley, D., Keshishian, H., Shankland, M., and Toroian-Raymond, existence of early specified energids. If early specified en- A. (1979). Quantitative staging of embryonic development of the ergids are involved, either they do not preferentially accu- grasshopper, Schistocerca nitens. J. Embryol. Exp. Morphol. 54, mulate Vasa protein or they remain deep within the egg 47–74. Braat, A. K., Zandbergen, T., van de Water, S., Goos, H. J., and until gastrulation. Zivkovic, D. (1999). Characterization of zebrafish primordial germ cells: Morphology and early distribution of vasa RNA. Dev. Dyn. 216, 153–167. ACKNOWLEDGMENTS Carre´, D., Djediat, C., and Sardet, C. (2002). Formation of a large Vasa-positive germ granule and its inheritance by germ cells in We thank Chuck Cook for assistance with PCR methodologies; the enigmatic Chaetognaths. Development 129, 661–670. Nan Hu for assistance with Drosophila staining; colleagues in the Carrera, P., Johnstone, O., Nakamura, A., Casanova, J., Ja¨ckle, H., Akam Lab (including Jim and Lynn) for advice and assistance with and Lasko, P. (2000). VASA mediates translation through inter- embryological methods; members of Joardan Raff and Daniel St. action with a Drosophila yIF2 homolog Mol. Cell 5, 181–187.

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