Germ cell development in the Honeybee (Apis mellifera); Vasa and Nanosexpression
© Dearden; licensee BioMed Central Ltd. 2006
Received: 03 October 2005
Accepted: 17 February 2006
Published: 17 February 2006
Studies of specification of germ-cells in insect embryos has indicated that in many taxa the germ cells form early in development, and their formation is associated with pole plasm, germ plasm or an organelle called the oosome. None of these morphological features associated with germ cell formation have been identified in the Honeybee Apis mellifera. In this study I report the cloning and expression analysis of Honeybee homologues of vasa and nanos, germ cell markers in insects and other animals.
Apis vasa and nanos RNAs are present in early honeybee embryos, but the RNAs clear rapidly, without any cells expressing these germ cell markers past stage 2. These genes are then only expressed in a line of cells in the abdomen from stage 9 onwards. These cells are the developing germ cells that are moved dorsally by dorsal closure and are placed in the genital ridge.
This study of the expression of germ cell markers in the honeybee implies that in this species either germ cells are formed by an inductive event, late in embryogenesis, or they are formed early in development in the absence of vasa and nanos expression. This contrasts with germ cell development in other members of the Hymenoptera, Diptera and Lepidoptera.
The formation and placement of primordial germ cells (PGCs) in animal embryos is of interest in developmental biology, evolutionary biology and biotechnology. In Diptera, including the well studied Drosophila melanogaster, PGCs form as morphologically and molecularly distinct pole cells, in the posterior of the embryo, early in development [1, 2]. In insects outside the Diptera PGCs can develop in regions of pole or germ plasm, a distinct form of cytoplasm in early eggs , or are associated with the oosome [4–6] a specialised subcellular structure that appears to define PGC fate early in embryogenesis. These specialised structures and/or cytoplasm are widely distributed amongst insects but are not found in all insect embryos [1, 7].
In the honeybee (Apis mellifera), little is known about the formation of PGCs in the embryo. In late embryonic and larval stages PGCs have been identified in the genital ridge situated near the dorsal surface of the abdomen where the gonads develop . Previous authors have stated that there is no evidence for PGCs arising early in development  as is seen in other insects.
In other insects PGC placement and development has been studied using molecular markers of germ cell fate [7, 9, 10]. Two genes, vasa and nanos, have proved useful markers for PGCs in a broad range of species.
The vasa gene encodes a DEAD box RNA helicase that is expressed in the PGCs of all major groups of animals examined [11–17]. In mice, vasa related genes are expressed in PGCs and are required for PGC development . In Drosophila vasa is required for multiple processes in the development and maintenance of PGCs (reviewed in ) including the localisation of nanos RNA .
Nanos genes encode zinc finger transcription factors that have been shown to be expressed in the PGCs of Diptera [21–23], Caenorhabditis elegans , Cnidarians [25, 26], Leech [27, 28], mice  and humans . In Drosophila nanos is required for PGC migration and fate  and acts to repress somatic cell fate in PGCs by repressing differentiation [31, 32]. In Zebrafish, nanos is required for PGC migration and survival . Mice have three nanos-like genes, two of which are expressed in PGCs and required for their maintenance .
Vasa expression, assayed with in-situ hybridisation or antibodies, has been used most generally as a PGCs marker in arthropod embryos. Using both techniques, PGC specification has been studied in the orthopteran Schistocerca gregaria. In this species PGCs form at the dorsal boundaries of the germband in the abdomen and migrate dorsally to the gonads . Vasa expression has also been examined in the lepidopteran Bombyx mori . In this species, vasa RNA is first expressed in the presumptive embryo and then comes to be located in cells at the posterior of the germ band. In later stages these vasa positive cells populate the abdomen in regions consistent with the forming gonads. In non insect arthropods, vasa expression has also been examined in the two-spotted spider mite (Tetranychus urticae) , and the crustaceans Daphnia  and Parhayle . In Tetranychus, vasa RNA marks a population of cells underlying the cellular blastoderm that migrate into the posterior of the embryo and are incorporated into the gonad. In Daphnia, vasa protein is localised in a subcellular compartment in 8 cell stage embryos and is partitioned into a single blastomere at the 16-cell stage. This vasa positive cell is the progenitor for the germ line in this species . In Parhayle, vasa protein expression becomes apparent at the 8-cell stage in the g-micromere. Manipulation of the embryo, however, indicates that localised determinants for PGC development exist at the 2 cell stage .
