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. Author manuscript; available in PMC: 2011 May 3.
Published in final edited form as: Dev Dyn. 2008 Jul;237(7):1851–1861. doi: 10.1002/dvdy.21582

Polycomb group gene expression in the sea urchin

Eric A Gustafson 1, Gary M Wessel 1,*
PMCID: PMC3086543  NIHMSID: NIHMS287594  PMID: 18521949

Abstract

Embryonic development in metazoan animals involves a remarkable degree of both genetic and epigenetic regulation. Polycomb group (PcG) genes are essential for initiation and maintenance of epigenetic developmental regulation. This report identified homologous PcG genes representing the core components of three known Polycomb repressive complexes in the sea urchin Strongylocentrotus purpuratus. Quantitative, temporal and spatial analyses revealed two significant aspects of their transcript expression during embryonic development. First, almost all showed a localized expression pattern in mesenchyme blastula and gastrula-stage embryos. As the embryo specifies its three germ layers, it may require it PcG genes transcripts to remain in localized stem cell populations, but turn-over in differentiating tissues. Second, their transcript levels gradually decreased during larval pluteus stages. This is consistent with progressive stem cell differentiation in the embryo. Taken together, these results suggest that PcG genes are conserved in S. purpuratus and are actively expressed during early embryogenesis.

Keywords: Polycomb group proteins, sea urchin, polycomb repressive complexes

Introduction

Cell-type specific gene expression requires precise developmental regulation. This epigenetic regulatory process coordinates spatial and temporal cell differentiation within the developing embryo. A large body of evidence in multiple organisms suggests that the conserved family of Polycomb group (PcG) proteins is essential for this process. PcG proteins initiate and maintain heritable transcriptional repression of multiple genes involved in cell differentiation during development. The first PcG genes were genetically identified in Drosophila as a group of repressors essential for proper Hox gene expression (Lewis, 1978; Struhl, 1981; Duncan, 1982; Jurgens 1985). Extensive analysis in several animals, including mammals, suggests that while as many as five different complexes may exist, PcG proteins form three general biochemically and functionally distinct chromatin-binding complexes, PhoRC, PRC2 and PRC1 (Schuettengruber et al., 2007). Although it is not completely understood how all three PcG complexes functionally cooperate in gene silencing, several models suggest that PhoRC targets transcriptional repression, PRC2 initiates the repression and PRC1 maintains the repressive conditions (Muller and Kassis, 2006; Rajasekhar and Begemann, 2007; Cao et al., 2005). However, the actual mechanism is likely more sophisticated with each PcG complex contributing some overlapping functions.

In Drosophila, the Pho-repressive complex (PhoRC) contains Pleiohomeotic (Pho) or Pho-like and dSfmbt (Klymenko et al., 2006). Pho and Pho-like are the only PcG proteins known to bind DNA in a sequence-specific manner, through their conserved C-terminal Zn-fingers, and specifically target PhoRC to Polycomb response elements (PREs) as found in Drosophila Hox genes (Strutt et al., 1997; Mohd-Sarip et al., 2002; reviewed by Muller and Kassis, 2006). Yin-Yang was identified as the mammalian homolog to Drosophila Pho and can functionally compensate for loss of Pho (Brown et al., 1998; Atchison et al., 2003). The malignant brain tumor (MBT) domain repeats are essential for dSfmbt to selectively interact with mono- and di-methylated H3-K9 and H4-K20 (Klymenko et al., 2006). The human L3MBTL1 protein is thought to facilitate chromatin compaction by utilizing its 3 MBT domains for recognition of specific histone methylations and for binding two nucleosomes simultaneously (Trojer et al., 2007). Together these data suggest that the PhoRC recognizes and binds to PcG targets and may promote silencing directly or by recruiting other Polycomb-repressive complexes (PRCs).

