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J. Biol. Chem., Vol. 281, Issue 39, 28782-28793, September 29, 2006
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1
From the
Departments of Molecular Genetics and Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, April 7, 2006 , and in revised form, August 3, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Because of the important role that apoptotic pathways play in normal and neoplastic development, relevant protein components are expressed constitutively, and their activity is maintained by many regulatory proteins (reviewed in Refs. 3 and 7). Well-known regulatory proteins include the Bcl2 protein family, the IAPs, SMAC/DIABLO, and the ubiquitin/proteasome pathway (reviewed in Refs. 7 and 8). A recently characterized, putative regulator of apoptosis is Rybp (also known as DEDAF), a zinc finger-containing protein that belongs to a family of proteins that also includes Yaf2 (9, 10). Rybp has been shown to interact with death effector domain (DED)2-containing proteins including FADD, caspase 8, caspase 10, and DEDD (11). The first three of these interacting proteins are components of the extrinsic or death receptor apoptotic pathway, while DEDD is a nucleolar protein that promotes apoptosis in the nucleolus as well as in the cytosol after its translocation (reviewed in Ref. 12; also see Ref. 13 and references therein). A pro-apoptotic role for Rybp was suggested because, it could promote formation of the DISC (death-inducing signaling complex comprising Fas, FADD, and pro-caspase 8) in 293T cells, and enhance Fas- and caspase 10 DED-mediated apoptosis in lymphoma cell lines (11). In a separate study, Rybp was shown to interact with the viral protein Apoptin, and to enhance apoptosis in transformed cells but not primary cells (14). Finally, Rybp has been shown to be up-regulated (with a corresponding decrease in a miRNA that may target it) in breast cancer cells that have been treated with a pro-apoptotic dose of the HDAC inhibitor LAQ824 (15).
Mice homozygous null for Rybp died around the time of implantation, and exhibited embryonic and extraembryonic defects (16). Of note, rybp–/– embryos were unable to trigger full decidualization in vivo, as evidenced by the lack of a normal apoptotic response following the initiation of implantation. Beyond this phenotype, a subset of the Rybp heterozygous null mice exhibited an exencephalic phenotype because of disrupted neural tube closure. This mouse model demonstrated that the effects of Rybp loss are dose-dependent, and that Rybp has a role in early development as well as in brain organogenesis. Moreover, even though Yaf2 is structurally and functionally related to Rybp, it cannot fully compensate for Rybp loss (16). To date, Yaf2 has not been studied in the context of a developing organism, and a link between Yaf2 and apoptosis has not been described.
In this study, we characterized the zebrafish ortholog of yaf2 and employed antisense morpholino oligomers to knock-down Yaf2 expression during zebrafish embryogenesis. Yaf2 morphant embryos exhibited a dramatic increase in the level of caspase-dependent cell death that was causal to a developmental arrest prior to the one somite stage. With decreased dosage of the morpholinos, the morphological defects became less severe and an essential role for Yaf2 in maintaining the anterior central nervous system (CNS) was revealed. Once again, the disruptions in the CNS resulted at least in part from excessive apoptosis. These findings implicate Yaf2 as a survival factor during early development and organogenesis. This function of Yaf2 may relate in part to its newly recognized ability to inhibit caspase 8-mediated apoptosis.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The one somite arrest morphants were scored based on the (i) lack of somite development and (ii) darkened, rounded up cells around the yolk. Moreover, the Yaf2 morphant embryos remained in this arrested state, until dying by 30 hpf. The hypomorphic embryos were first scored at the 10 somite stage where a dark band of dying cells could be seen beneath the eyes. In later stages, the hypomorphic phenotype was easily scored by the progressive anterior brain degeneration.