In Hymenoptera, the insect order containing the honeybee, vasa expression has been examined only in the polyembryonic parasitic wasp Copidosoma floridanum. In this species, an organelle, the oosome, stains for vasa RNA and is segregated into cells that then express vasa RNA and protein and form PGCs. Inheritance of PGCs appears to regulate both larval caste and axis formation [10, 34]. Oosomes that specify germ-cell fate are found in many hymenopteran species [4, 6] but are not present in Honeybee ovaries or embryos .
Nanos genes have a role in specifying posterior regions of insect embryos [22, 23, 36–38]. In Drosophila, nanos RNA is localised to posterior regions of the embryo, and nanos translation is repressed in the rest of the embryo by the binding of smaug protein to a RNA secondary structure in the 3'UTR of the nanos mRNA . Nanos protein then regulates Hunchback (Hb) translation by recruiting a cofactor, pumilio, that binds a 'nanos response element' in the 3'UTR of the Hunchback mRNA, thus restricting Hb protein expression to the anterior of the embryo . This translation repression activity in the posterior of insect embryos appears to be conserved in the orthopteran Schistocerca americana  where nanos RNA is also posteriorly localised. Posteriorly localised nanos expression is also found in mosquito embryos .
In this paper I have cloned cDNA fragments of Honeybee vasa and nanos and used in-situ hybridisation on embryos and ovaries to determine if honeybee PGCs are specified early in development, like many holometabolous insect embryos, or if they form late in development from an inductive event.
Identification of homologues of nanos and vasain honeybees
Reciprocal blast searches  of the honeybee and Drosophila melanogaster genomes with Drosophila vasa and nanos sequences identified a single region of the honeybee genome with homology to vasa, and a single region with homology to nanos. Gene predictions corresponding to each of these regions were collected and analysed.
A predicted transcript (GB14804-PA, DQ288391) located on group 1.64 has high homology to Drosophila vasa. I term this transcript Amvasa. The transcript is predicted to encode a protein containing all of the 8 domains conserved in DEAD box helicases. The GIVGXA motif, conserved in insect vasas, is also conserved but the EXRKF domain conserved among Vasa and PL10 proteins is not present. The function of this motif is not known. The full length of this transcript was cloned from cDNA.
The sequence with highest homology to Drosophila nanos in the Honeybee genome is included in a predicted transcript (GB14366-PA) that is annotated as similar to venom protein Vn50 (Located on group 8.7). Analysis of this transcript indicates that homology to nanos is restricted to two upstream exons almost 7 KB from the exons of the predicted transcript with homology to venom protein Vn50 (Figure 2A). Just downstream of the second exon corresponding to nanos is a gap in the genome sequence that may have caused an error with the gene prediction software. I have sequenced across this gap and identified an in-frame stop codon downstream of the second exon. Primers designed to amplify full-length nanos from cDNA indicate that the gene prediction is correct in this area. I designate the gene from which this sequence derives Amnos. This sequence is deposited in Genbank as DQ288392.
In Drosophila the 3'UTR of nanos contains a structural RNA motif required for translational repression [39, 41], no sequence homology to this region can be seen in the Amnos 3'UTR. An RNA structure prediction program (MFOLD) predicts the first 130 bases of the Amnos 3'UTR produces a similar RNA fold to that of the Drosophila 3'UTR (Figure 2A ii and iii). The Drosophila motif contains sequences similar to bearded boxes that are also associated with translation repression . Predicted bearded boxes are present downstream of the Amnos gene but not in the predicted secondary structure.
Expression of Amvasain honeybee embryos
The expression of Amvasa RNA seems to indicate that PGCs in Apis form late in embryogenesis. It is possible, however, that the distribution of the Amvasa RNA does not reflect the distribution of Amvasa protein, as in Drosophila . To determine if Amvasa protein is present in embryos stages 2–8, embryos were stained using an antibody raised against Schistocerca gregaria vasa that has been shown to cross react in a number of species [7, 13, 25]. No staining above background in Apis embryos or ovaries was detected using the antibody in conditions in which Drosophila melanogaster embryos did stain in the canonical vasa pattern, implying this antibody does not cross-react with Amvasa (data not shown).