The core Polycomb repressive complex 2 (PRC2) components are conserved throughout metazoans and include Enhancer of Zeste 2 (Ezh2), which is a SET-domain histone methyltransferase that catalyzes trimethylation of lysine 27 on histone H3, Suppressor of Zeste 12 (Suz12), Embryonic ectoderm (Eed), and Retinoblastoma binding protein 4 (Rbbp4)(reviewed by Schuettengruber et al., 2007; Levine et al., 2004). Suz12 is required for Ezh2 methyltransferase activity and Eed is necessary for PRC2 complex stability (Cao and Zhang, 2004; Pasini et al., 2004). Rbbp4 was originally identified as a retinoblastoma binding protein and is found in several different complexes involved in transcriptional regulation and may function as a scaffolding protein that mediates protein-protein interactions within the complex (Qian et al., 1993; Schuettengruber et al., 2007).

Genome analysis in a variety of metazoans suggests that Polycomb repressive complex 1 (PRC1) genes originated during animal evolution, since they are absent from fungi and plants (Springer et al., 2002; Schuettengruber et al., 2007). Biochemical reconstitution experiments have shown that the core PRC1 components are conserved between Drosophila and humans and consist of Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc) and Drosophila RING1 (dRING)(Francis et al., 2001; Levine et al., 2002). PRC1 recognizes tri-methylated lysine 27 on histone H3 through the Pc chromodomain (Fischle et al., 2003). While the exact molecular mechanism that PRC1 employs for transcriptional repression is not completely understood, it is generally thought that PRC1 maintains the selective transcription inhibition conditions during development (Levine et al., 2004).

Phylogenetic analysis of the sea urchin genome Strongylocentrotus purpuratus underscores the remarkable conservation of key developmental regulatory genes (Sodergren et al., 2006). While several S. purpuratus Hox genes have been characterized (Angerer et al., 1989; Arenas-Mena et al., 1998; Arenas-Mena et al., 2000; Arnone et al., 2006; Cameron et al., 2006), the conservation and expression of Polycomb group genes are unknown. To gain insight into their role in S. purpuratus embryonic development, we have identified several predicted PcG homologs and characterized their transcript expression. These genes represent the core members of all three general biochemical PcG complexes. S. purpuratus PcG transcripts are largely present throughout oocytes and early embryos, but progressively localize in a tissue-specific manner in later embryos. This is consistent with a ubiquitous epigenetic imprinting mechanism early in embryogenesis which may become restricted to distinct stem cell populations as other cells differentiate.

Results

Identification of S. purpuratus polycomb group protein homologs

PcG protein sequences from D. melanogaster, M. musculus and H. sapiens were used for a PcG homolog computational search in the S. purpuratus genome. The BLAST analysis (http://www.hgsc.bcm.tmc.edu/projects/seaurchin/) identified thirteen polycomb and polycomb-related homologous gene predictions (Table 1). These were concluded to be PcG genes based on amino acid sequence and similarity of domain architecture to known PcG genes (Figure 1 and Table 1). All the S. purpuratus PcG genes listed in Table 1 have BLAST e-values lower than 1e-15 (Table 1). Although most of the genes identified exhibit the highest degree of conservation in their predicted functional domains, the biochemical nature of these putative complexes in S. purpuratus are unclear. However, their Drosophila counterparts comprise the Pho Repressive Complex (PhoRC), Polycomb Repressive Complex 2 (PRC2), and Polycomb Repressive Complex 1 (PRC1). (Klymenko et al., 2006; reviewed by Levine et al., 2004).

Table 1.

S. purpuratus homologs of conserved Polycomb group proteins

Predicted
PcG Complex*
Gene Name Genbank
accession
Predicted
polypeptide length
Domains and function Homologs
(BLAST score)
PhoRC Sp-Pho XP_790188 400 C2H2 Zn-finger (4) transcription factor, binds Polycomb Responsive
Elements (PREs)
D. rerio (8e-112)
H. sapiens (2e-104)
Sp-Mbt1 XP_785195 1805 MBT-domain (3), Sterile α-motif (SAM) protein-protein interaction
domain
M. musculus (9e-83)
H. sapiens (4e-82)
Sp-Mbt2 XP_001202275
XP_001201293
1944 MBT-domain (4), SAM protein-protein interaction domain G. gallus (2e-142)
H. sapiens (4e-141)
Sp-L(3)mbt XP_792284 992 MBT-domain (3), C2H2 Zn-finger (2), SAM protein-protein
interaction domain
H. sapiens (1e-180)
M.musculus (3e-179)

