Centrosomin (Cnn) is a required core component in mitotic centrosomes during syncytial development and the presence of Cnn at centrosomes has become synonymous with fully functional centrosomes in Drosophila melanogaster. Previous studies of Cnn have attributed this embryonic function to a single isoform or splice variant. In this study, we present new evidence that significantly increases the complexity of cnn. Rather than a single isoform, Cnn function can be attributed to two unique classes of proteins that comprise a total of at least 10 encoded protein isoforms. We present the initial characterization of a new class of Cnn short isoforms required for centrosome function during gametogenesis and embryogenesis. We also introduce new evidence for a complex mix of Cnn isoforms present during early embryogenesis. Finally, we reexamine cnn mutations, in light of the short isoforms, and find previously overlooked differences attributable to allele-specific mutant phenotypes. This study addresses several questions surrounding Cnn function at the centrosome during embryogenesis and shows that cnn function cannot be ascribed to a single protein.

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THE animal centrosome is the major microtubule-organizing center (MTOC) in most cell types and is critical for the nucleation of astral microtubules and organization of normal spindles during mitotic divisions. The centrosome consists of a pair of centrioles surrounded by the pericentriolar material or matrix (PCM), which is composed of a complex and dynamic mix of structural, functional, and regulatory proteins (BORNENS et al. 1987; KALT and SCHLIWA 1993; MACK et al. 2000). Centriole pairs function to maintain the PCM as a coherent structure (BOBINNEC et al. 1998) and are essential for the precise reproduction of centrosomes at the onset of mitosis (SLUDER and RIEDER 1985; SLUDER 1989). Our understanding of the many functions of the PCM has advanced significantly over the last quarter of a century, but a clear and comprehensive picture of the function of this region of the centrosome remains elusive. Since the centrosome is essential for normal cellular inheritance of chromosomes and cellular organelles, and defects associated with centrosomes appear to play a critical role in the onset and progression of several cancers (SCHATTEN et al. 2000a,b; KRAMER and HO 2001; LINGLE et al. 2002; GISSELSSON 2003; PIHAN et al. 2003; SCHNEEWEISS et al. 2003), knowledge of the function of centrioles and the PCM is important from the perspective of both general biology and human disease studies.

The syncytial stage of embryogenesis in Drosophila melanogaster has provided researchers with a powerful system for the genetic and molecular dissection of centrosome function. This stage of development is characterized by 13 rapid and synchronized nuclear divisions that occur in a common cytoplasm, followed by the synchronous cellularization of 6000 nuclei at the cortex of the embryo (FOE et al. 1993). This system has also proven to be extremely useful for the analysis of centrosomin (Cnn), a core component of the PCM. In a recent RNAi screen in Drosophila S2 tissue culture cells, Cnn and Polo kinase were found to be the two major PCM proteins required for centrosome maturation (DOBBELAERE et al. 2008). Additionally, Cnn is required for maintaining the connection between centrioles and the PCM as well as the correct positioning of centrioles within the centrosome (LUCAS and RAFF 2007). This was consistent with earlier studies that found Cnn was required for the proper localization of the centrosomal proteins CP60 and CP190 (MEGRAW et al. 1999), as well as centrosomal localization of the microtubule nucleating protein -tubulin during syncytial development and in somatic mitoses (MEGRAW et al. 1999, 2001; VAIZEL-OHAYON and SCHEJTER 1999). Cnn is also required for the localization of the microtubule-stabilizing D-TACC/Msps complex at centrosomes (ZHANG and MEGRAW 2007). Studies on the defects associated with Cnn deficiency have shown that normal spindles, which lack astral microtubules, form during early cleavage divisions in cnn mutant embryos; mitotic defects increase as nuclei migrate to the cortex and embryogenesis aborts prior to cellularization. Additionally, while Cnn is essential for syncytial development, it appears to be dispensable for later stages of development. The maternal supply of Cnn from heterozygous females is sufficient for the development of adult homozygous mutant flies and Cnn is not required for mitosis in S2 tissue culture cells (MEGRAW et al. 2001). Does Cnn have a limited function at the centrosome, as these results imply, or is cnn more complex than a single gene product?