To confirm the timing of Amvasa RNA expression, RT-PCR was carried out on RNA from honeybee embryos of various stages (Figure 3P). A PCR product of the target length, consistent with Amvasa RNA, was found only in RNA from just laid eggs and embryos over stage 10. No Amvasa RNA could be detected in embryos of stages 3–7, consistent with our in-situ hybridisation results.
Expression of Amnosin honeybee embryos
To confirm the timing of Amnos RNA expression, RT-PCR was carried out on RNA from honeybee embryos of various stages (Figure 4K). A PCR product of the target length (257 bp), consistent with Amnos RNA, was found only in RNA from just-laid eggs and embryos over stage 10. These data are consistent with our in-situ hybridisation results in that Amnos RNA is not expressed between stages 3–7.
Expression of Amvasain queen and worker bee ovaries
In the queen ovary, both nurse and germ cells express Amvasa. Amvasa RNA is not localised to any particular area in either cell type. Amvasa RNA is present in oocytes in the vitellarium, but expression is absent, or very weak in the germarium and terminal filament. In the nurse cells, RNA is present in all cells in the vitellarium, but in the germarium, the expression of Amvasa RNA is reduced in the nurse cells closest to the oocytes (arrows in figure 6A). Expression of Amvasa RNA does not extend beyond the germarium in nurse cells. No expression of Amvasa is seen in follicle cells.
In worker ovaries a similar expression pattern can be seen (Figure 6B). Amvasa RNA is present in all oocytes, but, in contrast to queen ovaries, this expression extends into the terminal filament (Figures 6D, E, F, G, and 6H). Expression of Amvasa RNA in the nurse cells is reduced in worker ovaries. Expression can be seen in nurse cells in the vitellarium, and, faintly, in the nurse cells associated with the last oocyte in the germarium (Figure 6C). In the terminal filament (Figures 6D, E, F, G and 6H) Amvasa RNA expression marks germ cells in the entire structure. Comparison of the expression of Amvasa RNA and the location of nuclei stained with DAPI indicates that the germ cells in the start of the terminal filament are surrounded by disc shaped 'terminal filament cells'  that do not stain for Amvasa. However within these compartments, cells that do not express Amvasa are also present (Figure 6E and 6F). It is not clear if these are nurse cells or follicle cells. In the last sections of the terminal filament, cells positive for Amvasa RNA are interspersed with cells that are not (Figure 6G and 6H). The expression of Amvasa in the worker, but not queen terminal filament is a clear difference between castes.
Expression of Amnosin queen and worker bee ovaries
In worker bee ovaries, by contrast, Amnos RNA is weakly and uniformly distributed in oocytes in the vitellarium and germarium (Figure 7G) and, at higher levels, in the nurse cells of the oldest oocyte. Amnos RNA is not expressed in nurse cells, follicle cells or cells in the terminal filament.
Identification of Apis homologues of nos and vasa
Blast searches and phylogenetic reconstruction indicate that I have identified honeybee homologues of the evolutionarily conserved germ-cell markers nanos and vasa.
The Amvasa gene, also identified by , has all of the conserved sequences indicative of a vasa protein, and clusters with other insect vasa sequences to the exclusion of non vasa DEAD box helicases. The sequence does, however, have significant variation in the EXRKF motif, that is conserved in PL10 and vasa helicases. The function of this motif is unknown, thus the significance of its absence from Amvasa is not known.
The Amnos gene identified in this study is similar in C-terminal regions to nanos proteins from other species. This region contains the zinc coordination residues of the zinc fingers. These residues are completely conserved in Amnos. Drosophila nanos contains a sequence that forms a specific secondary structure in the 3'UTR that is required for translational repression . This secondary structure is bound by the smaug protein in early embryos . A similar secondary structure is predicted to form in the Amnos 3'UTR though its function, if any, is unknown.
The conservation and phylogenetic placement of these sequences is consistent with them encoding the conserved germ-cell markers vasa and nos.