PRC2 Sp-Ezh2 XP_790741 551 SET-domain histone methyltransferase, specific tri-methylation of
H3K27 recognized by the Polycomb (Pc) chromodomain
X. tropicalis (2e-179)
H. sapiens (5e-179)
Sp-Eed XP_786345 461 WD40-domains (5) coordinate multi-protein complex assemblies as
a scaffold for protein interactions
D. rerio (2e-157)
H. sapiens (2e-156)
Sp-Suz12 XP_788076 757 No defined domains, required for HTMase and silencing activity
of the EED-EZH2 complex
D.rerio (4e-123)
M. musculus (1e-122)
Sp-Rbbp4 XP_780271 430 WD40-domains (5) M. musculus (0.0)
H. sapiens (0.0)

PRC1 Sp-Pc1 SPU_20586 1393 Chromodomain, chromatin organization modifier domain involved
in promoting heterochromatin condensation
R. norvegicus (1e-22)
C. familiaris (2e-22)
Sp-Pc2 SPU_20946 456 Chromodomain D. melanogaster (7e-16)
R. norvegicus (2e-16)
Sp-Ph XP_789940 788 Zn-finger, SAM protein-protein interaction domain H. sapiens (5e-50)
M. musculus (9e-50)
Sp-Psc XP_783799 479 C3CH4 Zn-finger (RING-finger) domain M. musculus (3e-54)
H. sapiens (9e-54)
Sp-Ring1 XP_788976 306 C3CH4 Zn-finger (RING-finger) domain M. musculus (3e-93)
H. sapiens (4e-93)
*

The S. purpuratus polycomb group protein homologs are categorized based on genetic and biochemical evidence of complex association from their respective counterparts in various animals

Figure 1.

Figure 1

Predicted domain architecture of S. purpuratus polycomb group proteins. The schematic depiction for each polycomb group protein analyzed in this report, as well as each domain, are proportional to their predicted amino acid length. The domain location is representative of its position within each protein. The polycomb repressive complex groupings are based on the biochemical complex association of their homologs in other animals.

While the PhoRC was only recently characterized, it is clear that Pho and MBT-domain repeat proteins play an important role in heterochomatin formation, transcriptional silencing and, at least in Drosophila, target PREs (Klymenko et al., 2006; Trojer et al., 2007; Muller and Kassis, 2006). Therefore, for the purpose of this report, all S. purpuratus MBT-domain repeat and Pho genes are categorized as PhoRC genes. A genomic search identified Sp-Pho as the S. purpuratus Pho homolog due to its high sequence similarity to the human YY1 as well as the similar 4 C-terminal Zn-finger domain structure (Table 1 and Figure 1). The S. purpuratus genome contains 3 predicted genes with repeated MBT domains. Sp-L(3)mbt shares significant sequence similarity to both mouse and Drosophila L(3)mbt and shares a similar arrangement of its 3 MBT domains, 2 Zn-fingers and C-terminal SAM domain (Table 1 and Figure 1). Although Sp-Mbt1 and Sp-Mbt2 both contain 4 MBT domains and a C-terminal SAM domain, the lack of additional sequence similarity makes it unclear whether they represent S. purpuratus dSfmbt or Scml homologs.

All four PRC2 core genes are present in the S. purpuratus genome. Sp-Ezh2 was identified as the S. purpuratus Ezh2 homolog and has a C-terminal SET histone methyltransferase domain (Table 1 and Figure 1). Although Sp-Suz12 contains no known functional domains, it exhibits considerable sequence similarity to mouse and Drosophila Suz12 (Table 1 and Figure 1). The Sp-Eed is homologous to human and mouse Eed as well as with Drosophila Extra sex combs (Esc) and is predicted to have 5 WD40 domains (Table 1 and Figure 1). The Sp-Rbbp4 sequence is also predicted to have 5 WD40 domains (Table 1 and Figure 1).