The possibility that cnn is a complex gene seemed likely, as previous Northern and Western analyses had identified multiple alternatively spliced transcripts and protein isoforms, respectively (LI et al. 1998). The Drosophila genome project has recovered additional cnn cDNA clones (ADAMS et al. 2000) and there are now five alternative splice variants listed on FlyBase (http://flybase.org/). Alternative splicing may increase the protein complexity, but it does not explain why early anastral spindles are relatively normal. Thus it is possible that Cnn does not have an essential function during the early cleavage divisions. Alternatively, the cnn^mfs, cnn^E2, and cnn^B4 mutant alleles used in the above studies may produce low levels of partially functional protein. As suggested by others, a more detailed analysis of cnn mutations (VIDWANS and O'FARRELL 1999), and a more thorough investigation of cnn and its gene products, may further elucidate the function of Cnn at the D. melanogaster centrosome.

In this study, we have carried out a detailed analysis of cnn at the transcriptional and protein level, and present evidence for two classes of Cnn protein. In addition to the previously described isoforms, which we have termed "long forms," there is a second unique class of short isoforms present at the centrosome in early embryos. We have used a combination of Northern and Western analyses, and indirect immunofluorescence microscopy studies to provide a more detailed profile of the function of both isoforms and the overall complexity of cnn products during development. Additionally, we show that different mutant genotypes have differential effects on gametogenesis and embryogenesis. The severity of the phenotypes can be correlated with the severity of the mutation, demonstrating that several alleles do retain partial function at the centrosome. An important finding is that Cnn short forms function to maintain spindle poles during early cleavage divisions in the absence of Cnn long forms, and these short isoforms are only eliminated by the cnn^hk21 null mutation. We conclude both long and short Cnn isoforms contribute to centrosome function, and knowledge of the cnn mutant genotype used in any experiment is a significant factor when interpreting results. While this study is by no means exhaustive with respect to cnn, we hope it provides a more comprehensive background for future studies of cnn and other studies of centrosome function that utilize cnn mutations as a genetic background.

Drosophila stocks:

All flies used in this study were grown on standard corn meal agar medium at 25°. Wild-type flies were Oregon R. The following mutant alleles were used: cnn^hk21, cnn^mfs7, cnn^mfs3, and the deficiency Df(2R)cnn (HEUER et al. 1995). A P{ry^+t7.2=Acp70A^g.Yp1.hs}G10 strain was used as a source of unfertilized embryos for total protein extracts. To express the GFP::Cnn-PG fusion transgene during embryogenesis the Cnn-PG line GfpC7c8-111 (w; P{w^+mC=pUASP-GFP-Cnn-PG} was crossed to a nanos::Gal4 line (P{GAL4::VP16-nos.UTR}^MVD1) (VAN DOREN et al. 1998). Embryos collected from P{GAL4::VP16-nos.UTR}^MVD1; (w; P{w^+mC=pUASP-GFP-Cnn-PG} mothers were collected and stained for the presence of GFP to determine the pattern of accumulation of the chimeric protein.

Reproductive fitness:

To test male fertility individual males were mated with five newly eclosed virgin Oregon R females and kept together for 2 days. Males were mated to six sets of females over a 12-day period. All progeny that eclosed within 18 days of the initial mating date were counted. A total of 20 males were tested for each strain. To determine egg production 25 newly eclosed females were maintained with 50 Oregon R males on egg-lay plates with yeast paste. The total egg production was counted every 24 hr. Two replicates were performed for each strain.

Characterization of the cnn locus:

To characterize the cnn locus we screened ovarian, embryonic, and testes cDNA libraries, as previously described (HEUER et al. 1995). The cnn-RB and cnn-RC cDNA sequences were acquired from FlyBase (http://flybase.org/). Sequences were aligned with the genomic sequence and maps were determined with MacVector software (Accelrys). Northern analysis was performed as previously described (LI et al. 1998), using a 4.32-kb Cnn1a (Cnn-PA) cDNA and specific probes to coding exons 1a, 1b, and 1c (see Figure 1).

