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FoxY is a member of the forkhead transcription factor family that appeared enriched in the presumptive germ line of sea urchins (Ransick et al., 2002, Dev Biol 246:132). Here we test the hypothesis that FoxY is involved in germ line determination in this animal. We found two splice forms of FoxY that share the same DNA-binding domain but vary in the carboxy-terminal trans-activation/repression domain. Both forms of the FoxY protein are present in the ovary and in the early embryo, and their mRNAs accumulate to their highest levels in the small micromeres and adjacent non-skeletogenic mesoderm. Knockdown of FoxY resulted in a dramatic decrease in the Nanos mRNA and protein levels as well as a loss of coelomic pouches in the 2-week-old larvae. Our results indicate that FoxY positively regulates Nanos at the transcriptional level and is essential for reproductive potential in this organism.
Forkhead proteins are transcription factors characterized by their highly conserved forkhead (fkh) DNA-binding domains (Kaufmann and Knochel 1996). They have diverse functions in a wide variety of animals and fungi, including cell fate specification and differentiation, regulation of the cell cycle, and metabolism (reviewed in Carlsson and Mahlapuu 2002). Forkhead proteins are thought to bind to DNA as monomers, with binding sites consisting of 15–17 bp (reviewed in Carlsson and Mahlapuu 2002). The number of forkhead-box genes, or Fox genes, correlates with the anatomical complexity of the organism. For example, 4 forkhead transcription factors are found in the yeast Saccharomyces, 22 in the purple sea urchin Strongylocentrotus purpuratus (Tu et al. 2006), and 39 in humans (reviewed in Carlsson and Mahlapuu 2002).
The Strongylocentrotus purpuratus FoxY (FoxC-like) gene was first identified by Ransick et al. (2002) in a differential screen for endomesoderm effectors. Ransick et al. compared the mRNA of embryos treated with LiCl, known to “vegetalize” the embryo, to mRNA from “animalized” embryos over-expressing a dominant-negative cadherin that blocks the nuclearization of β-catenin (Miller and McClay 1997). This approach was used to search for transcription factors involved in endomesodermal specification. The FoxY mRNA localized prominently with small micromeres of the early embryo and in the coelomic pouch of the larvae, a pattern of special interest to us because it overlaps with the localization of both nanos and vasa mRNAs and proteins (Juliano et al. 2006; Juliano et al. 2010b; Voronina et al. 2008). Nanos and Vasa are genes involved in germ line function and in multipotency (Juliano et al. 2010a).
Nanos contains two CCHC zinc fingers and acts with Pumillio as a translational repressor by binding the Nanos response element (NRE) located in the 3′-untranslated regions (3′-UTRs) of Nanos-regulated mRNAs. The role of Nanos was first described in Drosophila, where Nanos and Pumilio are required together to translationally repress hunchback mRNA to promote posterior patterning (Murata and Wharton 1995; Wharton and Struhl 1991). In the sea urchin, Nanos is required to maintain the small micromeres and adult rudiment formation (Juliano et al. 2010b). While many studies focus on the function of Nanos as a translational repressor, its transcriptional regulation is not well understood. We tested FoxY as a potential regulator of nanos based on the previously reported FoxY in situ hybridization pattern (Ransick et al. 2002) and found that FoxY positively regulates nanos transcription.
We found two splice forms of foxy mRNA (Fig. 1, S1) that share the same DNA-binding region in the amino-terminus. Within the sixth exon, the short form has a segment of 76 nucleotides not present in the long form, resulting in a frame shift of the carboxyl terminus of the protein. As a consequence, the isoelectic point and molecular weight of FoxY-S (short splice form) and FoxY-L (long splice form) differ significantly: 6.56 / 62.7 kDa and 8.3 / 71 kDa, respectively.
Both splice forms are most abundant in early blastula and mesenchyme blastula and decrease in gastrula (Fig. 1), which is similar to a previous study detailing a high resolution time course for all forms of foxy mRNA (Materna et al. 2010). Using probes against both forms of foxy, we observed foxy mRNAs accumulation in cells of the small micromere lineage, as indicated by Vasa protein immunolabelng, and in the adjacent non-skeletogenic mesoderm (Fig. 2). By the larval stage, foxy mRNA decreases in abundance by real-time, quantitative PCR (QPCR), and is not detectable any longer by RNA in situ hybridization (Fig. 1 and data not shown).