The core PRC1 genes are also found in the S. purpuratus genome. Both Sp-Pc1 and Sp-Pc2 contain the conserved chromodomain that is known to specifically interact with the H3K27 tri-methylation mark (Figure 1 and Table 1). These genes show the greatest conservation within the predicted chromodomain and their flanking sequences are significantly divergent. Since the Sp-Pc1 and Sp-Pc2 gene predictions did not have assigned Genbank accession numbers, they are listed by the Baylor University Sea Urchin Genome project predicted gene model numbers (Table 1). The Sp-Ph sequence is considerably similar to both human and Drosophila and contains a Zn-finger and C-terminal SAM domain (Table 1 and Figure 1). The genome search also revealed the RING-finger domain containing-gene Sp-Psc as the posterior sex combs homolog due to its high similarity to Bmi1 polycomb ring finger proteins in mice and humans (Figure 1 and Table 1). Finally, the RING-finger domain gene prediction Sp-Ring1 was identified through sequence similarity to the PRC1-associated dRING1 in Drosophila and its mammalian counterparts in humans and mice (Table 1 and Figure 1).

Quantitative Sp-PcG transcript profiles

S. purpuratus PcG gene transcripts were analyzed by qPCR in order to address global changes in transcript levels during developmental. Two distinct expression profile groups are evident. In one group, a maternally loaded transcript stockpile is present in the egg, which decreases to around 40% that level by gastrula stage. Sp-Pho, Sp-Mbt1, Sp-Mbt2, Sp-Suz12 and Sp-Ezh2 exhibit this first type of expression profile (Figure 2). In the second group, relatively low levels of each transcript are present in the egg which then dramatically increases between 6-fold to 12-fold by blastula and mesenchyme blastula stages. Sp-L(3)mbt, Sp-Eed, Sp-Rbbp4, Sp-Psc, Sp-Pc1, Sp-Pc2 and Sp-Ph exhibit this second type of expression pattern (Figure 2).

Figure 2.

Figure 2

Quantitative RT-PCR (qPCR) measured relative S. purpuratus polycomb group mRNA levels. (A) Sp-pleiohomeotic, (B) Sp-mbt1, (C) Sp-mbt2, (D) Sp-L(3)mbt, (E) Sp-Retinoblastoma binding protein 4, (F) Sp-supressor of zeste 12, (G) Sp-enhancer of zeste 2, (H) Sp-embryonic ectoderm development, (I), Sp-posterior sex combs, (J) Sp-polycomb 1, (K) Sp-polycomb 2, and (L) Sp-polyhomeotic during several embryonic developmental stages. All values were normalized against Sp-ubiquitin RNA and represent RNA levels as the fold difference relative to the respective RNA levels in the egg. Error bars indicate standard deviation from triplicate qPCR reactions for each sample.

In situ RNA hybridization profiles

PhoRC

The spatial expression patterns for S. purpuratus PcG gene transcripts were analyzed by whole mount in situ RNA hybridization. Sp-Pho, Sp-Mbt1 and Sp-Mbt2 all displayed a very similar expression pattern which is consistent with their qPCR results. High levels for each of these transcripts were uniformly distributed from developing oocytes to blastula-stage embryos (Figure 3). While the Sp-L(3)mbt transcript was also uniformly distributed throughout oocytes, it was dramatically reduced in eggs as well as during cleavage and in blastula-stage embryos (Figure 3). By mesenchyme blastula-stage, however, all of the predicted PhoRC transcripts were enriched in the vegetal plate, primary mesenchyme cells and oral ectoderm. These transcripts were all localized to the tip of the archenteron, and oral ectoderm in gastrula and reduced levels for each were present in the pluteus gut. Sp-Mbt1 and Sp-Mbt2 transcripts also showed localization to the blastopore lip in gastrula embryos (Figure 3).