View larger version (32K): In this window In a new window Download PPT slide   FIGURE 1.— The transcriptional map of the centrosomin gene. The centrosomin gene is depicted (top line) showing the three promoters (P1, P2, and P3), the associated unique starting coding exons (1a, 1b, and 1c), the 5'- and 3'-UTRs (open boxes), and all coding exons (solid boxes and stippled, shaded boxes). The theoretical and the known alternatively spliced transcripts are shown below, with associated 5'- and 3'-UTRs (open boxes) and coding exons (shaded or stippled boxes), and are identified by "cnn" followed by an arbitrary number and a letter indicating the promoter and starting exon, and the FlyBase identifier below. Two of the coding exons (ICa and ICb) in the cnn5c and cnn-Theo transcripts use alternative exons in a different reading frame that overlap the exon 4bicid region (stippled boxes with asterisks above). An alternative internal 3' splice site in coding exon 5 is indicated by a prime mark. All coding start sites (ATG) and termination sites (TAA, TAG, and TGA) are indicated for each transcript. The three nonsense mutations used in this study and their effect on transcripts is shown (arrows). The hk21 mutation results in early termination of all transcripts and is a true cnn null mutation. The mfs7 and mfs3 mutations result in the early termination of all Cnn long isoform transcripts, but have no effect on short isoform transcripts.

 

Antibody production:

To produce Cnn short form protein in Escherichia coli we amplified bases 806–1383 of the Cnn7c Cnn-PG coding sequence with the following primers: Cnn-PGAb5'-X: AAG AAT TCA ACG AGG CCA TAG ACT CTC TTA AG and Cnn-PGAb3'-H: AAG GAT CCT ACT GGT GGT GGC CCT GAT GAT AAA C. PCR products were cloned into a Topo-TA pCR2.1 vector (Invitrogen) and their sequence was verified using ABI Big Dye 3 reagents on an ABI 3700 sequencer. The PCR product was cut with EcoRI, filled with Klenow, cut with XbaI, and ligated into a pWR590-1 lacZ fusion cassette that was cut with HindIII, filled with Klenow, and cut with XbaI. Ligations and protein production were done as previously described (MATTHEWS et al. 1989). Cocalico Biologicals produced antibodies in guinea pigs.

To differentiate between the antibodies to Cnn proteins used in this study we have designated each antibody by the antigen used. The previously reported guinea pig anti-Cnn.Ex1a2 was made against the first 574 amino acids of Cnn-PA, but tests indicate it only recognizes the peptide encoded by exons 1a and 2 of the Cnn-PA isoform (MEGRAW et al. 1999). The previously reported anti-Cnn.Ex456 was made against the last 20 amino acids encoded by exon 4, all of exon 5, and the first 679 amino acids encoded by exon 6 of the Cnn-PA isoform (HEUER et al. 1995). The guinea pig anti-Cnn.Ex4abicd short form antibody described above was made against the peptide encoded by exons 4a, 4bic, and the first 20 amino acids of exon 4d of the Cnn-PG isoform.

Western blots:

A centrosome-enriched fraction was prepared as previously described (MORITZ et al. 1995). To analyze phosphorylation, embryos were collected and lysed in calf intestinal phosphatase buffer. All other protein fractions and the Western analyses were performed as previously described (EISMAN et al. 2006) with the following modification: First dimension Immobiline linear strips, pH 4–7 (GE Healthcare) were loaded with 150 or 300 µg of protein, using the cup-loading method following rehydration or with active loading under 100 volts during rehydration as per the manufacturer's protocols (HEALTHCARE 2004). Isoelectric focusing was carried out on 18-cm pH 4–7 Immobiline strips according to the manufacturer's protocol (GE Healthcare). The strips were electrophoresed on an IPGphor apparatus (GE Healthcare) for a total of 50–60 kV/hr with the cup-loading method, 90–130 kV/hr with the active-loading method. After isoelectric focusing, the strips were first equilibrated in a reducing solution (2% SDS, 10 mM DTT) for 15 min at room temperature and then equilibrated in an alkylating solution (2% SDS, 25 mM iodoacetamide and bromophenol blue). The strips were then loaded on 8% SDS-polyacrylamide gels. Second dimension electrophoresis was performed at 4° at a constant current of 10–25 mA per gel. Proteins were immediately transferred to Hybond ECL nitrocellulose membrane (Amersham Bioscience). Guinea pig anti-Cnn.Ex1a2 and rabbit anti-Cnn.Ex456 were used at 1:2000 dilution, guinea pig anti-Cnn.Ex4abicd was used at 1:500 dilution, and secondary antibodies were used at 1:20,000 dilution (Jackson ImmunoResearch Labs). Proteins were detected using Pierce SuperSignal West Pico Chemiluminesence substrate and developed on Kodak film.