Expression Plasmids
The full-length IMAGE clone of zebrafish yaf23 was obtained from Open Biosystems. The full-length ORF of zyaf2 was amplified with the following primers: forward primer 5'-GGGGATCCGGAGGAATGGACTACAAGGACGACGATGACAAGGGAGACAAGAGGAGTCCGACGAGG-3' (introduced a BamHI site and an in-frame N-terminal FLAG tag) and reverse primer 5'-CCGAATTCCTAGTGAGATTCCCCGTTGAG-3' (introduced an EcoRI site). The FLAG-tagged zebrafish yaf2 insert was then subcloned into the pCS2+ and pcDNA3.1 expression vectors. Human YAF2 cDNA was obtained from Dr. Te-Cheung Lee, a C-terminal FLAG tag was introduced by PCR, and the PCR product was subcloned into pcDNA3.1. For the rescue experiments, mutations in the wobble position were made in amino acids 2–4 of the hYAF2 coding region by PCR mutagenesis on an hYAF2-FLAG template using the following primer, which also introduced a BamHI site: 5'-CGGGATCCCCAAGCCATGGGCGATAAAAAGAGCCCCACCAGG-3' (the changed nucleotides are in bold). The 3' primer was directed to the pCDNA3.1 vector (5'-CAGATGGCTGGCAACTAG-3'). The hYAF2-FLAG PCR product was then subcloned into pCS2+. Mouse rybp was cloned with a C-terminal FLAG tag by RT-PCR with 5' primer, 5'-GGAATTCCCGTCCATGACCATGGGCGACAAG-3' (introduced an EcoRI site) and 3' primer, 5'-GGAATTCATCATTCACTGCTGACATGTCG-3' (introduced an EcoRI site), and the PCR product was subcloned into pcDNA3.1 that carried a FLAG tag. Human caspase 8 DED was cloned into the pCDNA 3.1 vector using primers that added an N-terminal HA tag with the forward primer, 5'-GGCAAGCTTGCCATGGTTGGATACCCATACGACGTCCCAGACTACGCTGACTTCAGCAGAAATCCTTATG-3' (introduced a HindIII site) and reverse primer, 5'-CGCGGATCCCGGTCATTTGCTTTTCATTTGGTAAAC-3' (introduced a BamHI site and added a stop codon after amino acid 216). Human caspase 8 was cloned into pIRES2 EGFP (Clontech) by PCR with forward primer, 5'-ACGCGTCGACATGGACTTCAGCAGAAATCTTTATG-3' (introduced a SalI site) and reverse primer, 5'-CGGGGATCCGTCAATCAGAAGGGAAGACAAG (introduced a BamHI site). Zebrafish caspase 8 was cloned by RT-PCR from embryonic zebrafish mRNA using the forward primer, 5'-CGGAATTCGCCATGGTTGGATACCCATACGACGTCCCAGACTACGCTGATCCTCAGATCTTTCACGAG-3' (introduced an EcoR1 site) and reverse primer, 5'-CGGAATTCTCAGTCTATGGGCAGCACTAGTTTCTTGG-3' (introduced an EcoR1 site); the PCR product was subcloned into the pcDNA 3.1 expression vector. Additional details regarding construct generation are available upon request.
Whole Mount in Situ Hybridization
Embryos were fixed in 4% paraformaldehyde (PFA, Sigma) in PBS overnight and then dechorionated and stored in 100% methanol. Embryos older than 24 hpf were treated with 0.003% phenylthiourea (Sigma) to prevent pigmentation. Before probing, embryos were rehydrated and, if older than 24 hpf, treated with proteinase K (10 µg/ml) at room temperature for 5–45 min depending upon stage. The embryos were then refixed in 4% PFA for 1 h, and prehybridized in a 57% formamide hybridization buffer for 1 h at 70 °C. The buffer was replaced with 57% formamide hybridization buffer containing 1 ng/µl of the digoxigenin-labeled probe, and hybridization was performed at 70 °C overnight. Embryos were exposed to sheep anti-digoxigenin Fab antibody conjugated to alkaline phosphatase (Roche Applied Science) for 2 h at room temperature. The signal was detected using alkaline phosphatase NBT/BCIP staining, and the embryos were then cleared in a BB:BA 2:1 solution.