Figure 3.

Figure 3

Spatial expression of predicted PhoRC and MBT domain-containing S. purpuratus genes Sp-Pho, Sp-Mbt1, Sp-Mbt2, and Sp-L(3)mbt in oocytes, eggs and developing embryos using whole-mount in situ RNA hybridization (WMISH).

PRC2

Sp-Ezh2 had a uniform and very abundant transcript distribution in the oocytes and eggs which remained uniform, but gradually diminished later in embryogenesis (Figure 4). Sp-Suz12, Sp-Rbbp4 and Sp-Eed displayed expression patterns very different very different from Sp-Ezh2. While all their transcripts were abundant and uniformly distributed in the oocyte, Sp-Eed and Sp-Rbbp4 were considerably reduced in eggs, cleavage and blastula-stage embryos. Sp-Suz12 remains abundantly distributed throughout the egg and early cleavage stages, but barely detectable in blastula-stage embryos. However, Sp-Suz12, Sp-Rbbp4 and Sp-Eed transcripts reappear at mesenchyme blastula stage enriched at the vegetal plate, primary mesenchyme cells and oral ectoderm. These transcripts are then localized to the tip of the archenteron, the oral ectoderm and the lip of the blastopore during gastrulation. Sp-Rbbp4 transcript is absent in the aboral ectoderm of gastrula and presumably the aboral ectoderm in mesenchyme blastula-stage embryos. Sp-Suz12, Sp-Rbbp4 and Sp-Eed transcripts are then restricted to the left coelomic pouch of prism (black arrows). While the left coelomic pouch Sp-Eed and Sp-Rbbp4 transcript localization persists into the pluteus stage (black arrow), Sp-Suz12 is undetected in plutei. During metamorphosis, it is the cells of the left coelomic pouch that contribute to most of the adult rudiment and to the germline.

Figure 4.

Figure 4

Spatial expression of predicted S. purpuratus PRC2 genes Sp-Suz12, Sp-Ezh2, Rbbp4 and Sp-Eed by WMISH for the indicated developmental stages. Black arrows for Sp-Suz12 in prism, Sp-Eed and Sp-Rbbp4 in prism and pluteus-stage embryos indicate the left coelomic pouch.

PRC1

Sp-Pc2 and Sp-Psc appear to have similar transcript localization patterns from oocyte to mesenchyme blastula stage. The transcripts are uniformly distributed in the oocyte, but are weakly detected in eggs and early cleavage stages. Both transcripts are enriched in the vegetal plate and Sp-Pc2 appears also enriched in the oral ectoderm. By gastrula-stage, however, Sp-Pc2 and Sp-Psc transcript localization is significantly different. While Sp-Psc appears almost uniform in gastrula, Sp-Pc2 is enriched at the tip of the archenteron, blastopore lip and oral ectoderm. The Sp-Pc2 transcript staining is diminished in the pluteus and is exclusively in the gut, whereas the Sp-Psc is undetectable in the pluteus (Figure 5). Sp-Ph transcript is highly enriched throughout the oocyte but dramatically reduced in eggs and cleavage-stage embryos. Blastula through gastrula-stage embryos have Sp-Ph uniformly distributed in all cells, while plutei have reduced levels of transcript largely localized to the gut (Figure 5). Although Sp-Pc1 transcript detection was weak, Sp-Ring1 and Sp-Pc1 transcripts are uniformly distributed throughout development with a faint enrichment at the vegetal plate in mesehchyme blastula and reduced by pluteus stage (Figure 5).

Figure 5.

Figure 5

Spatial expression of predicted PRC1 S. purpuratus genes Sp-Pc2, Sp-Psc and Sp-Ph by WMISH in oocytes, eggs and the indicated stages of developing embryos.