Cloning and transformation:

To construct the vector used to express the GFP::Cnn-PG fusion protein we PCR amplified EGFP (Living Colors by Invitrogen) with the following primers: SpeGfp-5': ACT AGT ATG GTG AGC AAG GGC GAG and GfpMlu-3': ACG CGT CTT GTA CAG CTC GTC CAT G. We amplified the cnn7c (cnn-RG) transcript from a cDNA recovered from our library screen (Cnn4IIB) with the following primers: Mlu cnn7c (cnn-RG)-5': GGA CGC GTA TGA ATA GTA ATC GAA C and cnn7c (cnn-RG)stBgl2 X-3': GGA GAT CTC TAA GTG CCC CAG C using standard PCR techniques and added SpeI and EcoRI restriction sites in the primers. PCR products were cloned into a Topo-TA pCR2.1 vector (Invitrogen) as per the manufacturer's protocol and verified by sequencing. All other cloning was done using in-gel ligation techniques as previously described (KALVAKOLANU and LIVINGSTON 1991). PCR products were digested with appropriate enzymes and subcloned into a pBlueskript vector. The fusion protein fragment was cut with NotI and shuttled into a pUASP vector (RORTH 1998). Transformed Drosophila lines were generated as previously described (MILLER et al. 2002), except we used the pUC hsp 2-3 P-element helper plasmid, a gift from Joseph Duffy, and we transformed a w¹¹¹⁸ Drosophila stock. We recovered homozygous transgenic lines for these constructs on chromosomes I, II, and III (w; P{w^+mC=pUASP-GFP-cnn7c (cnn-RG)}. Stocks of these constructs are available from the Bloomington Stock Center (stock nos. 7254 and 7255).

Fixation and immunostaining:

Embryos were collected and fixed in 50% heptane: 50% MeOH/EGTA as previously described (EISMAN et al. 2006). Ovaries were dissected in 1x Robb's medium and fixed in buffer (100 mM potassium cacodylate; 100 mM sucrose; 40 mM NaAOc; 10 mM EGTA; pH 7.2) containing 8% formaldehyde. Embryos and ovaries were immunostained as previously described (GORMAN and KAUFMAN 1995). Specimens were mounted on glass slides in 90% glycerol and 10% PBS, with 0.2 mM n-propylgallate (Sigma). The following antibodies were used: guinea pig (whole sera) anti-Cnn.Ex4abicd at 1:50, guinea pig (whole sera) anti-Cnn.Ex1a2 at 1:500 (MEGRAW et al. 1999), or rabbit anti-Cnn.Ex 456 at 1:300 (HEUER et al. 1995), rabbit anti--tubulin 84B at 1:100 (MATTHEWS et al. 1989), and mouse MC anti-GFP at 1:100 (Santa Cruz). Actin was stained with rhodamine-phalloidin (Molecular Probes). DNA was stained with TOTO3 (Molecular Probes) at a final concentration of 1:1000. All fluorescent secondary antibodies were used at 1:200 (Jackson ImmunoResearch Labs).

Microscopy and imaging:

All images were captured on a Leica TCS confocal microscope, with a 63x HCX Plan Apo oil immersion objective, using TCSNT software. Images are all Z-series that range from 2.5 to 6 mm thick, composed of 0.5- to 0.9-mm-thick sections. Projected images were processed and assembled into figures with Adobe Photoshop version 7.0 software.

centrosomin is a complex gene:

Alternative splicing is known to increase the complexity of animal proteomes, but the functional significance of this complexity is uncharacterized for many genes. Previous work has shown that at least one product of D. melanogaster centrosomin (cnn) is a maternally supplied core component of the syncytial centrosome and is required for normal development during the preblastoderm and syncytial blastoderm stages of development (HEUER et al. 1995; MEGRAW et al. 1999; VAIZEL-OHAYON and SCHEJTER 1999). A second isoform that differs at the amino terminus was shown to be required for mitotic and meiotic divisions during spermatogenesis, as well as organization of the sperm axoneme (LI et al. 1998). In an attempt to identify all the alternatively spliced variants of cnn, we screened databases, cDNA libraries, and EST collections (see MATERIALS AND METHODS). Our screen identified three promoters associated with unique starting exons and recovered nine full-length cDNA transcripts that translate into nine unique protein isoforms of Cnn (Figure 1). Cnn isoforms can be grouped into two distinct protein families that we refer to as long and short forms, suggesting that the functional complexity of Cnn may be greater than previously thought.