To generate the probes, the expression constructs were linearized and purified by phenol/chloroform extraction. Antisense RNA was generated using an in vitro transcription kit (Promega) and labeled with a digoxigenin NTP mix (Roche Applied Science). The probes were purified over G-50 Sephadex RNA columns (Roche Applied Science) and the concentration determined by spectrophotometry. The zyaf2 probe comprised the full open reading frame subcloned into pCS2+. The zCaspase 8 probe in pCS2+ contained its open reading frame from amino acid 131 to the end (amino acid 476). SK-zopl1 was provided by Dr. Hazel Sive, pCS2+ zotx2 was provided by Dr. Shuo Lin, KS+ zkrox20 was provided by Dr. Yu Chen, and pSPORT1 zpax2.1 was obtained from the ZFIN zebrafish data base (ID: ZDB-EST-021126-10).
Morpholinos
Two phosphorodiamidate morpholino oligomers were generated (by Open Biosystems and GeneTools) against the Yaf2 sequence. The ATG morpholino (ATG MO) (5'-CGTCGGACTCCTCTTGTCTCCCATG-3') is complementary to –1 thru +24 relative to the zYaf2 translation initiation codon. The splice site morpholino (SS MO) (5'-ATTGCAGCTTCACCTCATTTTCTTG-3') is targeted to the splice donor in the long intron following the penultimate exon in the zebrafish yaf2 genomic locus.3 BLAST searches predicted that the morpholinos were specific for yaf2. The three zyaf2 mRNA isoforms that have been identified3 would all be targeted by the splice site morpholino, which gives the same phenotype as the ATG morpholino that would only affect the predominant isoform that is the focus of this report. A morpholino (5'-ATATCCATCACACTGGCGGC-3'), based on the pBluescript vector (Stratagene), was used as a control in the injections. Morpholinos were injected into fertilized eggs at the 1–2 cell stage in a total volume of 2.3 nl.
Reverse Transcription-PCR
RNA was isolated using Trizol reagent (Invitrogen) and purified by phenol/chloroform extraction. First-strand cDNA was generated using the Superscript First-strand PCR kit (Invitrogen) with random hexamer primers, and then amplified by PCR using Platinum Taq (Invitrogen). The primers used to amplify yaf2 were the same primers used for the subcloning into pCS2+. The same first strand cDNA samples were amplified with primers for the housekeeping gene 
-actin: forward primer, 5'-AAGCAGGAGTACGATGAGTCTG-3' and reverse primer, 5'-GGTAAACGCTTCTGGAATGAC-3'.
Apoptosis Assays and Caspase Inhibitor Treatment
TUNEL Assay—Injected embryos were staged and fixed overnight in 4% PFA. After washing with PBST, they were dechorionated and dehydrated into 100% methanol. Prior to staining, the embryos were rehydrated into PBST, post-fixed for 1 h in 4% PFA, blocked for 1 h in 1 x TdT buffer, and incubated with a mixture of the TdT enzyme (150 units/ml; Invitrogen) and digoxigenin-labeled dUTP (0.5 µM; Roche Applied Science) overnight at room temperature. The embryos were then washed first in PBST/1 mM EDTA at 65 °C for 2 h and then in PBST/bovine serum albumin. Embryos were exposed to anti-digoxigenin Fab antibody conjugated to alkaline phosphatase (Roche Applied Science) overnight at 4 °C. The signal was detected using alkaline phosphatase NBT/BCIP staining and the embryos were then cleared in a BB:BA 2:1 solution.