Discussion

We have identified the core PcG component genes in the sea urchin S. purpuratus and documented their temporal and spatial transcript expression during early embryonic development. While the exact functions of these genes in basal deuterostomes are unknown, a large body of evidence has elucidated their roles in the epigenetic regulation of several developmental processes. In particular, PcG expression is critical to prevent differentiation in both embryonic and adult stem cells through targeted transcriptional repression (Spivakov and Fisher 2007; Kamminga et al., 2006). Global analysis of PcG binding in both human and mouse embryonic stem (ES) cells suggest that they maintain silencing of genes associated with differentiation (Lee et al., 2006; Boyer et al., 2007). Therefore, the principal function for PcG proteins in stem cells appears to be maintenance of pluripotency.

S. purpuratus PcG transcripts exhibited two general expression profiles. One group showed high levels of expression throughout the eggs and in early embryos which gradually became localized and less abundant by mesenchyme blastula and gastrula-stage embryos. The second group of transcripts were present in low levels in eggs and early embryos, but were abundant and localized by mesenchyme blastula and gastrula-stage embryos. Interestingly, Sp-Suz12 displayed characteristics of both expression profiles. These expression patterns may reflect the progressive cellular differentiation as the embryo develops. In cell culture as well as developing mouse embryos, Eed and Ezh2 levels steadily decrease upon stem cell differentiation (de la Cruz et al., 2005; Kuzmichev et al., 2005; Silva et al., 2003). Consistent with these observations and despite any difference in transcript levels in the early embryos, all showed a gradual decrease during larval development.

The restricted transcript localization for many of the genes during mesenchyme blastula and gastrula stages may also reflect localized stem cell populations in the embryo. The Psc mouse homolog Bmi1 was shown to control neuronal stem cell proliferation and renewal (Molofsky et al., 2003; Zencak et al., 2005). The Sp-Psc transcript appeared upregulated in mesenchyme blastula embryos and localized to the vegetal plate. It should be noted that every S. purpuratus PcG gene transcript examined was enriched at the vegetal plate in mesenchyme blastula embryos. Recent evidence has shown that Eed transcription is specifically upregulated in mouse embryonic stem cells by two essential stem cell factors Oct3/4 and STAT3 (Ura et al., 2008 in press). While the Sp-Eed transcript is largely absent in the early embryo stages, it is dramatically upregulated and localized in mesenchyme blastula and gastrula embryos. While its transcript level is reduced in larvae, it remains prominent in the left coelomic pouch. This group of cells contributes to the adult rudiment during metamorphosis and expresses the conserved germ cell markers vasa and piwi (Juliano et al., 2006; Voronina et al., 2008). Since the adult sea urchin must inherit a stem cell population from the larval embryo, Sp-Eed expression may help maintain pluripotency in these cells.

In addition to stem cell identity, PcG proteins are associated with several other developmental processes. For example, the mouse Eed was first identified as essential for proper cell movements during gastrulation (Faust et al., 1998). Interestingly, several of the S. purpuratus PcG gene transcripts, including Sp-Eed, were localized to vegetal plate just prior to gastrulation and to the lip of the blastopore in gastrula. This may reflect a morphogenic role for Sp-Eed and other PcG genes in sea urchin gastrulation. Aberrant expression of PcG genes is also associated with several human cancers (reviewed by Rajasekhar and Begemann, 2007). Finally, the epigenetic transcriptional silencing by PcG proteins is implicated in developmentally regulated X-chromosome inactivation (reviewed by Yang and Kuroda, 2007).

This report identified homologous PcG genes representing the core components of three known Polycomb repressive complexes in S. purpuratus. Quantitative, temporal and spatial analyses revealed two significant aspects of their transcript expression. Most exhibited a localized expression pattern in mesenchyme blastula and gastrula-stage embryos. This suggests that S. purpuratus may require it PcG genes transcripts in local stem cell populations during germ layers specification. Also, their transcript levels gradually decrease during pluteus stages. This is consistent with progressive stem cell differentiation in the embryo. Altogether, these results suggest that PcG genes are conserved in S. purpuratus and are dynamically expressed during early embryogenesis.