To describe each transcript and protein, we assigned each identified full-length cnn transcript an arbitrary number followed by a letter indicating the promoter and first coding exon, as well as the FlyBase identifier when available. We have also utilized the FlyBase identifiers for each CDS when these are available and have expanded both the cnn-RX and Cnn-PX designations for those transcripts and encoded proteins, which are identified in this work but not yet specified in FlyBase. On the basis of the original cnn1a (cnn-RA) transcript, the 5'-UTR and most distal coding exon were designated exon 1a, followed by coding exons 2–7. Additional coding exons in other alternatively spliced transcripts that lie between the cnn1a (cnn-RA) exons are designated by the cnn1a (cnn-RA) upstream exon number followed by a letter (see Figure 1). The only exceptions to this exon numbering system are two coding exons in the cnn5c (cnn-RC) transcript, which are designated ICa and ICb, for Isoform Cnn-PC. The open reading frames for these two exons and exon 4bicid overlap, but the two reading frames are shifted by a single base and utilize unique frame-shifted codons. Additional alternative splicing mechanisms in cnn include alternative internal 3' splice sites, intron retention/exclusion, and cassette exons (see Figure 1 for details). We have adopted this nomenclatural system in case more transcripts and coding exons are identified in cnn.

The three promoters in cnn raise the possibility that different transcripts are expressed in specific spatial or temporal patterns during development. The long and short protein products are similar in size and structure within each family, but the predicted isoelectric point (pI) of each protein is variable (Table 1), a protein property that could be critical for optimal function in different subcellular environments (SCHWARTZ et al. 2001). To determine the pattern of cnn transcripts during development, we analyzed northern blots of extracts from early and late embryos, third instar larvae, pupae, ovaries, and testes, using a cnn1a (cnn-RA) cDNA probe and specific probes to the coding regions of exons 1a, 1b, and 1c (Figure 1). Although these probes do not necessarily distinguish every potential transcript, the Northern analysis does reveal the complexity of cnn expression during development.

View this table: In this window In a new window   TABLE 1 cnn transcript size and predicted protein size and pI

 
The cnn1a (cnn-RA) cDNA probe (Figure 2A), which should recognize all long form cnn transcripts, shows that there are at least two major transcripts present in embryos, third instar larvae, ovaries, and testes (although the upper band in testes is weak). The lower band in embryos, larvae, ovaries, and testes is similar in size to that predicted for cnn1a (cnn-RA), cnn2b (cnn-RE), cnn3b (cnn-RB), and cnn4c (cnn-RD) transcripts while the upper band correlates with the cnn5c (cnn-RC) transcript. Interestingly, there are two bands in third instar larvae that are present in low abundance. Only the lower band is detected by our exon 1b-specific and 1c-specific probes, suggesting that there may be a fourth promoter, a fourth initiating coding exon, or a combination of these two possibilities.

View larger version (60K): In this window In a new window Download PPT slide   FIGURE 2.— Northern analysis reveals the transcriptional complexity of cnn. RNA extracts from different development stages were identified with a Cnn1a cDNA probe and probes specific to each of the three first coding exons. Exposure times for each lane are shown below each Northern blot. (A) The Cnn1a cDNA probe detects two bands in all lanes except testes. The lower 4.40-kb bands are consistent with Cnn1a, 2b, 3b, and 5c transcript size and the upper band is the approximate size of the Cnn4c transcript. The upper band in larvae may represent an unknown transcript. (B) The exon 1a-specific probe identifies an intense band above 4.40 kb in embryos and ovaries and a less abundant band at 4.40 kb that is likely to be the Cnn1a transcript. This band is barely detected in larvae and testes. The upper band may represent an abundant and unknown transcript that is similar to Cnn4c. Additionally there is a single weak band near 2.37 kb in late embryos, larvae, pupae, and testes that is the size of the Cnn6a transcript. (C) The exon 1b-specific probe identifies a single band at 4.40 kb at all stages except oogenesis, and is the size of the Cnn2b and 3b transcripts. The weak band near 7.46 kb in embryos is likely unprocessed RNA, and the band between 4.40 and 2.37 kb may be an unknown transcript. (D) The exon 1c-specific probe detects a weak band just below 4.40 kb during all developmental stages except late embryogenesis. This is likely to be the Cnn5c transcript. The band below 2.37 kb is abundant at all stages, especially spermatogenesis, and is the size of Cnn7c, 8c, and 9c transcripts. This probe does not detect the Cnn4c transcript. E 04, embryos 0–4 hr; E 0-22, embryos 0–22 hr; L3, third instar larvae; P, pupae; O, ovaries; T, testes.