To identify FoxY protein, we generated two different peptide antibodies against the FoxY splice forms and tested them by both immunoblotting and in situ immunolabeling. By immunoblot analysis, we detected both forms of the FoxY proteins of the expected sizes present in the ovary, blastula, and gastrula stages (Fig. 3). Unfortunately, these antibodies do not label embryos in situ, so we do not know the spatial distribution of the FoxY protein. At this point we cannot decipher if FoxY-L and FoxY-S have different mRNA and/or protein localizations, since the in situ RNA probes of whole mount embryos do not distinguish against these splice forms and the antibodies do not work in whole mount immunolabeling.
The peptide sequences used for antibody generation were designed outside of the conserved Forkhead domain. We blasted these peptide sequences in the annotated genome databases (SpBase.org) and do not find similarities to any other Fox protein (data not shown), suggesting that it’s unlikely that these antibodies recognize other Fox protein family members. By immunoblot, we observed a 48% decrease in the level of FoxY protein in foxy morpholino antisense oliognucleotide (MASO)-knockdown embryos, indicating that the antibodies are largely selective, that the knockdown is not complete, and/or that the FoxY protein accumulates stably in the embryo (Fig. 4A). We also tested the efficacy of a foxy MASO with reporter constructs containing a complementary sequence to the foxy MASO (Fig. 4B,C). Results indicate that only the mCherry reporter construct with the antisense foxy MASO sequence was repressed by the foxy MASO, whereas a control eGFP reporter construct without the target foxy sequence co-injected into the embryo was not affected by the presence of the MASO (Fig 4B,C). This result suggests that the injected foxy MASO is specifically binding to the target sequence and is functional in the embryo.
To test the function of FoxY, we knocked down FoxY in embryos. We first assayed the level of foxy mRNAs in these embryos at 11, 17, and 24 hours post-fertilization (hpf) by QPCR, and observed that FoxY positively regulates its own mRNA (Fig. 5).
Next, we examined the effect of knockdown on Nanos2, which is present exclusively in the small micromere lineage. nanos mRNA was not significantly altered with FoxY knockdown at 11 and 17 hpf; by 24 hpf, however, nanos mRNA was significantly decreased by QPCR (Fig. 5A). As a fluorescent dye was coinjected with the foxy MASO as a marker for injection and also as a proxy of the amount of foxy MASO injected into the newly fertilized embryo, we were also able to determine by in situ hybridization that nanos mRNA quantities remaining in an embryo depended on the amount of foxy MASO introduced (Fig. 5B). foxy knockdown embryos also showed a significant decrease in Nanos protein by 24 hpf (Fig. 6).
FoxY depletion in the embryo resulted in no obvious developmental defects up to the pluteus stage. After 2 weeks of culturing, however, the overall size of the larvae was significantly smaller than the mock-injected embryos of the same age (Fig. 7). In addition, coelomic pouches that were present in the pluteus stage regressed and eventually were lost. This foxy-knockdown phenotype is similar to the nanos-knockdown phenotype (Juliano et al. 2010b), suggesting that FoxY is epistatic to Nanos.
Results indicated that the two splice forms of foxy are differentially expressed. In the ovary, it appears that the FoxY-S form is more abundant than FoxY-L, whereas in later embryonic stages, the FoxY-L is more abundant (Fig. 3). These two splice variants have different carboxy-terminal amino acid composition, thus each variant has its own trans-activation/repression domains and may differentially regulate its target genes. The functional difference between these two FoxY isoforms is currently not known, although previous studies have shown that the function of forkhead proteins may depend on their phosphorylation sites (Chen et al. 2009; Singh et al. ; Tan et al. 1998). As FoxY-L has 7 additional predicted serine phosphorylation sites and 1 additional threonine and tyrosine phosphorylation sites, we speculate that the FoxY transcription factor isoforms may recruit different cofactors or have its activity modified depending on the status and location of its phosphorylation sites.
Both forms of foxy mRNA peak around the blastula stage (Fig. 1), and FoxY protein is present in great abundance in the ovaries (Fig. 3). From previous studies, nanos mRNA and protein are also abundant in the oocytes and later restricted to the small micromere descendants in the embryo (Juliano et al. 2006; Juliano et al. 2010b). Although several studies have focused on the post-translational regulation of Nanos, the cis-regulatory transcriptional control of the nanos gene is mostly unknown. Recent study on the Drosophila nanos identified an enhancer element located between −108 and +97 of its transcription start site that is sufficient to drive Nanos-GFP expression in the germline stem cells (Ali et al. 2009). Maternal transcription factors Ovo and initiation complexes bip2 and Trf2 are required for germline-specific gene expression of nanos and vasa in the germ plasm in Drosophila (Yatsu et al. 2008). Ovo is a transcriptional activator involved in female germline maintenance in Drosophila (Mevel-Ninio et al. 1991; Oliver et al. 1987; Staab and Steinmann-Zwicky 1996). The sea urchin ovo mRNA is localized to the vegetal plate of the mesenchyme blastula, but is undetectable later (Juliano et al. 2006). Since nanos mRNA is upregulated in the early blastula stage and maintained at the same levels until the pluteus stage, other transcription factors are likely involved in the transcriptional activation of nanos. The majority of forkhead proteins bind to a seven-nucleotide core consensus sequence (RYMAAYA [(R=A or G; Y = C or T; M + A or C]) that is found several times in the nanos promoter, suggesting that forkhead transcription factors can directly regulate nanos (Kaufmann et al. 1995; Overdier et al. 1994; Pierrou et al. 1994). nanos is also subjected to post-transcriptional control, such as translational regulation by RNA localization, as observed in Drosophila (Gavis and Lehmann 1994) and by Deadend RNA binding protein in zebrafish (Kedde et al. 2007); microRNA-mediated deadenylation (Mishima et al. 2006); and translational repression by a RNA secondary structure element as in Xenopus (Luo et al. 2011).