Germ cell development in Apis mellifera
Expression of Amnos and Amvasa RNA occurs in two phases of honeybee embryonic development. Both genes are expressed early in development, Amvasa in the entire embryo, and Amnos in a gradient pattern with highest concentrations in the posterior of the embryo, both probably due to maternal RNA contribution. These expression patterns disappear by stage 2. In later development, at and after stage 9, both genes are expressed in a subset of cells that lie close to the dorsal boundary of the embryo proper and the extraembryonic membranes. These cells eventually come to be located in the dorsal-most regions of the embryo consistent with the genital ridges and forming gonads. That both genes are not expressed in embryos between stage 2 and stage 9 implies that during these stages there are no PGCs in the honeybee embryo, and that these cells form via an inductive event at, or just before, stage 9.
Neither morphological studies, nor the use of molecular markers has found any evidence for pole-cells, pole plasm or early segregating PGCs . This is unlike the formation of PGCs in Drosophila, which has a similar long-germ form of embryo to that of Apis.
The expression of these two PGC markers implies that germ-cells form very close to the boundary of the embryo proper and the extra-embryonic membranes. The cells form in A3-A6 and no sign of migration of the PGCs can be seen. As dorsal closure occurs, the cells appear to be moved, passively, to the dorsal surface of the larva, close to the dorsal midline.
These findings indicate two possible scenarios for the origin of PGCs in the honeybee embryo. It is possible that the appearance of PGC markers at a late stage of embryogenesis, similar to the situation in mice [48, 49] implies that an inductive event at stage 9 specifies PGCs. The organisation of the cells, in a single line, is what might be expected if the cells are being induced by a signal released from an almost linear source. Just dorsal of the PGCs are the extra-embryonic membranes that cover the dorsal opening. It is possible that the putative inducing signal leading to the formation of PGC fate emanates from the extra-embryonic membranes.
It is also possible that the Amvasa and Amnos RNA are not expressed in germ-cells that do form early in development, and only come to be expressed when these cells reach the abdomen. Drosophila vasa RNA expression is indeed absent from PGCs until stage 12  and nanos expression is down-regulated in Drosophila after stage 10 . In the crustacean Parhayle PGCs are specified very early in development, without the expression of vasa 
This study is unable to determine which of these two possible mechanisms underlies germ-cell formation in Honeybees.
Localisation of nos RNA and posterior development in Apis mellifera
In Drosophila and other insects nanos acts to regulate posterior development . Nos expression has been studied in both Drosophila [38, 41], other dipterans [22, 23] and Schistocerca . In each studied species nos RNA is localised in posterior regions in early development. In Drosophila most nos RNA is localised very tightly to the posterior, and its translation is repressed in anterior regions . In Drosophila nos acts as a key regulator of posterior development, translationally repressing, in concert with pumilio, the anterior gene hunchback in the posterior [37, 50]. In Schistocerca, the expression of nos is coincident with a clearance of hunchback expression from the very posterior regions of the embryo . Hunchback mRNA from Schistocerca is predicted to contain a nanos response element, similar to that which is bound by Drosophila nanos to repress hunchback translation.
In Honeybees Amnos RNA is present in the posterior of the early embryo (appearing in just laid eggs and clearing in stage 2) This domain is broader that that in Drosophila where the highest levels of nos RNA form, and appears to represent a concentration gradient, with highest RNA expression in the very posterior of the embryo. The Amnos 3'UTR does not contain an element with sequence similarity to the stem-loop structure in the 3'UTR of Drosophila nos, but a similar RNA structure is predicted to be formed by it.
In the ovary of queen bees, Amnos RNA expression is found in a localised patch in oocytes, that appears to move, during oocyte maturation, from anterior to posterior. In Schistocerca , and mosquitoes nanos RNA is also localised tightly to posterior regions of the oocyte. The localisation of Amnos RNA in the oocyte is consistent with a role in posterior patterning.
I have used Amvasa and Amnos as molecular markers of PGC fate in the honeybee Apis mellifera. The timing of placement of their expression provides no evidence for early specification of germ-cells but implies that honeybee PGCs form from an inductive event late in embryonic development. It is also possible that germ-cells form early in the Honeybee embryo but do so in the absence of vasa or nanos expression. Both these possibilities differ from PGC specification in Drosophila, where morphologically distinct PGCs appear early in development and are marked by the expression of vasa and nanos, or the more closely related Copidosoma, where a cellular organelle determines PGC fate and contains vasa protein .