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Acridine Orange/Ethidium Bromide—HEK293T cells were plated at a density of 1 x 106 cells per 10-cm dish and transfected with 3 µg of each expression construct using calcium phosphate precipitation. The cells were harvested after 24 h by trypsinization (Invitrogen), resuspended in 1 ml of PBS, and then a 50-µl aliquot was removed and labeled in a double blind manner. The aliquots were stained by adding 4 µl of an acridine orange (100 µg/ml)/ethidium bromide (100 µg/ml) solution. 10 µl of each sample was dotted on a glass slide, covered with a coverslip, and counted using x180 magnification on a Nikon SM1500 fluorescent microscope per Ref. 19. A minimum of 200 cells were counted per point, and grouped into four categories based on nuclear morphology and stain: viable (green with intact nuclei), necrotic (orange with intact nuclei), early apoptotic with intact membrane (green with fragmented nuclei), and late apoptotic with damaged membrane (orange with fragmented nuclei). Whole cell lysates were made from the remaining cells to ensure expression of the introduced proteins.
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Rescue Experiments
Capped hYAF2 mRNA was made by linearizing the pCS2+ hYaf2 FLAG construct with NotI and transcribing with Sp6 using the mMessage Sp6 kit (Ambion), according to the manufacturer's instructions. To confirm the integrity of the capped mRNA, in vitro translations were performed with rabbit reticulocyte lysate (Promega) and resolved by SDS/PAGE followed by Western blotting. The membranes were incubated overnight with primary antibody (1:1000 FLAG antibody (Sigma, M2)) at 4 °C and probed with a 1:3000 dilution of the horseradish peroxidase-conjugated anti-mouse secondary antibody (Amersham Biosciences) for 1 h at room temperature. Bound antibodies were detected by chemiluminescence (Amersham Biosciences). Titration of hYAF2 mRNA injected into embryos revealed that injection of up to 200 pg did not cause any phenotypic changes. For rescue, a mixture of 5 ng of ATG MO, 200 pg of hYAF2 mRNA, and 100 mM KCl or 2.5 ng of ATG MO, 200 pg of hYAF2 mRNA, and 100 mM KCl was injected into fertilized eggs at the one cell stage, and the injected embryos were observed for appearance of the morphant phenotypes.
Immunoprecipitations
HEK293T cells were plated at a density of 1 x 106 cells per 10-cm dish and transfected with 5 µg of each expression construct using Superfect (Qiagen) according to the manufacturer's instructions. 24 h post-transfection, cells were lysed in PG buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 5 mM EDTA) in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were incubated on ice for 30 min and centrifuged at 14,000 rpm at 4 °C for 10 min. The supernatant was then precleared by rotating for 20 min with a 50% slurry of protein A-Sepharose beads (Sigma). An 80-µl aliquot was removed, and the rest of the sample was added to a 50% protein A-Sepharose bead slurry that had been preincubated for 20 min with anti-HA antibody (12CA5; kind gift from Dr. Matthew Scharff; 50 µl per point). Immunoprecipitations were performed for 2 h at 4 °C, and then the immunocomplexes were washed five times in PG buffer with protease inhibitors at 4 °C. Lysates and immunoprecipitates were resolved by SDS/PAGE and analyzed by Western blotting overnight at 4 °C with anti-FLAG HRP (M2, Sigma) 1:5000. Bound antibody was detected using the Super Signal West Pico Luminol enhancer (Pierce) per the manufacturer's instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Embryos Blocked for Yaf2 Expression Undergo an Early Developmental Arrest—To determine the physiological role of Yaf2 in vivo, we designed a translation interference antisense morpholino and injected it into embryos at the 1–2 cell stage. Seventy percent of embryos derived from fertilized eggs injected with 5 ng of this morpholino arrested development before somitogenesis (
16 hpf) (Fig. 3A, ATG MO). These embryos developed normally through gastrulation, but failed to progress to the one somite stage (for staging see Ref. 21). The developmental arrest was preceded by the appearance of "dark regions" within the embryo (Fig. 3B) that have been shown previously to indicate the presence of dying cells (22). Of the morphant embryos that did not arrest, many displayed a less severe phenotype similar to that seen consistently with injection of lower doses of morpholino (described below). Injection of control morpholinos did not cause any abnormal phenotypes (Fig. 3, A and B).