 
The exon 1a-specific probe (Figure 2B) reveals two long form transcripts in embryos and ovaries, and a single long form transcript expressed at low levels in testes. The lower and weaker band in embryos and ovaries correlates with the cnn1a (cnn-RA) transcript, while the upper band appears to be a splice variant for which we have not identified a full-length cDNA. The most likely explanation for this transcript is that there is a cnn5c (cnn-RC)-like form (cnn-Theo in Figure 1) that does not initiate at exon 1c but rather initiates at exon 1a and includes exon 2. This transcript would have a predicted size of 5.02 kb consistent with the size of the band observed in the Northerns. Additionally, there is a weak short form band in late embryos, third instar larvae, pupae, and testes, which correlates in size with the cnn6a (cnn-RF) transcript. The absence of detectable signal in ovaries and 0- to 4-hr embryos indicates that this isoform may not be expressed or is accumulated at very low levels at these times. There are also several weak bands that run well below 2.37 kb that may correspond to transcripts that as of yet have no recovered cDNAs or represent degradation products.

The exon 1b-specific probe (Figure 2C) identifies a single long form transcript in embryos, third instar larvae, pupae, and testes, which corresponds to cnn2b (cnn-RE), cnn3b (cnn-RB), or a combination of both transcripts. This transcript is abundant in late embryos and is most likely the predominant somatic version of cnn present after cellularization. The most parsimonious explanation for the larger bands present at both embryonic stages is that these are unprocessed primary transcripts. There are weak bands at both embryonic stages that run between 4.40 and 2.37 kb that may represent cDNA-less transcripts. Consistent with our transcriptional map of cnn there are apparently no short isoforms transcribed from the second promoter.

The exon 1c-specific probe (Figure 2D) recognizes a weak band running just below 4.4 kb in early embryos and testes, and very weak bands in third instar larvae, pupae, and ovaries, which are the same size as the cnn5c (cnn-RC) transcript. We do not see a band corresponding to the cnn4c (cnn-RD) transcript, so this transcript is either expressed at low levels or is absent during these stages of development. This probe also recognizes a single short form band present at all stages investigated, which is the approximate size of the cnn6a (cnn-RF), cnn7c (cnn-RG), cnn8c (cnn-RH), and/or cnn9c (cnn-RI) cDNA transcripts, although we have no way to differentiate among these splice variants. There are also several weak smaller bands, similar to the exon 1a-specific probe results, which again may represent unknown transcripts or degradation products.

As noted, Northern analysis of cnn shows there are apparently long form transcripts present during the development of D. melanogaster that have not yet been identified in cDNA libraries. The most abundant of the cDNA-less transcripts is present in ovaries and throughout embryogenesis, is larger than the cnn1a (cnn-RA) transcript, and utilizes the most distal promoter and exon 1a. There is also a large cDNA-less transcript expressed in third instar larvae that is detected by the full-length cnn1a (cnn-RA) probe, but is not detected by the three exon-specific probes. This suggests that this cDNA-less transcript is the product of a fourth promoter, a unique starting exon associated with a known promoter, or a combination of both possibilities. Thus, the current map of cnn transcripts represented by recovered cDNAs apparently does not yet provide a comprehensive picture of the alternative splice variants encoded by this gene, and the complexity of cnn at the RNA level is likely to increase with further investigation.

The complex protein map of Cnn during early embryogenesis:

The above Northern analysis of cnn suggests there may be several protein isoforms present during embryogenesis in D. melanogaster. Although there are clearly multiple transcripts present, if they are not translated these splice variants have problematic functional significance. Our previous work suggested there is a single abundant protein isoform present in embryonic extracts (HEUER et al. 1995; MEGRAW et al. 1999) and in Drosophila S2 cells (MEGRAW et al. 2001), although Western analysis of immunoprecipitated Cnn preparations from embryonic extracts apparently detected two phosphorylated isoforms (LI and KAUFMAN 1996). Additionally, it has been shown that at least one isoform of Cnn is the most abundant protein in purified embryonic centrosome preparations, but is not abundant in total embryonic protein extracts (LANGE et al. 2000). To characterize the isoforms present during early embryogenesis we used a combination of 1D and 2D Western analysis, and antibodies specific to either the long or short Cnn protein isoforms predicted from the Northern analyses and/or recovered cDNA clones (Table 2). Our goal was to gain insight into both the qualitative protein profile of Cnn, and a semiquantitative measure of different Cnn isoforms present in early embryos. We employed several methods to enrich our preparations for Cnn proteins. This analysis reveals a complex pattern of Cnn isoforms that is similar to the complexity suggested by our Northern analysis during early embryogenesis.