Any putative induction process implied by these results appears similar to the segregation of PGCs in Schistocerca, where early segregation is also not seen. The placement of PGCs, forming close to the border between extra-embryonic and embryonic tissue and inside the epidermal layer, seems similar in both species.
This mode of PGC formation indicates that the evolutionary history of PGC segregation in insects is not simple, with different mechanisms acting in different species. It is unclear, at this point, what the ancestral mechanism is, and how the different mechanism of PGC specification in extant insects may have arisen.
Apis mellifera were cultured using standard techniques in Dunedin, New Zealand. Honeybee embryos were collected from frames removed from nucleus boxes containing small honeybee colonies.
Gene identification and phylogenetics
Homologues of the Drosophila melanogaster vasa and nanos genes were identified in the Honeybee genome sequence (version 2) using tBlastN searches . Regions of the genome with significant blast hits were extracted and blasted back to the Drosophila melanogaster genome. Gene predictions from either NCBI (using gnomon) or ENSEMBL were then examined in the regions with reciprocal top blast hits for vasa and nanos. Primers for amplification from cDNA were designed to the regions with the highest homology.
Multiple alignments of the predicted Apis genes with homologous genes from other species were carried out using ClustalX . Phylogenetic analysis was performed on these multiple alignments using MrBayes 3.1  or Phylip 
Ovaries were dissected from mated queen bees in PBS and poly A+ RNA extracted using a quickprep RNA extraction kit (GE Biosciences). cDNA was generated from this RNA using superscript II reverse transcriptase (Invitrogen) and an oligo-DT primer following the manufacturers instructions. PCR was performed on this cDNA using primers for vasa (tggcaatgtaacgataaaaagacc, AmvasaRNA5' tgggcgacacgatgacaac, AmvasaRNA3') or nanos (gtctccacacgcaccacaa, AmNanos5' acgccgcaagaaaaataagaaac, AmNanos3'). PCR products of the correct size were cloned into pGEM-T-Easy (Promega) following the manufacturers instructions. Plasmid DNA was isolated from these clones and sequenced at the Allan Wilson Centre for Molecular Ecology and Evolution.
RNA for RT-PCR experiments was extracted with an RNAeasy kit (Qiagen), treated with RNAse free DNAse (Invitrogen) for 2 hours at 37 degrees, heated to 65 degrees for 30 minutes, and precipitated with ammonium acetate and isopropanol. First strand synthesis and PCR were performed as above with primers for Amvasa (gcgtttccacccatcatc and gttgtcatcgtgtcgccca and Amnos (as above).
In-situ hybridisation and antibody staining
In-situ hybridisation was performed on ovaries and embryos as described in Osborne and Dearden  and antibody staining after in-situ hybridisation using the 4D9 anti-engrailed-like antibody  as described in Osborne and Dearden .
Ovaries were dissected from mated queen bees and fixed in a mixture of 4% formaldehyde in PBS: Heptane overnight. Ovaries were washed in methanol and rehydrated in PTw (PBS + 0.1% Tween 20). The ovaries were treated with 1 μg/ml RNAse A and PTw for one hour, and then treated with 0.5 μg/ml Propidium iodide and 0.33 mM Alexa-Fluor 488 Phalloidin (Molecular probes) overnight in PTw. The ovaries were washed in PTw four times over 30 minutes and individual ovarioles dissected from the ovary mass. Individual ovarioles were mounted on microscope slides in 70% glycerol. Confocal imaging was carried out using a Leica confocal microscope.
I would like to thank Cassandra Extavour and Michael Akam for kind provision of their anti-vasa antibody, Sue Heath and Prof. Alison Mercer for their help with Honeybee rearing and Megan J Wilson, Andrew G Cridge and E P Dearden for their critical reading of this manuscript. I would also like to thank Dr Craig Marshall and the Chemistry and Botany Departments, University of Otago for providing space for Honeybee culture. The anti-engrailed monoclonal antibody developed by Corey Goodman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by a University of Otago Research Grant and a Royal Society of New Zealand Marsden Fund Grant (UOO0401).
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