To confirm the specificity of this phenotype, and to control for the possibility that the ATG MO might unexpectedly target another transcript, a second morpholino was designed against a zyaf2 splice junction (the splice site morpholino, or SS MO) and used similarly for injection into fertilized eggs. Injection of the SS MO resulted in a phenotype identical to that seen with the ATG MO (Fig. 3, A and B, SS MO), albeit that higher concentrations of the SS MO were required to cause the developmental arrest (20 ng of SS MO in comparison to 5 ng of ATG MO).
To ensure that the SS MO was indeed effective in preventing production of a mature zyaf2 message, RT-PCR assays were performed using primers that amplify the entire yaf2 coding region and flank the splice junction to which the SS MO is targeted. These assays showed the absence of the normal yaf2 message (Fig. 3C, compare lane 6 to lane 5), a result that was confirmed by in situ hybridization assays on morphant embryos that had been injected with the SS MO (Fig. 3D). The fact that two morpholinos of entirely distinct sequences, and that function by distinct mechanisms, generate identical phenotypes indicates that the specific loss of Yaf2 is incompatible with normal embryonic development.
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As also suggested by acridine orange studies (data not shown), the TUNEL positivity was associated with caspase-dependent apoptosis and thus was significantly reduced by treating the injected embryos with 200 nM pan-caspase inhibitor zVAD-fmk (Fig. 4A; embryos in 200 nM zVAD columns). zVAD-fmk treatment also had an impact upon the developmental arrest phenotype of the morphants (Fig. 4B). At 16 hpf, 80% of the untreated morphant embryos were arrested at the presomitic stage, whereas only 28.5% of the zVAD-fmk-treated embryos were arrested (Fig. 4B, left graph). The rescue achieved by caspase inhibition was partial, because it did not entirely eliminate the pre-somitic arrest, and since the majority of the treated morphants that did survive exhibited developmental abnormalities (Fig. 4B, right graph; hypomorphic phenotype discussed next). Nevertheless, taken together our findings suggest that the phenotype caused by zyaf2 depletion can be attributed at least in part to a significant increase in the level of caspase-dependent apoptosis.
A Dose-response Assay with the Morpholinos Reveals a Hypomorphic Phenotype—As alluded to above, 13% (n = 522) of embryos derived from fertilized eggs injected with the ATG MO or 4.5% (n = 597) of embryos derived from fertilized eggs injected with the SS MO survived the pre-somitic arrest and displayed instead what we term a "hypomorphic phenotype" (Fig. 5A, left graph). This phenotype became more predominant when the dosages of the morpholinos were halved (Fig. 5A, right graph; 75.7% with 2.5 ng of Yaf2 ATG MO (n = 229) and 73.0% with 10 ng of Yaf2 SS MO (n = 155)). The hypomorphic phenotype is first evident around the 10 somite stage. Whereas the posterior somites and tail region appear to have developed normally in these morphants, the anterior region is malformed and marked by dark cells with pebbled morphology (Fig. 5B, compare panels c and d to a and b; SS MO hypomorphic embryos not shown). This dark region of apparent cell death expands as the embryo develops, and by 29 hpf an overall deterioration of the brain/head structures can be seen (Fig. 5B, compare panels g and h to e and f). Although there is a range in expressivity, the characteristic defects include malformed forebrain, eyes, and ears, and disrupted/contorted neural tube and notochord. There are also frequent disruptions in the periderm around the yolk (data not shown). Most of these hypomorphic embryos do not move, fail to hatch, and die around 72 hpf.
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To determine whether changes in the level of apoptosis underlie the phenotypes of the hypomorphic embryos, we performed TUNEL assays on embryos that had been injected with the lower morpholino dosage in comparison to controls. Indeed, there was a significant increase in cell death in the anterior region of the hypomorphic embryos from the 10 somite stage through 29 hpf (Fig. 6B). This increase was apparent even at 20 hpf, when there is a normal developmental surge in apoptosis in the brain related to neurogenesis (23) (data not shown). Taken together with the gross morphology and the marker gene expression patterns (Figs. 5B and 6A), our findings suggest that depletion of Yaf2 causes increased apoptosis resulting in degeneration of anterior structures of the zebrafish embryo.
