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Boule Is Present in Fish and Bisexually Expressed in Adult and Embryonic Germ Cells of Medaka

Background The DAZ family genes boule, daz and dazl encode RNA binding proteins essential for fertility of diverse animals including human. dazl has bisexual expression in both mitotic and meiotic germ cells, whereas daz has male premeiotic expression, and boule is largely a unisexual meiotic regulator. Although boule has been proposed as the ancestor for dazl/daz by gene duplication, it has been identified only in invertebrates and mammals. It has, however, remained unclear when and how the DAZ family has evolved in vertebrates. Methodology and Principal Findings This study was aimed at identifying and characterizing the DAZ family genes in fish as the basal vertebrate. We show that boule and dazl coexist in medaka and stickleback. Similar to the medaka dazl (Odazl), the medaka boule (Obol) is maternally supplied and segregates with primordial germ cells. Surprisingly, Obol is expressed in adult germ cells at pre-meiotic and meiotic stages of spermatogenesis and oogenesis. However, the maximal meiotic Obol expression in spermatocytes contrasts with the predominant pre-meiotic Odazl expression in spermatogonia, and the diffuse cytoplasmic Obol distribution in early oocytes contrasts with the Odazl concentration in the Balbinani's body. Conclusions The identification of fish boule and dazl genes provides direct evidence for the early gene duplication during vertebrate evolution. Our finding that Obol exhibits bisexual expression in both embryonic and adult germ cells considerably extends the diversity of boule expression patterns and offers a new insight into the evolutions of DAZ family members, expression patterns and functions in animal fertility.

Introduction
Germline development and gametogenesis proceed in multiple processes that are complex and apparently divergent among metazoans. Understanding of the mechanisms underlying these processes is crucial for fertility and reproductive medicine in human as well as for germline engineering in animals. The DAZ (Deleted in Azoospermia) gene family offers one of the few lines of evidence that argue for evolutionary conservation of these processes at the molecular level. The DAZ family comprises daz, dazl and boule. DAZ family proteins are characterized by a conserved ribonucleoprotein (RNP)-type RNA recognition motif (RRM) and one (Boule and Dazl) or multiple repeats (Daz) of the DAZ motif. Historically, the DAZ family was established with the identification of the founder member Daz. In human, multiple copies of Daz genes cluster on the Y chromosome [1,2]. Deletion of the Daz cluster is associated with azoospermia and oligospermia, making Daz a strong candidate for the Azoospermia factor. In addition, the human has an autosomal Daz-like gene, termed Dazla [3,4]. The Y-chromosomal Daz is limited to catarrhine primates, whereas the autosomal Dazl homologue has been found in mouse [5]. Accordingly, the autosomal Dazla has been proposed to be the ancestor of the Daz cluster, with its duplication followed by transposition to the Y chromosome having occurred during the evolution of primates [2]. Dazl homologues have been identified also in all major groups of non-mammalian vertebrates including chicken [6], Xenopus [7], axolotl [8], zebrafish [9], medaka [10] and gibel carp [11]. The third family member boule was found in Drosophila as a gene essential for the germline development [12]. Similarly, in the nematode Caenorhabditis elegans, a gene named as daz-1 was identified as the boule/dazl counterpart [13]. In all organisms examined, the DAZ family genes are exclusively expressed in, and required for, the germline development. The notion that daz, dazl and boule are evolutionary homologues is supported by observation that the human Dazl can rescue the Drosophila boule mutant to some extent [14]. Because vertebrates have both boule and dazl, and invertebrates merely have boule, it has been proposed that boule is the ancestor of the DAZ gene family. During the vertebrate evolution, boule underwent gene duplication, producing the autosomal dazl, which duplicated further, leading to additional copies that were translocated to the Y-chromosome in catarrhine primates and amplified to multiple daz genes [15]. Indeed, boule has recently been described in few mammals including human [15], mouse [15], sheep [16] and cattle [17].
In spite of germline expression, different DAZ family genes have considerable diversity in sex-and stage-specific expression. Human Daz and Drosophila boule are transcribed specifically in the male germline [1,2,18]. Human Dazla and its mouse, zebrafish, and Xenopus homologues are expressed in the germline of both sexes [7,9,19,20,21]. In the nematode daz-1 (boule/dazl homologue) mRNA is expressed in germ cells mostly in female [22], albeit its protein is detectable in germ cells in male as well [23]. In sheep, boule is also highly expressed in both male and female gonads undergoing meiosis as analyzed by RT-PCR [16].
The role of the DAZ family genes in the production of gametes is still largely unknown, partly due to the apparent diversity of phenotypes caused by their defects. A human male missing the DAZ cluster shows a varying range of defects in spermatogenesis, from no germ cells at all to less severe spermatogenic arrest generating some mature spermatids [1], indicating that the DAZ cluster is not absolutely necessary for the entry into meiosis and sperm production. Targeted disruption of mouse Dazla results in the complete absence of gamete production in both sexes, with Dazla-defective mice having female germ cells arrested at prophase of meiosis I and male germ cells being affected at the proliferating stage [19]. In contrast, the Drosophila boule is essential for the meiotic progression in spermatogenesis but not oogenesis, as mutant flies have male germ cells arresting at the G2/M transition in meiosis I, exhibiting limited postmeiotic differentiation [12]. In the nematode, mutations of the boule homologue daz-1 completely abolishes fertility in hermaphrodites due to arrest at meiotic prophase in oogenesis, while daz-1-defective males exhibit no significant defect and are fully fertile [13,22,23]. Thus, although the DAZ family genes encode regulators of gametogenesis, their sex specificity varies considerably depending on the family member and species.
With the identification of human and mouse boule in addition to dazl and daz, Xu et al [15] proposed a model for the DAZ gene family evolution. According to this model, boule was the ancestor gene that underwent the first gene duplication, generating autosomal dazl; dazl during primate evolution underwent the second gene duplication and chromosomal translocation to the Y chromosome, resulting in Daz; and two more Daz gene duplication produced the present-day Daz clusters in human. There are two puzzles in understanding of the DAZ family evolution. First, boule usually has unisexual meiotic expression in both invertebtates and mammals, whereas dazl has bisexual expression in both premeiotic and meiotic germ cells, raising an interesting question as to whether the first vertebrate boule homologue has unisexual or bisexual germline expression. The second puzzle is concerned with the timing of the first boule gene duplication leading to dazl. If this gene duplication indeed occurred early in vertebrate evolution as hypothesized [15], all vertebrate species must have both boule and dazl. However, boule in vertebrates has so far been identified only in mammals, raising the question as to whether boule is restricted to mammals by lineage-specific gene duplication. Since the separation between the fish and tetrapod lineages ultimately leading to mammals occurred at the beginning of vertebrate evolution approximately 450 million years ago, fish represent an ideal system to explore the DAZ family evolution in the basal vertebrates.
Previously, we have identified dazl in the fish medaka and revealed its expression in embryonic and adult germ cells of both sexes [10]. This study was aimed at the identification of boule in medaka (Oryzias latipes), and both boule and dazl in a second fish species, namely the three spined stickleback (Gasteroteus aculeatus). Furthermore, we examined the medaka boule RNA expression in developing embryos and adult gonads. Medaka is an excellent model for investigating germline development and gametogenesis in vivo and in vitro. It has a completely sequenced genome facilitating gene identification and reverse genetics, male germ stem cell line capable of sperm production in vitro [24], the ease of observation of germline development and robust genetic manipulations in embryos [25] and stem cell cultures [26]. We showed that the medaka boule has a previously unidentified expression pattern, which may provide important implications on the DAZ gene family evolution and boule gene function.