View this table: In this window In a new window   TABLE 2 -Cnn antibodies

 
The long form antibodies anti-Cnn.Ex456 (HEUER et al. 1995; LI and KAUFMAN 1996) and anti-Cnn.Ex1a2 (MEGRAW et al. 1999), hereafter referred to as anti-Cnn.Long, both identify a single band of similar size that runs below the 170.8-kDa marker on Western blots of total embryonic protein (TEP) (Figure 3A, lane 1). The apparent size of this band is 20 kDa larger than the predicted size of the Cnn-Theo (Cnn-PC-like) isoform and 50 kDa larger than the Cnn-PA isoform (Table 1). The presence of a single Cnn long isoform suggests only one transcript is translated in embryos, or other isoforms were undetectable due to low concentrations. To differentiate between these two possibilities, we ran protein fractions enriched for centrosomal components (CEF) (MORITZ et al. 1995). Western analysis shows the CEF is enriched for long isoforms of Cnn, and short exposure time identifies at least two bands when the amount of CEF protein loaded is equal to the TEP amount loaded. One of the detected forms comigrates with the band seen in the TEP as well as another that migrates below (Figure 3A, lanes 2 and 3). These same two bands are also detected when a threefold dilution of CEF is used (Figure 3A, lanes 4 and 5), or when Cnn is immunoprecipitated from the CEF using the anti-Cnn.Long antibody (Figure 3A, lanes 6 and 7). However, the presence of additional bands of Cnn at lower molecular weights, especially in the high concentration lanes (e.g., Figure 3A, lane 3), suggests the centrosome preparation method may result in a low level of degradation of Cnn. These results suggest there are two long isoforms of Cnn in early embryos and the lower molecular weight band is the least abundant form of the protein.

View larger version (63K): In this window In a new window Download PPT slide   FIGURE 3.— Embryos have a complex mix of Cnn isoforms at the centrosome. Cnn isoforms from TEP fractions, centrosome-enriched fractions, and unfertilized oocytes were detected with specific antibodies to the long and short isoforms. Each lane was loaded with 50 µg of protein, except the 3x dilution lane (17 µg). Molecular weight markers are indicated on the left. (A) The long form antibody anti-Cnn.Long detects a single band in the TEP fraction, and two bands in the CEF fractions and the immunoprecipitated CEF fraction after short exposure times. The upper band is always more abundant than the lower band. The additional bands may be other isoforms, degradation products, or an artifact of the centrosome enrichment method. (B) Anti-Cnn.Long staining of ovarian and TEP fractions have a single Cnn band, except the 2-hr fraction, which has an additional upper band due to an unknown PTM (see text). -Tubulin staining is shown as a loading control. (C) In Acp70A unfertilized oocytes the predominant form of Cnn detected by anti-Cnn.Long is similar to the band seen in all TEP extracts (Cnn-Theo) but over time a smaller isoform accumulates (Cnn1a) similar to the lower band present in 4- to 5-hr TEP extracts. -Tubulin staining is shown as a loading control. (D) The short form antisera anti-Cnn.Short detects two bands in the TEP fraction and three bands in the CEF fraction. The lower band is the most abundant, although short isoforms are much less abundant than long forms. The molecular weight for all long and short bands is higher than the predicted size.

 
The failure to detect the lower molecular weight band of Cnn in TEP fractions could mean this isoform is always present at low concentrations or may only be present during very early stages of syncytial development. To test this possibility, we analyzed total protein from mated female ovaries and from embryos at different time points during syncytial development. One dimensional Western analysis detects a single Cnn band in ovaries and all embryonic time points except the 2-hr embryonic extracts, which have an additional band that migrates above the main TEP band (Figure 3B). This band was also detected in the 1- to 2-hr extracts with longer exposure time but not at the earlier time points (data not shown). Immunostaining of embryos from these sample collections shows the apparent discrepancy between the 1- to 2- and 2-hr samples is due to a higher percentage of cellular embryos present in the 2-hr sample. We hypothesize that this upper band is a relatively unstable post-translational modification (PTM) to Cnn, as the band is absent in protein samples which were stored for extended periods of time, have undergone repeated freeze–thaw cycles, or have been exposed to temperatures above 25° for extended periods of time. These results suggest the larger Cnn-Theo (Cnn-PC-like) isoform is maternally loaded and is the most abundant Cnn protein during syncytial development.