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Co-injection of 200 pg of hYAF2 with 5 ng of the ATG MO rescued the developmental arrest; only 26.9% of the embryos (n = 93) arrested prior to the one somite stage as compared with 80% when the ATG MO was injected alone at this dosage. Of note, we did not observe complete rescue of morphant embryos to survival under these rescue conditions, as the majority presented as hypomorphs (Fig. 7A). Finally, coinjection of 200 pg of human YAF2 mRNA with 2.5 ng ("half-dose") of the ATG MO was able to rescue partially the hypomorphic neural phenotype (Fig. 7B). Specifically, this coinjection gave rise to 36% of embryos displaying the hypomorphic phenotype and dying by 72 hpf, 48.5% of embryos with normal heads and surviving up through 5 days (but displaying cardiac edema), and 9.4% of embryos appearing normal (n = 203). This is in contrast to 76% of embryos developing the hypomorphic phenotype when the half-dose of the ATG MO is injected alone (Fig. 7B). Taken together, the findings of these rescue studies suggest that the specific loss of Yaf2 results in developmental defects and increased apoptosis, and that the molecular and cellular functions of Yaf2 are highly conserved throughout evolution.
Yaf2 Interacts with Caspase 8 and Inhibits Caspase 8-mediated Apoptosis—Our data from the zebrafish Yaf2 morphants suggest that Yaf2 may function normally to inhibit apoptosis/promote cell survival. However, there are repeated examples in the literature of apoptotic regulators that when disregulated lead to unexpected apoptotic phenotypes (for example see Ref. 24). As a first step toward addressing whether Yaf2 could be anti-apoptotic, we examined the relationship between Yaf2 and caspase 8; the rationale for this stemmed from the previously reported interactions of the Yaf2-related family member Rybp with caspases 8 and 10 (11). As shown in Fig. 8A, co-immunoprecipitation experiments were performed upon lysates from HEK293T cells transfected with an HA-tagged version of the human caspase 8 DED domain and Flag-tagged human Yaf2, zebrafish Yaf2, mouse Rybp, or control proteins. As expected, a strong interaction was seen for Rybp and the caspase 8 DED (Fig. 8A, lane 5). Strong interactions with the caspase 8 DED also were observed for human and zebrafish Yaf2, respectively (Fig. 8A, lanes 1 and 3). This is consistent with the fact that the regions on Rybp/DEDAF important for interaction with the DED of caspase 10 (human DEDAF amino acids 24–45 and 144–180; (11)) are highly conserved (77% identity) within Yaf2 (corresponding residues in zYaf2 are amino acids 22–43 and 102–137; see Fig. 1A).
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The Zebrafish Yaf2 Morphant Phenotype Is Caspase 8-mediated—Encouraged by the findings from the cell culture-based assays suggesting that Yaf2 can inhibit caspase 8-mediated apoptosis, we returned to the zebrafish to assess whether the apoptosis seen in the Yaf2 morphants was linked to caspase 8. First, a caspase 8 activation assay was performed upon lysates made from the morphants, using a synthetic caspase 8-specific substrate (LETD) conjugated to luciferin. As shown in Fig. 9A, left graph, there was a 3.7-fold increase in luciferin (indicative of caspase 8 activation) in the Yaf2 morphant embryos injected with 5 ng of ATG MO at 14 hpf as compared with stage-matched control-injected embryos. When these morphants were sampled at slightly later time points, this increase in caspase 8 activity was less impressive (1.6-fold), suggesting that the caspase 8 activation is an early event (data not shown; see also Ref. 26). For the hypomorphic phenotype, when embryos that had been injected with the half dose of the Yaf2 ATG MO were sampled at the 18 somite stage, there was a 2.3-fold increase in luciferin as compared with stage-matched control-injected embryos (Fig. 9A, right graph). The caspase 8 activation also appears to be an early event in the hypomorphs, because at 29 hpf there was minimal caspase 8 activation observed (data not shown).