Fish Possess both boule and dazl
The DAZ family proteins show apparent sequence variations in diverse organisms except for the RRM and DAZ motif. To determine whether boule was present in the fish lineage, we exploited molecular cloning and bioinformatics approach and chose the medaka as the first model to identify boule and dazl homologues in fish. Since Odazl, the medaka dazl, has previously been cloned [10], here we focused on cloning boule gene in the strain i 3 medaka. We cloned the boule gene before the medaka genome sequence became available. A multiple sequence alignment of invertebrate and mammalian Boule proteins led to the identification of two conserved sequences for degenerate primers (see below). RT-PCR with the degenerate primers resulted in a partial sequence that was found to be most similar to the human BOULE. Rapid amplification of cDNA ends (RACEs) by using gene-specific primers led to the composition of a full length cDNA that was cloned by using terminal primers and verified by sequencing. This cDNA appears to be the medaka boule, termed Obol. Obol is 2,097 nt in length, contains a 74-nt 59 untranslated region (UTR), a 1,134-nt 39 UTR and a 885-nt open reading frame (ORF) encoding a protein of 295 amino acid residues (aa) (Fig. 1). The predicted protein OBol possesses a conserved RRM and a DAZ repeat (Fig. 1). OBol is 41% and 42% identical in sequence to the mouse and human BOULE, respectively, and 28% to the human DAZL (data not shown), indicating it is more similar to Boule than to Dazl. The conserved positions usually reside within the RRM ( Fig. 2A).
The cloning of Obol in this study and Odazl in our previous work enabled us to search for boule and dazl homologues in other fish species. Blast search against sequenced fish genomes (www. ensembl.org) resulted in the identification of single boule (ENSGACP00000019047) and dazl (ENSGACP00000009272) genes also in stickleback (Gasterosteus aculeatus). A multiple sequence alignment of the RRM reveals that Boule differ from Dazl proteins in 20 invariant and/or conserved positions, besides 27 invariant and/or conserved positions common to both Boule and Dazl ( Fig. 2A). On a phylogenetic tree, fish and mammalian Boule proteins are clustered together, whereas all Dazl forms a separate clade (Fig. 2B). As illustrated in Fig. 2C, the stickleback belongs to the Percomorpha, separating about 100 million years ago from the Beloniformes to which the medaka belongs. Together, Percomorpha and Beloniformes are within the lineage Acanthopterygii and separated approximately 180 million years ago from the Ostariophysii to which the zebrafish belongs [27]. Interestingly, the branching into the Boule and Dazl clades coincides with separation between fish and tetrapod lineages. This, together with the fact that fish Boule is more similar to mammalian Boule than to fish Dazl, strongly suggests the early appearance of vertebrate boule before fish-tetrapod separation about 450 million years ago.
Fish boule and dazl resemble their mammalian counterparts in gene structure consisting of 12 and 11 exons, respectively (Fig. 3). To further determine whether fish boule and dazl were the evolutionary orthologues of human Boule and Dazl respectively, we analyzed the chromosomal syntenic relationships. Obol is adjacent to stoml2 gene on Ultracontig 140 that is syntenic to a small region of stickleback chromosome/group 1. This region contains eight genes including boule and is fully syntenic to the Boule-bearing region on human chromosome 2 (Fig. 4A). On the other hand, the dazl-bearing regions on different fish chromosomes (medaka Chr11, stickleback group X and zebrafish Chr19) exhibit a conserved synteny to the Dazl-containing human chromosome 3 (Fig. 4B). Taken together, fish have both boule and dazl genes, demonstrating that the gene duplication leading to boule and dazl must have taken place before the separation between the fish and tetrapod lineages. Furthermore, the DAZ gene family in fish very probably has only two members, namely boule and dazl, because blast searches by using boule and dazl queries failed to detect any additional DAZ family genes in fish cDNA databases and all of the five sequenced genomes (data not shown). This situation is reminiscent of non-primate mammals and indicates that either boule or dazl has undergone no or rare additional gene duplication after fish-tetrapod separation. In accordance with this notion is the presence of a putative boule also in chicken (XP_421917), in addition to a described chicken dazl [6] Boule has a Novel Expression Pattern in Medaka In all species examined so far, dazl has bisexual germline expression, while boule usually has unisexual expression. Moreover, boule also shows sex specificity in different organisms, namely male fly [12] and mammals [15], but female worm [22]. RT-PCR analyses revealed that the adult Obol RNA expression is absent in somatic tissues but high in adult gonads of both sexes (Fig. 5A), suggesting that adult Obol expression may be restricted to germ cells. Amazingly, the Obol RNA is easily detectable in early developing embryos (Fig. 5B) before the midblastula stage when bulk zygotic gene transcription takes place [28], demonstrating that Obol RNA is maternally supplied.
In both fly and mammals, boule expression occurs in male meiotic germ cells, and its deficiency causes male sterility by meiotic arrest in Drosophila. We performed section in situ hybridization (SISH) to examine Obol expression during spermatogenesis and compared with Odazl and Olvas, the wellstudied germ cell markers in diverse animals [8,10,29,30,31]. The adult medaka testis is composed of seminiferous lobules, each comprising cysts of germ cells synchronously at various stages of spermatogenesis. Cysts of spermatogonia are located at the most peripheral region. Spermatogenesis proceeds synchronously within each cyst, and cysts of germ cells at progressively advanced stages of development reside closer to the efferent duct, which is in the central region. This allows unambiguous definition of individual spermatogenesis stages. SISH by using an antisense RNA probe revealed that the Obol transcript was most abundant in meiotic cells, namely spermatocytes. A faint signal was observed also in the meiotic products spermatids. Interestingly, spermatogonia at the testicular periphery also exhibit a weak but detectable signal (Fig. 5C). In contrast, the Odazl signal peaks in spermatogonia and reduces in spermatocytes and disappears in post meiotic stages (Fig. 5D). On the other hand, Olvas displays an expression pattern overlapping with, but distinctive from, Obol and Odazl (Fig. 5E). Sense probes did not detect reproducible staining (data not shown). To precisely compare the spatial expression pattern between Obol and Odazl or Olvas, we performed sensitive fluorescent in situ hybridization (FISH). As shown in Fig. 5F-I, Obol is clearly detectable in certain spermatogonia, and its maximal meiotic expression in spermatocytes alternates with the preferential premeiotic expression of Odazl and Olvas in spermatogoina. These results demonstrate that Obol owns a distinct expression pattern from Odazl and Olvas, and its expression occurs in both premeiotic and meiotic stages of medaka spermatogenesis.
We then examined Obol expression in the ovary. In medaka, the adult ovary comprises a small number of oogonia and oocytes, and oogenesis proceeds in 10 stages [32]. By chemical SISH, Obol expression persists throughout oogenesis, in both premeiotic and meiotic stages (Fig. 6A), with the strongest signal being found in early meiotic cells, namely stage II-V oocytes, and a weak but detectable signal being seen in oogonia. This stage-preferential Obol expression is similar to Odazl (Fig. 6B) and Olvas (Fig. 6C), except that Olvas also has high expression in oogonia.
A salient difference in RNA localization between Obol and Odazl was found in stage III-V oocytes. In these oocytes, Obol is diffuse in the ooplasm (Fig. 6A and D), whereas Odazl predominantly concentrates in a cytoplasmic structure, the so-called Balbiani's body (BB; Fig. 6B, D-F). Diffuse distribution was seen also for Olvas (Fig. 6C) and medaka dead end [33]. Double color co-localization by FISH reinforced and further strengthened this distinction between Obol and Odazl in subcellular localization into the BB. Specifically, Obol is undetectable in the BB but abundant in the remaining ooplasm, whereas Odazl is highly abundant in the BB but rare in the remaining ooplasm. In addition, premeiotic expression in oogonia is clearly detectable for both Obol and Odazl. Taken together, Obol resembles Odazl and Olvas in premeiotic and meiotic expression during oogenesis, but differs from Odazl in the absence of distribution into the BB.
The BB is a membraneless spherical structure in close contact with the nuclei of early developing oocytes [32,34] and contains mitochondria, endoplasmic reticulum and several germ plasm components including the dazl RNA during early oogenesis in Xenopus and zebrafish [11,34,35,36]. Specifically, the BB is characterized by highly concentrated mitochondia called mitochondrial cloud, and mitochondrial staining by e.g. the MitoTracker reagent represents a standard procedure to validate the BB identity in the early oocytes of several organisms including zebrafish [11,34,35,36]. FISH and mitochondrial staining clearly revealed that the spherical structure with localized Odazl RNA was indeed within mitochondrial cloud, namely the BB (Fig. 6G-I).
In Xenopus [7,21] and fish [9,10,37], dazl is maternally supplied and expressed in primordial germ cells (PGCs). In contrast, boule expression in invertebrates and mammals is restricted to adult germ cells, but absent or unknown in early embryos or PGCs [15]. Our experiments described so far demonstrated that the Obol shows an expression pattern distinct from what reported for invertebrate and mammalian boule, in that Obol has bisexual premeiotic and meiotic expression. These observations, together with early embryonic expression analyzed by RT-PCR (Fig. 5B), provoked us to examine the Obol temporospatial expression during embryogenesis. By chemical whole mount in situ hybridization, Obol was easily detectable in early developing embryos such as the 4-cell stage (Fig. 7A), conforming to its maternal supply as revealed by RT-PCR (Fig. 5B). When embryogenesis proceeds, Obol becomes restricted to putative migrating PGCs at stage 18 and post-migratory PGCs at stage 27 ( Fig. 7D and G). This Obol expression pattern is similar to Odazl (Fig. 7B, E and H) [10] and Olvas (Fig. 7C, F and I) [29,38]. To verify that Obol-expressing cells were truely PGCs, we performed double color FISH to see whether Obol-positive cells were positive also for Odazl and Olvas. As illustrated in Fig. 7J-P, Obol indeed co-localizes with Odazl and Olvas in the genital PGCs at stage 27. Therefore, we conclude that Obol expression occurs in medaka PGCs.