Experimental Procedures

Animals

Gravid Strongylocentrotus purpuratus adults were obtained from the wild and kept in artificial seawater at 16°C. Gametes were acquired by injecting the animals with KCl (0.5M). Whole ovaries were dissected from females. Following fertilization, embryos were cultured in artificial seawater at 16°C, collected at various developmental stages and either fixed for in situ hybridization or used for RNA extraction.

Sea urchin polycomb group homologs

Computational gene sequence predictions for Sp-pleiohomeotic, Sp-mbt1, Sp-mbt2, Sp-L3mbt, Sp-polycomb1, Sp-polycomb2, Sp-polyhomeotic, Sp-posterior sex combs, Sp-enhancer of zeste 2, Sp-suppressor of zeste 12, Sp-embryonic ectoderm development and Sp-nucleosome remodeling factor 55 were identified in the S. purpuratus genome (http://www.hgsc.bcm.tmc.edu/projects/seaurchin/) by BLAST analysis using polycomb group protein sequences from D. melanogaster, H. sapiens, M. musculus and D. rerio. Predicted conserved protein domains for each S. purpuratuspolycomb group gene were identified using the pfam program (http://pfam.janelia.org/)(Finn et al., 2006).

Cloning

RNA was purified from whole S. purpuratus embryos and ovaries using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) and as described previously (Bruskin et al., 1981). Approximately 1-2kb fragments of the S. purpuratus polycomb group protein homologs were cloned by RT-PCR (Promega, Madison) into pGEM-T EZ (Promega, Madison) using gene-specific primers and RNA purified from various developmental stages (Table 1). The clones were also verified by sequencing.

Quantitative PCR (qPCR)

Template cDNA was prepared using the TaqMan® Reverse Transcription Reagents kit (Applied Biosystems, Foster City) and RNA purified from S. purpuratus ovaries, eggs and embryos (described above). Primer sets were designed to amplify 100-175 base pair-sized products using the primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Table 1). The 7300 Real-Time PCR system (Applied Biosystems, Foster City) and the Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad) were used for qPCR data acquisition. Data from each gene was normalized to ubiquitin mRNA levels and represented as a fold change relative to RNA levels in the egg as described previously (Juliano et al., 2006). Each qPCR reaction was run in triplicate and averaged. Error bars indicate the standard deviation resulting from the triplicate qPCR reactions.

In situ RNA hybridization

DIG-labeled antisense RNA probes specific for each gene were generated using a DIG RNA labeling Kit (Roche, Indianapolis) and the cloned PCR fragments (described above) as templates (Table 1). Whole-mount in situ RNA hybridizations (WMISH) were performed as previously described (Arenas-Mena et al., 2000). Non-specific DIG-labeled RNA probes complementary to the pSPT 18 vector were used as negative controls for each WMISH experiment. Images were taken using a 40X oil immersion objective on a Zeiss Axiovert 200 M microscope.

Table 2.