Finally, to further demonstrate that the apoptosis seen in the Yaf2 morphant embryos is caspase 8-mediated, we treated these embryos with a caspase 8-specific competitive inhibitor, zIETD-fmk (Fig. 9B). These studies showed that zIETD-fmk treatment rescues the phenotype resulting from Yaf2 deficiency (from the one somite arrest to the hypomorphic phenotype) to the same extent as zVAD-fmk treatment (compare with Fig. 4B). Taken together with the results of Fig. 8, these results suggest that Yaf2 plays a role in inhibiting caspase 8-mediated apoptosis, which may in turn relate to the cellular and organismal phenotypes observed in the morphants.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The pre-somitic arrest phenotype of Yaf2 morphant embryos classifies zyaf2 among the "early arrest" genes that when mutated give rise to general phenotypes during the first day of development (22). These mutants have been subdivided into two classes. Class I mutants have increased cellular abnormalities (e.g. lysis) before morphological defects are observed. Class II mutants have morphological defects before cell death, and many of these exhibit extensive degeneration of the CNS by 30 hpf. Because dying (i.e. dark, spherical, acridine orange-, and TUNEL-positive) cells are observable in the Yaf2 morphant embryos at the tailbud stage prior to the arrest, we assign zyaf2 to the Class I group along with other genes required for general cell maintenance (22). This assignment is also supported by the finding that the cellular death appears causal to the embryonic death since treatment with either the pan-caspase inhibitor zVAD-fmk or the caspase 8-specific inhibitor zIETD-fmk allows the embryos to survive the pre-somitic arrest (Figs. 4B and 9B).
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A later developmental role for Yaf2 in cell survival in the CNS was uncovered in our studies employing lower dosages of the morpholinos. Beginning at the 10 somite stage when brain morphogenesis is initiating and continuing through 29 hpf when the brain is partitioned into its individual lobes (21), morphological changes in the head region of Yaf2 hypomorphs are apparent including the presence of dark patches of dying cells (Fig. 5B). TUNEL analysis indicated that these changes correlate with significantly increased levels of apoptosis in the hypomorphs in comparison to stage-matched controls (Fig. 6B). The end result of this massive apoptosis is the degeneration of anterior head structures including the anterior brain (for other reports on zebrafish neural degeneration mutants see Refs. 31–33). It is interesting to note that a dose-dependent role in the CNS for the Yaf2-related protein Rybp was uncovered in Rybp-deficient mice (16). Specifically, a subset of the Rybp heterozygous null mice showed an exencephalic phenotype due in part to defective neural tube closure. In addition, in rybp–/– <-> rybp+/+ diploid embryo chimeras, the presence of Rybp-deficient cells in the developing murine CNS resulted in chaotic forebrain overgrowth, among other abnormalities (16).
Our study provides support for a possible physiological mechanism of Yaf2 action in cell survival that relates, at least in part, to its ability to inhibit caspase 8-mediated apoptosis (Fig. 8B). It also appears that caspase 8 pathways are relevant to Yaf2 function in the morphants because (i) caspase 8 activation appears to be an early event therein and (ii) specific inhibition of caspase 8 by zIETD-fmk treatment rescues the morphant phenotype (Fig. 9). Notably, this rescue was similar to that seen with pan-caspase inhibition (Fig. 4B), as well as that seen with coinjection of YAF2 mRNA (Fig. 7). Whereas the caspase 8 inhibition studies suggest that Yaf2 inhibits apoptosis at the level of caspase 8 itself, we cannot rule out that Yaf2 also may inhibit apoptosis upstream of caspase 8 or even other types of apoptosis.