Conclusions
In the present study, we report the identification of both boule and dazl in fish as an ancient vertebrate and the analysis of their expression in medaka embryonic and adult development. Several lines of evidence, including phylogenetic sequence comparisons, protein structure, genomic organization and most convincingly, chromosome synteny, support the notion that the fish boule and dazl are the orthologues of mammalian boule and dazl, respectively. With the identification of boule in mouse and human, Xu et al. [15] proposed that the DAZ gene family has evolved from boule by gene duplication and translocation. Our work corroborates and extends this report by demonstrating direct evidence for ancient gene duplication during early vertebrate evolution, prior to the separation between fish and tetrapod lineages approximately 450 million years ago (Fig. 8). The identification of fish boule indicates that boule and dazl coexist widely in the ancient vertebrate. Meanwhile, they become paralogs of each other since the Daz gene family underwent the first duplication.
We have made four striking observations that convincingly demonstrate a totally novel expression pattern for boule in medaka. The boule RNA expression has been reportedly restricted to meiotic germ cells of male flies and mammals or mainly in female worms but rarely in both sexes, and boule deficiency causes sterility by meiotic arrest rather than by abolishing other processes of germline development. Consequently, boule is best known as an ancient meiotic gene conserved in metazoans [15]. Here we firstly demonstrate that boule in medaka also exhibits a maximal meiotic expression, but this occurs in both sexes, in contrast to its unisexual expression in all other organisms well examined so far. Second, we show that the medaka boule also has premeiotic and post-meiotic expression in the adult ovary and testis. Third, we have revealed that medaka boule is a maternal message and expressed throughout embryonic germ cell development. Finally, we present evidence that in medaka, boule differs from dazl in subcellular distribution to the Balbiani's body of early developing oocytes. Taken together, our investigations have revealed a previously unidentified bisexual boule expression in embryonic and adult mitotic and meiotic germ cells, further underscoring the expression diversity of DAZ family genes (Fig. 8). Interestingly, boule expression in medaka is in a   . It is noteworthy that fluorescent dyes DAPI and propidium iodide do stain nuclei of oogonia but do not stain nuclei of oocytes in medaka, a similar situation has been described also in zebrafish [43]. doi:10.1371/journal.pone.0006097.g006 sharp contrast to its unisexual meiotic expression reported in mammals and invertebrates, but is similar to that of medaka dazl [10], despite clear differences in preferential expression and stagespecific subcellular localization in the oocytes. The striking finding that boule and dazl have overlapping but distinct expression patterns provides a new insight into the evolution of DAZ family genes and their expressions as well as functions. Since fish separated from tetrapods at the earliest stage of vertebrate evolution, it is tempting to speculate that the medaka boule expression may represent the prototype of an ancient vertebrate boule. This notion is supported by the identification of putative boule and dazl genes in a marine fish, and more importantly by similar expression patterns to their medaka counterparts (data not shown).
The identification of both boule and dazl in medaka and stickleback in this study will facilitate the further investigations in other fish species. Further, examination of boule and dazl expression patterns in other vertebrate species including ancient fishes will help to elaborate the evolution, expression and function of the conserved DAZ family genes in (in)fertility of vertebrates including the human.
This study has focused on the RNA expression patterns of both boule and dazl. It deserved to note that RNA expression pattern may differ from that of the protein products [19,22,23,39,40]. Therefore, future work is needed to produce antibody against the medaka Boule protein for analyzing its expression and subcellular localization by immunohistochemistry.