The small micromere descendants that become part of the coelomic pouches contribute to the adult rudiment. In adult tissues, foxy mRNA is present in the gonads and its protein is present in the ovary (data not shown and Fig. 3). FoxY may function during oogenesis (in adult tissues) and in early development by regulating nanos. It may also regulate endomesodermal genes. Previous studies indicated that foxy mRNA is found only in the small micromeres (Materna and Davidson 2012; Ransick et al. 2002); however, using Vasa protein as a reference for small micromeres (Voronina et al., 2008), we find that foxy mRNA has a broader distribution within the small micromeres and in the adjacent non-skeletogenic mesoderm in the blastula and gastrula stages (Fig. 2). This suggests that FoxY may potentially regulate other genes within other lineages, although one dominant function appears to be the transcriptional regulation of nanos.
Adult Stronylocentrotus purpuratus were obtained from Patrick Leahy (Point Loma Invertebrate Lab, Lakeside, CA) and kept in 15°C artificial seawater until needed. Artificial seawater was generated from Instant Ocean (Aquarium Systems, Mentor, OH). Eggs and sperm were collected by intracoelomic injection of 0.5M KCl. Eggs were collected in filtered seawater and sperm was collected dry.
Nested foxy primers were used in the cloning of full-length foxy in a PCR reaction using gastrula cDNA. Primers used in the first and second rounds of PCR were: FoxY5′-103For (5′-CTGCACTGACTCTGCCTACA) with FoxY-3′+2038Rev (5′-TGTCTGTTGGATCCAGCAGT) and FoxY5′-77For (5′-GCTTCACAAATCTCGCCTCA) with FoxY-TGAend (5′-TCACATACTGTGTATTCGTGT). Splice forms were detected using nested primers FoxY-F1 (5′-CATCCTAACTTGCCATGCAC) with 3′FoxY-3′end (5′-TCACATACTGTGGTATTCGTGT) and FoxYF2 (5′-TTTGATCAGTGCGATCGACTC) with FoxY R1 (5′-CTTGAGATCGTCCTGATGTC) using cDNAs from the ovary, egg, 4-cell, 32-cell, early blastula, mesenchyme blastula, gastrula, and pluteus. PCR products were run on 1.5% agarose gel. Bands were cut out, gel purified using QiaQuick columns (Qiagen, Valencia, CA), and cloned into pGEM T-easy (Promega). Recombinant clones were sequenced.
foxy antisense probes were labeled with digoxigenin (DIG RNA Labeling Kit (SP6/T7); Roche Diagnostics Corporation, Indianapolis, IN, USA). PCR primers used to clone the RNA probe were Fox-1F (5′-ATGGAGGACCAGGAGGATGATG) and Fox3′Rev (5′-GAATTCACTTTGATGATGTACGCTCAGGTC). The plasmid was linearized with BamHI and in vitro-transcribed with Sp6 RNA polymerase. This probe was used to detect native transcript in eggs and embryos at a concentration of 0.1 μg probe/mL, according to previously published protocols (Minokawa et al. 2004).
A total of 500 uninjected or 100 injected embryos were collected at various time points, and total RNA were extracted from them using the Qiagen RNeasy microkit according to manufacturer’s instructions (Qiagen Inc., Valencia, CA). cDNA was amplified using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City). QPCR was performed using the 7300 Real-Time PCR system (Applied Biosystems, Foster City). QPCR primers were designed using the Primer3 program (Rozen and Skaletsky 2000). Ten uninjected or two injected embryo-equivalents were used for each QPCR reaction with the SYBER Green PCR Master Mix (Invitrogen, Carlsbad, CA). The estimated numbers of transcripts are calculated based on the level of ubiquitin in various developmental stages, as described previously (Materna and Davidson 2012). For injected embryos, results were first normalized to ubiquitin levels and expressed as fold-difference compared to the uninjected eggs. Primers QPCR-L-For (5′-CTTCTGATCGAATCCATGCC) and QPCR-exonRev (5′-TTGTCGGATGTGTGTCTGGA) were used to detect FoxY-L and primers QPCR-S-F (5′-GATGATGAGAGACAACCAAC) and QPCR-exonRev were used to detect FoxY-S.