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Although our data implicate Yaf2 as a survival factor during early zebrafish development and organogenesis and suggest that Yaf2 can influence caspase 8 pathways, it is important to note that Yaf2 (and Rybp) most often have been reported to interact with transcriptional regulators. These regulators include DNA-binding proteins as well as Polycomb Group (PcG) transcriptional repressors (9, 10, 34–41). Originally classified as regulators of differentiation during embryogenesis, PcG proteins have now been implicated in the processes of cell renewal, cell cycle control, and cell survival (for recent reviews see Refs. 42–46). We have shown that, in addition to binding to caspase 8, zYaf2, like its mammalian counterpart (37, 41), is capable of interacting with the mammalian Ring1A and Ring1B PcG proteins (data not shown). Accordingly, the apoptosis resulting from Yaf2 deficiency may relate in part to the Yaf2 interaction with PcG proteins and their involvement in these cellular processes. The contributions of Yaf2-PcG interactions to the observed morphant phenotypes remain to be determined. Finally, it is possible that functions of Yaf2 in apoptosis and in Polycomb group protein biology could interrelate. One model that warrants further investigation is that Yaf2 (and Rybp) may be playing more general cellular roles in regulating processes such as post-translational modifications and/or subcellular localization.
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FOOTNOTES |
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Data in this paper are from a thesis (S. E. S.) to be submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1 To whom correspondence should be addressed: Dept. of Molecular Genetics, 1300 Morris Park Ave., Ullmann 809, Bronx, NY 10461. Tel.: 718-430-3216; Fax: 718-430-8778; E-mail: agus{at}aecom.yu.edu.
2 The abbreviations used are: DED, death effector domain; AO, acridine orange; BAC, bacterial artificial chromosome; CNS, central nervous system; hpf, hours post-fertilization; MO, morpholino; PcG, Polycomb Group; SS, splice site; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; zIETD-fmk, benzylozycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone; zLETD-aminoluciferin, benzylozycarbonyl-Leu-Glu-Thr-Asp-aminoluciferin; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; PBS, phosphate-buffered saline; HA, hemagglutinin; PFA, paraformaldehyde.
3 The BAC clone containing the zebrafish yaf2 genomic sequences can be accessed through the NCBI Nucleotide Database under GenBankTM accession no. AL929504. The amino acid sequence of the zYaf2 protein can be accessed through the NCBI Protein Database under NCBI accession no. CAI11643. The amino acid sequences of the other zYaf2 protein isoforms can be accessed through the NCBI Protein Database under NCBI accession no. CAI11644 and NCBI accession no. CAI11645. Sequences corresponding to the purchased IMAGE clone (7222218) for zyaf2 can be accessed through the NCBI Nucleotide Database under GenBankTM accession no. CN016939.
4 The amino acid sequences of the human, mouse, and zebrafish Yaf2 proteins can be accessed through the NCBI Protein Database under NCBI accession no. Q8IY57; NCBI accession no. AAH02192; and NCBI accession no. CAI11643, respectively. The amino acid sequence for human and mouse Rybp can be accessed through the NCBI Protein Database under NCBI accession no. NP_036366 and NCBI accession no. NP_062717, respectively.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(a member of the Myc/Max/Mad network that should not associate with caspase 8). Lysates probed with the FLAG and HA antibodies are shown below the IP blot. Molecular mass is shown in kDa on the left. B, HEK293T cells were transfected with human caspase 8 (hC8) alone or with either mRybp, hYaf2, or zYaf2 (left graph) or with zebrafish caspase 8 (zC8) alone or with either mRybp, hYaf2, or zYaf2 (right graph). Apoptosis was determined by nuclear morphology using an acridine orange/ethidium bromide assay. The amount of apoptosis (early+late) is graphed relative to that obtained with caspase 8 only, which is taken to be 100%. At least 200 cells per sample were counted, and the studies were performed in a double blind fashion. Error bars represent S.D. for two independent experiments. m, mouse; h, human; z, zebrafish.