Fish
The medaka was maintained under an artificial photoperiod of 14-h light to 10-h darkness at 26uC. All procedures carried out with medaka fish are conformed to animal care guidelines (Guidelines on the Care and Use of Animals for Scientific Purposes) as outlined by the National Advisory Committee For Laboratory Animal Research in Singapore. Embryogenesis was staged according to Iwamatsu [41]. Oogenesis was according to the 10-stage series of Iwamatsu [32].

Isolation of cDNA Sequence
Total RNA was isolated from embryos and adult tissues of strain i 3 medaka by using the Trizol Reagent (Invitrogen). To eliminate genomic DNA contamination, RNA samples were treated with RNase-free DNase (Promega). SMART cDNA libraries were synthesized by using the RACE cDNA Amplification Kit according to the manufacturers' instructions (BD BioSciences). To clone a fragment of the medaka boule cDNA, RT-PCR was performed with degenerate primers inferred from conserved amino acid sequences ( Fig. 2A). After sequencing, this cloned fragment was used to design gene-specific primers for 59 and 39 RACEs as described previously [30]. A full length cDNA sequence was obtained by using terminal primers (ggatccTTCGCGG-GCGGGGGAAATGAAGTG and aaaaccgcggACCCTTGACG-TTTAAC CACAG; lower case letters, introduced restriction sites for cloning).

Sequence Analysis
Blast searches were run against public databases by using BLASTN for nucleotide sequences and BLASTP for protein sequences. Multiple sequence alignment was conducted by using the Vector NTI suite 11 (Invitrogen). Phylogenetic tree was constructed by using the DNAMAN package (lynmon Biosoft). Genomic organization and chromosomal locations were investigated by comparing the cDNA and corresponding genomic sequence (http://genome.ucsc.edu/).

RT-PCR Analysis
The synthesis of cDNA was described [10]. PCR was performed by using two Obol-specific primers (bouleF2, aaaactcgagG-CAGGCCGATGGCGCCTCC; bouleR2, aaaaccgcggACCC TTGACGTTTAACCACAG). As a control, b-actin was amplified from the same set of cDNA samples using MA1 (TTCAA-CAGCCCTGCCATGTAC) and MA2 (CCTCCAATCCAG ACAGAGTATT). PCR was run for 30 cycles (b-actin) 35 cycles (boule) of 20 s at 94uC, 20 s at 58uC and 60 s at 72uC. The PCR products were separated on 1.2% agarose gels and documented with a bioimaging system (Synoptics).  [7,10,12,15,19,21,23,40,44,45,46]. Genes (in italics) refer to RNA expression while Proteins represent the protein expression profiles. Major stages of germline development are diagramed as a timeline for DAZ family gene expression. Expression of dazl is detected in many or all stages of germline development in both sexes. Daz has premeiotic male expression. Meiotic expression of Boule occurs in male fly, mouse and human (most abundantly in primary spermatocytes) and mitotic and meiotic in female worm. Data obtained in this study from medaka clearly demonstrate that boule is also expressed in embryos at the earliest stage, in primordial germ cells (PGCs) and adult premeiotic and meiotic germ cells of both sexes. The expression of medaka boule is similar to that of dazl in medaka and other organisms, despite of clear differences as detailed in the text. doi:10.1371/journal.pone.0006097.g008 Fish DAZ Gene Family PLoS ONE | www.plosone.org RNA in situ Hybridization RNA in situ hybridization by chemical staining with BCIP/NBT substrates on whole mount samples (WISH) and sections (SISH) were performed as described previously with minor modifications [30]. Briefly, pGEM vectors containing the 1.3 kb partial regions of Obol ORF and 39 UTR (see Fig. 1), 1.8-kb Olvas ORF [38] or 984-bp Odazl [10] were linearized with Xho I and Sac II and used for the synthesis of sense and anti-sense RNA probes from Sp6 or T7 promoter by using the digoxigenin (DIG) or FITC RNA Labeling Kit (Roche); the RNA probes were treated with RNasefree TURBO DNase and purified (cat# AM1340, Ambion). Multiple color fluorescent in situ hybridization (FISH) was performed by using the TSA TM Plus Fluorescence Systems according to the product manual (NEL756, NEN Life Science). Nuclear staining was done by using 49-6-Diamidino-2-phenylindole (DAPI) in the Gold Antifade reagent (Invitrogen).