Primers used for qPCR and generation of in situ RNA hybridization probes

Gene (SPU_#) Primers for qPCR Primers for in situ probe RNA in situ probe gene location
Sp-Pho (10295) F: TGTCAACCCATCTCGATTCA
R: CAGGGACAGAGTCTGCATCA
F: GAGGTGGAGATTGAGAGCATACCAATGG
R: AAAGGCAACGTGACTACCACTGTCTG
1100 nucleotides, includes 4 C2H2 Zn-finger
domains
Sp-Mbt1 (13689) F: ACTCGATGGATGGCAGTACC
R: TACACCAGGGTGGGAAGAAG
F: CAGATGTCGGAGGAAGATGAACTGAAGC
R: TAGGAATTGAACCACCTCAGCGATTCC
1104 nucleotides, includes C-terminus
Sp-Mbt2 (21547) F: CTGATCAACGCTGCCTACAA
R: ATCATGGCACGGTAGCTCTT
F: GCTTCTCTGGTCCTTACCTGAACAAGG
R: TGAATACTTTGGCAAGACTGGCACAGTC
1102 nucleotides, includes C-terminus
Sp-L(3)mbt3 (17786)➔(21123)* F: TCACTTTGATGGTTGGGACA
R: GCCTGATTGCCCAGTGTTAT
F: CAGTTGGACTGCTTACCTAAAGATGACCAG
R: GGACGTCTTATGATGACTGGTGTACTTCC
922 nucleotides spanning both gene predictions,
includes 2 MBT and 2 C2H2 Zn-finger domains
Sp-Pc1 (20586) F: TCGCTCCGAACTTATCCCTA
R: GTTTTCGTTCTTCGCTTTCG
F: TTATAGATGAGAAACCGTTGAGCAAACCCG
R: TCAGATCCAGTGGCGAATCCACC
1075 nucleotides, includes C-terminus
Sp-Pc2 (20946) F: AACAATGAGGATGGGGATGA
R: TCAACATCGACATTGGGAGA
F: CTCTGGGAAGACCTCCGTTGTTTCG
R: CTACGAGGCGGACTTTATAAGTGATGACG
1064 nucleotides, includes C-terminus
Sp-Ph (00345) F: GCTCCAAGAGACTGGGTCTG
R: TCCAAGGTTGTGCCACATTA
F: CAGCTCTACAACCATTCCAACACAGTCC
R: GCTCAAGGCTGTCATCAAATGATCCTCC
1042 nucleotides, includes 70% of SAM domain
Sp-Psc (23424) F: AGTAGCTGCGGTCCGATAAA
R: CAGCTGCAGGACATCTCAAA
F: CGCAAAACGCGGCTGAAGATAACG
R: GGGCATTGTAGGTATGCTCAACGC
1404 nucleotides, includes C3CH4 Zn-finger domain
Sp-Ezh2 (23366) F: TGCTACCTCGCTGTGAGAGA
R: TTTCCATCCTCGTTGGAGAC
F: ACTGTTCTTCACAACATTCCTTACATGGGC
R: TGGCATGTTGGAATGATTGGCAAAACG
1587 nucleotides, includes SET domain
Sp-Eed (19607)➔(26082)* F: CCCCTACAAGCATGGTGACT
R: CTTCCGATACGGGTTGAAGA
F: ATAAACAAAGCGGGTGTTTCAAACGTCGG
R: CTTGATCATCTCTACAGGGTCATGAATGCC
1083 nucleotides spanning both gene predictions,
includes 4 WD domain repeats
Sp-Suz12 (13163) F: TTCCAGAGGAATGGACCTTG
R: GGGTGCGGAAATACGTTCTA
F: ATGGCTCCCCACAAACATTCTCGG
R: TCCATTCCTCTGGAAGGCATAGCC
1509 nucleotides, includes N-terminus
Sp-Rbbp4 (26263) F: ATCTTGCCAAAGCGAGTCAT
R: CAGATCCCACAATGCAACAG
F: ATGAGCAGAACCACCTAGTCATAGCCAG
R: CAGTCTGTTGTTGGCATTCTAAATCAGCC
1062 nucleotides, includes 5 WD domain repeats
*

These genes each consist of two overlapping gene predictions

Ubq F: CACAGGCAAGACCATCACAC

Ubq R: GAGAGAGTGCGACCATCCTC

Primers are written in the 5′ to 3′ direction

Abbreviation List

PcG

Polycomb group

PhoRC

Pho-repressive complex

Pho

Pleiohomeotic

PRE

Polycomb response element

MBT

Malignant brain tumor

PRC

Polycomb repressive complex

Ezh2

Enhancer of Zeste homolog 2

Suz12

Suppressor of Zeste 12

Eed

Embryonic ectoderm

Rbbp4

Retinoblastoma binding protein 4

Pc

Polycomb

Ph

Polyhomeotic

Psc

Posterior sex combs

YY1

Yin Yang 1

SAM

Sterile alpha motif

Esc

Extra sex combs

WMISH

Whole mount in situ hybridization

DIG

Digoxigenin

References

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