Antiserum was raised in rabbits against polypeptides indicated in Fig. S1. N-term (DAIRATDVTAESRHC) and C-term (CPPSPFTTVSPPDTH) peptide polyclonal antibodies were generated by Sigma Genosys (Woodlands, Texas). For affinity purifications, peptides were immobilized using the Pierce AminoLink Plus Immobilization Kit (ThermoFisher Scientific; Rockford, IL) according to the manufacturer’s instructions. Heat-inactivated serum was passed over the antigen-immobilized column, and bound antibodies were eluted with 1 mL 100 mM glycine (pH2.5) into 50 μl of 1 M Tris (pH 9.5).
Confocal Z-stack images were collected on an LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc.; Thornwood, NY). Projection images of the Z-stacks were generated for quantitative analysis. Quantitative pixel-intensity analysis of whole embryos was conducted using the Metamorph program (Molecular Devices, Downingtown, PA) to determine the levels of Nanos2 immunolabeling in untreated, mock, and foxy MASO-injected embryos.
Embryo extracts were prepared in 2X sample buffer with 1 mM dithiothreitol and boiled for 5 minutes. Samples were separted by one-dimensional polyacrylamide gel electrophoresis (PAGE) in pre-cast 4–20% polyacrylamide Tris-Glycine gels (Nu-Sep Incorporated, Lawrenceville, GA, USA). Total protein was transferred onto nitrocellulose membrane (Fisher Scientific, Pittsburgh, PA, USA) and blocked with Blotto (3% non-fat dry milk (w/v), 170 mM NaCl, 50 mM Tris, 0.05% Tween20 (v/v) pH 8). Ten micrograms of each affinity purified N-term and C-term primary antibody were diluted separately in __? mL Blotto, followed by either alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibodies diluted in Blotto (1:5000 dilution (v/v); Jackson Immunoresearch Laboratories Incorporated, West Grove, PA, USA). Blots were washed 2 times with Blotto for 15 min, followed by one wash in 1X phosphate-buffere saline (PBS)-0.5% Tween20. Immunocreactivity was detected in developmental series by the alkaline phosphatase colormetric reaction using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). For the foxy MASO Western blots, proteins were detected by horseradish peroxidase–dependent chemiluminescence using luminol / hydrogen peroxide. The FoxY protein was normalized to the yolk protein YP30 and quantitated by densitometry using Image J (Abràmoff et al. 2004).
Morpholino antisense oligonucleotides (MASO) against foxy, (5′-CATGGCTCCAAGTGCAGAACACTAC) was ordered from GeneTools (Philomath, OR). Microinjections were performed as previously described (Cheers and Ettensohn 2004), with modifications. MASO oligonucleotides were resuspended in sterile water and heated to 60°C for 10 minutes prior to use. Injection solutions contain 20% sterile glycerol, 2 mg/ml 10,000MW Texas Red lysine charged dextran (Molecular Probes, Carlsbad, CA), and varying concentrations of MASO. Eggs were dejellied in acidic seawater (pH 5.2) for 10 minutes on ice, followed by seawater washes. Dejellied eggs were rowed onto protamine sulfate-coated (4% w/v) 60×15 mm petri dishes. Eggs were fertilized with sperm in the presence of 1 mM 3-amino-triazol (Sigma, St. Louis, MO). Injections were performed using a Femto Jet® injection system (Eppendorf; Hamberg, Germany). Injection needles 1×90 mm glass capillaries with filaments (Narashige; Tokyo, Japan) were pulled on a vertical needle puller (Narishige; Tokyo, Japan). The injected foxy MASO corresponds to approximately 12 nM.
Fig. S1 Amino acid sequence comparison of the predicted FoxY-L and FoxY-S isoforms. Two different polyclonal peptide antibodies were generated against FoxY. The N-term antibody (shown in blue) recognizes both FoxY-L and FoxY-S forms, whereas the C-term antibody (shown in red) specifically recognizes the FoxY-S.
We gratefully acknowledge support from the National Institutes of Health (HD028152) and the National Science Foundation (IOS-1120972).
We thank present and past members of PRIMO for helpful discussions. This work is supported by NRSA awarded to JLS and NIH grant awarded to GMW.