Mitochondrial Staining
The sections were stained for mitochondria by using the MitoTracker reagent at 100 nM according to the supplier's instruction (M22425, Invitrogen), followed by FISH with the Odazl antisense RNA probe as described above and DAPI staining for nuclei before visualization.

Microscopy and Photography
Observation and photography on Leica MZFIII stereo microscope, Zeiss Axiovertinvert and Axiovert upright microscopes with a Zeiss AxioCam M5Rc digital camera (Zeiss Corp) were as described previously [10]. Confocal images were photographed on Olympus FV500 microscope with the Olympus confocal image system (Olympus). Multiple color fluorescent WISH samples were scanned in a series of 0.5-mm slices and the 3dimensional images were reconstructed by Z-stacking 150-170 slices on the Huygens (Bitplane AG; Germany).


extfieldsofstudy: Biology Medicine

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Divide and Conquer: Progress in the Molecular Stratification of Cancer

Cancer remains an outstanding cause of global morbidity and mortality, despite intensive research and unprecedented insights into the basic mechanisms of cancer development. A plethora of clinical and experimental evidence suggests that cancers from individual patients are likely to be molecularly heterogeneous in their use of distinct oncogenic pathways and biological programs. Efforts to significantly impact cancer patient outcomes will almost certainly require the development of robust strategies to subdivide such heterogeneous panels of cancers into biologically and clinically homogenous subgroups, for the purposes of personalizing treatment protocols and identifying optimal drug targets. In this review, I describe recent progress in the development of both targeted and genome-wide approaches for the molecular stratification of cancers, drawing examples from both the haematopoietic and solid tumor malignancies.

prognosis of these cancers, often associated with overall 5year survival rates of < 20%, can be ascribed to several reasons. First, there is currently no effective screening methodology to detect these cancers at early disease stages, leading to most patients presenting with advanced stage disease, which is traditionally associated with poor prognosis. 7 Highlighting the importance of early detection, the five-year survival rate of patients in the US with operable early stage gastric cancer is 50%, but decreases to < 10% in individuals with late stage disease. 8 Second, conventional strategies for clinically treating these conditions, usually involving a combination of surgery, radiotherapy and chemotherapy, are far from optimal. In gastric cancer, chemotherapy response rates can range for 20% for single agent therapies to 40% for combination therapies, [9][10][11] and even if short-term responses are observed, most tumors will eventually develop drug resistance and relapse. The shortcomings of existing therapies for these conditions has understandably fueled intense interest in the use of so-called "targeted therapies", which are antibodybased or small-molecule drugs designed to selectively inhibit particular oncogenes and oncogenic pathways, such as HER2, EGFR, and the pro-angiogenic VEGF pathways. 12,13 Results from recent clinical trials evaluating these drugs are starting to confirm that they can indeed convey a significant, albeit modest, increase in overall survival and lifespan when administered in combination with conventional therapy. 14,15 Third, as we now argue in this review, another likely reason for the high mortality rate of these cancers is that these tumors are in reality highly heterogeneous, frequently differing between individual patients in their fundamental biology and reliance on specific cellular pathways. We will present a case for why considering cancer heterogeneity is likely to be important for improving patient outcomes in these diseases, and describe a variety of approaches currently being used to dissect cancer heterogeneity and subclassify cancers at the molecular level. For clarity, it is also important to note that for this Review, the term "cancer heterogeneity" or "tumor heterogeneity" will be used to refer to variations in tumors between individual patients, and not to the populations of different cell types within a given tumor ( e.g., cancer cells, stromal cells, infiltrating immune cells, etc).
An ample body of research over the past decade supports the notion that many cancers are likely to display significant variability at the clinical, histopathologic, and molecular level between individuals. For example, in non small cell lung cancer (NSCLC) clinical trials evaluating the EGFR inhibitor gefitinib, clinical responses were observed only in a small proportion of patients. 16,17 The existence of such 'high-responder' patient subpopulations provides compelling evidence of clinical heterogeneity in NSCLC. In terms of histopathology, tumors can also exhibit significant differences in histologic architecture, tumor microenvironment, tumor grade and differentiation status. In stomach cancer, it is well known that tumors can be subdivided into at least two distinct histological types -an intestinal variant where cancer cells develop as tubular structures exhibiting features of well-differentated glandular tissue, and a diffuse subtype where tumor cells are poorly differentiated and spread along the lining of the stomach, sometimes resulting in the classical 'linnitis plastica' phenotype. 18 Such phenotypic variability logically suggests that different biological programs are likely to be acting in individual tumors, even when they are derived from the same target organ. Reinforcing this notion, recent comprehensive genomic analyses of lung, breast, and brain tumors have confirmed that individual tumors can frequently exhibit heterogeneous patterns of somatic mutations, gene amplifications and deletions, epigenetic profiles, and gene expression portraits. [19][20][21][22][23] Given this remarkable heterogeneity however it is perhaps surprising that with some notable exceptions (discussed below) such patterns of variability are usually not considered in the specific management of cancer patients. Even today, most clinical trials involving many of the major solid cancers are still performed on unselected patient populations without prior stratification. 24,25 However, the ongoing identification of specific genes and proteins playing important functional roles in cancer development has recently led to advancements in the use of molecularlytargeted assays to stratify and classify tumors. One good example of a protein currently used for molecular stratification in breast cancer is the estrogen receptor (ER). Commonly measured by immunohistochemistry, overexpression of ER in breast tumors has been associated with improved patient prognosis and less aggressive cancers. 26 Perhaps more importantly, ER expression is also used as a predictive biomarker for treatment of breast cancer patients with anti-hormonal therapies such as tamoxifen or aromatase inhibitors. 27 At the genetic level, several assays are now available to test for the presence of specific oncogenic mutations in cancers. For example, in the afore-mentioned lung cancer gefitinib trials, subsequent genetic analysis revealed that patients exhibiting good responses to gefitinib all exhibited tumor somatic mutations in the tyrosine kinase domain of the EGFR gene. 28,29 Besides gene mutation, oncogene dysregulation can also occur through the processes of genomic amplification and deletion. Such aberrations, commonly detected either by techniques such as comparative genomic hybridization (CGH or alternatively by higher resolution array-CGH) or flouresence in-situ hybridization (FISH), can involve a wide range of sizes ranging from whole chromosomes and chromosomal arms, to specific genes. Examples of chromosomal aberrations with clinical significance include losses of chromosome 18q in colon cancer, commonly associated with poor prognosis, 30 and gain of chromosome 8q in pancreatic cancer. 31 At the single-gene level, high-level amplifications of the HER2 gene have been reported in 30% of breast tumors, identifying a subtype of patients with particularly poor prognosis. 32,33 Furthermore, with the development of anti-HER2 therapies such as traztuzamab (Herceptin), HER2 gene amplification is also currently used as a predictive marker for anti-HER therapy. 14,34 Besides conventional molecular platforms that typically target only one or two genes/proteins at a time, newer genomic approaches are also being evaluated with the capability of stratifying cancers at the level of multiple biomarkers. For example, gene expression profiles, where the expression level of every single gene is measured simultaneously, have been used to identify subclasses of breast cancer. 35 An important finding enabled by this technology has been the discovery that the expression profiles of distinct breast cancer "molecular subtypes" (e.g. ER positive and ER negative) differ not simply in their expression of single genes like ER, but are in fact strikingly different from one another at the level of hundreds if not thousands of differentially expressed mRNA transcripts. 36 The molecular distinctiveness of these subtypes is consistent with proposals that these subtypes are indeed distinct biological and clinical entities. This finding, which has been replicated in several centers across the world including ours, is just one example of how using genomewide information may provide greater accuracy and insights than relying on single biomarkers alone. [37][38][39] More recently, several groups have published reports describing how global gene expression profiles can be computationally deconvoluted to provide information regarding the activation levels of different oncogenic pathways in tumors. 40,41 For example, by identifying gene expression signatures associated with the activation of various oncogenic pathways in vitro, Bild et al., were able to predict the oncogenic status of these pathways in a variety of solid tumors such as breast, lung and ovarian cancers. 40 Excitingly, interpreting gene expression profiles in terms of pathway activation patterns allows such information to be readily linked to the selection of specific targeted therapies designed to inhibit these pathways. Another advantage of genomic assays is that they allow multiple pathways to be interrogated without requiring multiple tests to be performed, as would be the case in immunohistochemistry. Such "high-throughput" pathway profiling could reveal previously unanticipated interactions between different pathways, and even suggest possible strategies for combining different pathway inhibitors. Besides gene expression profiling, the introduction of newer deep-sequencing technologies may also permit individual tumors to be characterized at single-nucleotide resolution resolution, which may provide further insights into the repertoire of genetic aberrations and structural variations in solid cancers. 42,43 While the availability of these molecular platforms renders the ability to classify cancers technically feasible (i.e. "we can do it"), it is perhaps worthwhile at this juncture to formally consider why the practice of tumor stratification, whereby heterogenous cancers are subdivided into clinically and biologically homogenous groups, might be useful for the improvement of caner patient outcomes (i.e. "we should do it"). We believe that there are several reasons why this might positively impact the clinical management of cancer patients. First, the ability to stratify patients, at the point of diagnosis, into either good or poor prognosis categories might allow clinicians to personalize standard therapeutic regimens for individual patients through doseescalation or dose-reduction. In the former (dose-escalation), additional risks associated with the poor prognosis status of the patient might justify the enhanced possibility of adverse side effects caused by intensified therapy, while in the latter (dose-reduction), a priori knowledge that the patient may not require additional therapy might allow clinicians to scale down treatments, thereby minimizing toxic side effects, yet ultimately preserving the same level of clinical benefit. A good example of how such prognostic biomarkers might prove useful can be seen in the management of patients with early stage breast cancer. Retrospective analyses has established that a certain proportion of women with early stage breast cancer are likely to be sufficiently treated with surgery only, requiring no further adjuvant therapy. 44,45 However, without an assay capable of pre-identifying these good prognosis patients, most early stage breast cancer patients are treated by default with adjuvant chemotherapy, and it has been estimated that at least 70 -80% of women with early stage breast cancer are likely overtreated. To address this problem, researchers from the US and Netherlands described in 2002 a 70-gene expression signature capable of subclassify such early stage breast cancer patients into good and bad prognosis categories respectively. 46 Since then, the testing of this 70gene signature has undergone extensive independent validation and is now the subject of a major European clinical trial (MINDACT, see below). 47,48 A second reason for the importance of tumor stratification involves the prediction of response to specific therapies (predictive biomarkers). While we have already cited examples where tumors with specific genetic features have been shown to respond to particular therapies, such as EGFR mutations and EGFR inhibitors, or HER2 gene amplifications and traztuzamab, it should be noted that these cases likely represent only the fortuitous minority where the predictive biomarker corresponds to the drug target itself (e.g. trastuzamab and HER2). In many other cases, identification of the relevant predictive biomarker may not be so clear cut. For example, it has been reported that tumors with mutations in the B-RAF gene may impart sensitivity to MEK inhibitors, 49 or that mutations that constitutively activate the RAS oncogene may impart sensitivity to histone deactylating agents. 50 Besides biomarkers that predict drug sensitivity, there also exists a separate class of mutations in particular oncogenes that impart drug resistance. Examples of such "anti-predictive" factors include the lack of efficacy of EGFR inhibitors in colon cancers with k-ras mutations, 51 and resistance to traztuzamab in HER2-amplified breast cancers with concomitant activation of the PI3K pathway. 52 Beyond targeted therapies, several predictive biomarkers for conventional cytotoxic agents have also been proposed, such as mutated β-tubulin for taxanes and TS for 5-FU. 53,54 A third important justification for the implementation of tumor stratification is that it maximizes the opportunity for therapeutic benefits to be observed, particularly in the conduct of clinical trials evaluating novel therapies. Analyses by the pharmaceutical industry of current drug trials have revealed that most anti-cancer drugs typically fail during late stage evaluation (i.e. Phase II or III) typically due to lack of detectable efficacy or toxicity. 55 However, because such trials are typically performed in unselected patient populations, it is quite possible that certain drugs may elicit dramatic responses in a small population of patients, but that this beneficial effect may be diluted when such patients are intermixed with the larger population. Indeed, drugs such as herceptin and gefitinib would likely have not been approved if tested in an unselected set of cancer patients.
Thus, examples such as these provide a strong motivation for the practice of tumor stratification in the management and evaluation of cancer patients. 56 One example of a major cancer type where patient outcomes have been significantly impacted by molecular stratification has been childhood acute lymphoblastic leukemia (cALL). Prior to 1972, cALL was largely regarded and treated as a "single disease", with a dismal cure rate of only 5%. 57 Since that time, however, progress in cytogenetic and molecular technologies has revealed that cALL is in fact a heterogen eous collection of distinct diseases. 58 Through detailed comparisons of individual cALLs with their normal counterparts in the hierarchy of hematopoietic development, we now know that distinct subtypes of cALL are likely to originate from blood progenitor cells originally committed to differentiate via a T or B-cell lineage, but in cALL these progenitor cells have acquired mutations causing both deregulated cell proliferation and stage-specific developmental arrest. 59 Currently, cALLs are classified into at least seven distinct subgroups, with each subgroup being either associated with a particular stage of hematopoietic differentiation or a specific cytogenetic aberration such as hyperdiploidy or signature chromosomal translocations including TEL-AML1 (otherwise known at ETV6-RUNX1) or BCR-ABL. 60 Importantly, supporting the notion that these cALL subtypes are likely to represent distinct biological entities, in a large series of hundreds of cALL patients followed up over 15 years, it has been established that each of these subtypes is associated with a distinct course of disease progression. 58 For example, patients having TEL-AML1 or E2A-PBX1 fusions are more likely to exhibit relatively favorable clinical outcomes compared to patients with BCR-ABL or MLL-AF4 fusions. The biological distinctiveness of these subgroups has also been further supported by genome-wide gene expression profiling studies, confirming that the different cALL subtypes are each associated with characteristic patterns of gene expression. 61,62 Knowledge regarding the existence of these cALL subtypes has revolutionized treatment for cALL in several ways. First, it permitted clinicians to adapt and modify standard therapy regimens, once used to treat all cALLs, specifically to the different subtypes. For example, while most cALL cases are treated with a three phase regimen of remission-induction, intensification/consolidation, and continuation therapy, patients with mature B-cell ALL are further treated with short-term intensive chemotherapy, 63,64 while patients with T-Cell ALLs commonly are additionally treated with cyclophosphamide and asparagine during the remission induction phase. 65 Second, knowledge of the Progress in the Molecular Stratification of Cancer

IMPACT OF CANCER HETEROGENEITY -THE EXAMPLE OF CHILDHOOD ALL
specific molecular aberrations in each subtype allowed drug discovery efforts to focus on these aberrations as potential pharmacologic targets. This is best seen in BCR-ABL positive cALL, a subtype that has traditionally been associated with very poor prognosis. 66 However, with the development of imatinib mesilate, a small molecule inhibitor of the ABL kinase, there is now an effective treatment for BCR-ABL positive cALL, with initial response rates being close to 100% in this specific subgroup of cALL. 67 Through the tailoring of generic regiments and the availability of subtype specific targeted therapies, the cure rate today for childhood ALL exceeds 80%. While cALL remains a powerful testament to the utility of tumor stratification, the impressive advances in our ability to manage cALL, however, should not be taken as a sign that this disease has been truly conquered. There is still a significant fraction of cALL patients (20%) who are not cured with existing therapies, and our ability to manage ALL in older patients in still far from optimal. 66 One possible reason is that additional genetic and molecular heterogeneity may exist in this disease that has previously been undetected, and that further genetic subdivisions will be required to further differentiate between these various seven subclasses. Fortunately, more comprehensive and high resolution technologies are now available that are capable of identifying genetic and epigenetic aberrations on a genome wide scale. For example, a recent genome wide SNP analysis of ALLs associated with B-cell phenotypes identified several mutations, deletions, and structural rearrangements in various regulators of B-cell development, such as PAX5 and TCF3. 68 Future work will then allow ALL treatments to be individualized to the specific genetic makeup of each tumor cell.
In contrast to the hematopoietic malignancies, similar attempts to stratify many of the major solid cancers such as lung, gastric, and liver cancer have been hampered by many challenges. One major difficulty is that solid tumors are often typically characterized by highly aberrant chromosomal karyotypes that are considerably more complex than those observed in the hematopoetic malignancies. 69 With few exceptions, the heightened complexity of solid tumor karyotypes has thwarted attempts by conventional cytogenetics to identify signature aberrations associated with different solid tumor subtypes; for example, recurrent chromosomal translocations that may produce important fusion genes and transcripts. Indeed, it is only in the past few years that recurrent fusion genes such as TMPRSS2-ERG and EML4-ALK in solid tumors have been identified in prostate and lung cancer, respectively. 70,71 In both these cases, these fusion genes were not identified through conventional chromosomal cytogenetics, but through innovative high-resolution genomic approaches. Nevertheless, these examples provide important proof that recurrent fusion transcripts do indeed exist in solid tumors, and it is expected that identification of novel fusion genes will continue, particularly with the use of next-generation deep sequencing methods. 72 Besides increased chromosomal complexity, a second challenge faced in the molecular stratification of solid tumors lies in the traditional reliance of diagnostic platforms such as immunohistochemistry or gene sequencing that typically measure single biomakers (either genes or proteins). While such techniques are amenable to the detection of highly recurrent aberrations, recent large scale comprehensive sequencing analysis of various solid cancers, including pancreatic, brain, breast and colon cancers, has demonstrated that many solid tumors are likely to have a preponderance of mutations in multiple genes with low rates of recurrence. 22,73,74 However, when such genes are grouped together by their respective pathways of activation, analysis has shown that such mutations tend to target a series of common core signaling pathways. 73,75 These findings suggest that these oncogenic pathway, rather than the gene itself, is likely to represent the unit of mutational selection in solid cancers. Thus, the molecular stratification of these cancers will require analytical platforms that can measure multiple genes simultaneously, rather than one gene or protein at a time. Unfortunately, the expense and complexity of such genomic platforms are still prohibitively expensive and insufficiently robust to validate their regular use in clinical practice.
A third issue of complexity facing the molecular stratification of solid tumors is that unlike blood disorders, solid tumors comprise a diverse mix of many different cell types including cancer cells, stromal cells, infiltrating immune cells, and endothelial vasculature. 76 Recent evidence has shown that in many cases, disease progression and treatment response for many solid tumors likely involve a combination of interactions between these different cell types. For example, studies in experimental models have shown that tumor-associated fibroblasts, while nonmalignant in their genetic makeup, can play a profound role in the overall rate of tumor growth, 77 and studies have shown that colon cancer disease prognosis can be affected either by the rate of immune cells, 78 or by the presence of tumor stroma. 79 The necessity to consider interactions between such entities raises another level of complexity that is overtly less of an issue in the blood disorders, which are commonly thought of as clonal diseases.

CHALLENGES FOR THE MOLECULAR STRATIFICATION OF SOLID TUMORS
Besides the biological challenges of increased genomic complexity, pathway directed mutational profiles, and interactions with various cell types, it is also worth noting that a number of practical and logistic issues have also challenged research into the molecular stratification of solid tumors. Most solid tumors are collected through some invasive means such as surgery or biopsy, and so analysis of these tumors is limited only in clinical scenarios where such invasive procedures are an option. In the case of NSCLC, since late stage lung tumors are not treated with surgery, our knowledge of the molecular factors at play in late stage NSCLC is still relatively poor, despite the majority of NSCLC patients being diagnosed with advanced inoperable disease. 80 In such cases, samples are typically limited to material through diagnostic methods such as fine-needle aspiration (FNAs), which has led some investigators to devise novel ways to analyze such so-called 'low-volume' samples. 81,82 The challenge of obtaining large numbers of surgical samples has also impeded the establishment of large-scale molecular databases of these cancers to definitely establish overall levels of molecular diversity for many tumor types. Addressing this problem has required the formation of large multi-center consortia, or the development of novel statistical meta-analysis protocols to combine genomic data from multiple data sets to identify conserved molecular patterns. 83,84 Despite these significant challenges, progress is indeed being made towards the molecular stratification of solid cancers. One obvious example of a major solid cancer where attempts at molecular stratification are showing progress is carcinoma of the breast. As mentioned above, breast cancers are already routinely subdivided into ERpositive and ER-negative subtypes, which are associated with distinct prognosis and responses to anti-hormonal treatment. Another important subtype of breast cancer for which routine testing is performed are tumors that overexpress the HER2 growth factor receptor, as such tumors can be targeted with traztuzamab. In addition to these three well-recognized subgroups, results from more comprehensive expression profiling analysis has also revealed the existence of further subgroups. For example, it is now clear that ER+ tumors sometimes called "luminal" tumors, reflecting the fact that they are likely to arise from luminal cells in the mammary epithelium) can be further subdivided into two more subtypes -Luminal A and Luminal B, that show very distinct patterns of patient prognosis. 35,85 Genomic signatures have also been identified that can predict the mutational status of important cancer genes such as p53 in breast cancer, 86 and evidence is further emerging that these breast cancer subtypes could also exhibit distinct responses to various forms of conventional cytotoxic therapy as well as targeted therapies such as MEK inhibitors. 87,88 Besides subtypes based solely on gene expression patterns, recent work has also demonstrated that combining gene expression-based subtypes with DNA copy number information can further subdivide patients into even more homogenous populations. 89 Such "integrative genomic" analyses will likely be required to address the rampant genomic complexity of solid tumors.
In contrast to breast cancer, consensus regarding specific molecular subtypes in many other solid tumors has yet to gain general acceptance. In our own specific research area of gastric cancer, several distinct molecular subtypes of gastric cancer have been reported, and correlative analysis of these subtypes to clinical information has identified expression signatures associated with differing histological subtypes and in some cases prognosis. 90,91 However, few of these genomic predictors have been independently validated in reasonably large independent patient cohorts, and attempts at validation are confounded by differences in the patient populations from different countries and centres, including differences in treatment protocol. Furthermore, with the increasing number of genomic biomarkers linked to cell lineage, drug sensitivity and pathway activation status reported in the literature, there is also the oft-cited concern that different studies ostensibly targeting the same biological phenotype (e.g. prognosis in early stage breast cancer) tend to identify non-overlapping signatures. [92][93][94][95] Such concerns, however, should be evaluated in the light of recent studies showing that apparently 'distinct' signatures tend to nevertheless target the subpopulations of tumors, and that consensus can be ultimately established through the rigorous cross-comparison of different data sets, as has recently been demonstrated for breast cancer and NSCLC. 48,96 Ultimately, the acid test for the clinical robustness of such stratifications will have to occur in the form of well-designed randomized prospective control trails designed to robustly test the actual clinical utility of such proposed schema. In fact, there are already several trials that are already underway or being proposed. For example, the MINDACT trial involves the recruitment of 6,000 women across multiple centers with early stage breast cancer whose treatment will depend upon their expression profiles of the 70-gene prognostic classifier described above. 97 Another exciting series of trials involves the use of genomic signatures to guide chemotherapy treatment in early-stage lung cancer. 98 This is another clinically challenging disease where the standards of chemotherapy have not been established, and thus treatment of NSCLC Progress in the Molecular Stratification of Cancer

PROGRESS IN THE MOLECULAR STRATIFICATION OF SOLID CANCERS
patients is largely due to physician preference. Results from these trials will definitely establish if genomic technologies are at the stage where they are sufficiently robust and reproducible for the information to be interpreted in such a fashion that they actually guide clinical treatment.
We close this review with a brief survey of some anticipated future challenges facing clinicians and researchers interested in the molecular stratification of cancer. With the increasing ability to characterize tumors at multiple levels including mutations, expression signatures, copy number aberrations, and epigenetic profiles, it will be critical to develop robust analytical systems whereby such multi-dimensional data can be integrated, even possibly with host genotype information, to define robust biological subtypes of cancer. Analysis of the independence and relationship between different genetic aberrations, such as the mutual exclusivity of EGFR and k-ras mutations in lung cancer, 99 could also prove very useful in delineating major oncogenic signaling pathways and their subcomponents in primary tumors. Another challenge arising from the notion that most, if not all, cancers comprise multiple subtypes is how to transform currently existing drug discovery programs, that traditionally have focused on identifying single drugs with efficacy in the majority of cancers, to identifying many drugs targeted for distinct cancer subtypes, in a rapid and cost-efficient manner. A third challenge arises when one considers that many of these studies have relied upon the molecular analysis of tumors, which can only be obtained through invasive means such as surgery or biopsies. It would be exciting to assess if similar patterns of cancer stratification could be inferred using profiles derived from more easily obtainable body fluids from cancer patients such as blood or stool. Finally, one also needs to grapple with recent findings suggesting that there exists in tumors a rare stem cell-like population that can prove highly drug resistant. 100 Techniques to detect and classify such cancer stem cells will represent a key challenge in cancer classification, and success in doing so may represent the pivotal shift in our ability to treat cancer in a way that tangibly affects overall cancer outcomes.