The failure to detect the lower bands present in centrosome and Cnn-enriched protein fractions may be due to differential accumulation and/or degradation of the smaller isoform during each cell cycle, or due to the differential translation of isoforms throughout syncytial development. To test the later possibility, we used a P{ry^+t7.2=Acp70A^g.Yp1.hs}G10 strain as a source of oviposited unfertilized oocyte protein from 6-hr and 18-hr oocytes. Additionally, we compared unfertilized oocyte protein to 2-hr and 5-hr embryonic protein. On the basis of our Northern data, the 5-hr embryonic proteins should include the Cnn-PE isoform, which is similar in size to the Cnn-PA isoform. Western analysis of 6-hr oocytes detects an abundant upper band similar to the band in ovarian and embryonic extracts and a very weak lower band (Figure 3C, lane 1). However, in the 18-hr unfertilized sample this lower band increases in abundance relative to the upper band (Figure 3C, lane 2) and migrates at the same rate as the lower band in 5-hr embryos (Figure 3C, lane 4), supporting our prediction that the lower band in unfertilized oocytes is the Cnn-PA isoform. Taken together, these data show the Cnn-Theo (Cnn-PC-like) isoform is maternally loaded and is the predominant Cnn isoform at the syncytial centrosome. The Cnn-PA isoform is apparently present in embryos and likely at the centrosome at low levels. The translation of this isoform is initiated following egg activation and continues to be accumulated even in unfertilized eggs. Clearly the accumulation pattern of Cnn at the syncytial centrosome is more complex than previously appreciated.

In addition to Cnn long forms, Northern analysis of early embryos predicts at least one short form transcript. However, all short form transcripts are similar in size and alternatively spliced transcripts may not be detectable at the resolution of the Northern blots used. To detect all known short protein isoforms of Cnn and distinguish them from the long isoforms, we made antibodies to the peptide encoded by exons 4abic and the first 20 codons of exon 4d (anti-Cnn.Short). Although this antibody potentially could cross-react with Cnn-PD and similar isoforms, on Western blots it appears to be specific to short isoforms. Western analysis of TEP fractions detect two bands that are present in relatively low abundance and migrate more slowly than the predicted sizes of exon 1c short isoforms (Figure 3D, lanes 1 and 2). The CEF is enriched for these two bands, and shows an additional higher molecular weight band, although the concentration of short isoforms in embryos is clearly lower than the long isoforms (Figure 3D, lanes 3 and 4). Additionally, the same Western blot of TEP and CEF extracts shows the migration pattern of proteins differs before and after centrosomal enrichment. Thus for consistency we used only TEP fractions for the remainder of our analyses. However, both methods show an abundant short isoform similar in size to Cnn-PG, and a less abundant isoform likely to be Cnn-PF.

Long Cnn isoforms are post-translationally modified in embryos:

Although the above results support the presence of two distinct Cnn long isoforms in early embryos, PTM of a single isoform could also explain the two bands on 1D Western blots. However, if the two bands are indeed Cnn-PA and Cnn-Theo (Cnn-PC-like) protein isoforms, 2D Western analysis, which separates proteins on the basis of their isoelectric focusing point (pI) and size, should separate these isoforms into two discrete populations. We analyzed TEP fractions and unfertilized P{ry^+t7.2=Acp70A^g.Yp1.hs}G10 total oocyte protein extracts to minimize any differences due to sample preparation methods and to reproduce conditions used in 1D Western results. We also used cup loading and active loading for the first dimension (see MATERIALS AND METHODS) of TEP fractions to optimize our Western blots for Cnn. The cup loading results in better isoelectric focusing, but protein precipitation may reduce the amount of protein loaded into the gel, which is minimized when samples are loaded under low voltage (HEALTHCARE 2004).

The two different loading methods produce strikingly different results for Cnn on 2D Western blots. When the TEP fraction is cup loaded, a single long Cnn isoform is initially detected as 8 foci (Figure 4A, upper left panel), and with longer exposure times, 10 discrete foci (Figure 4A, lower left panel), whereas an equal amount of protein actively loaded detects multiple isoforms and a much more complex pattern of post-translational modification (Figure 4B, left panel). The signal in the area corresponding to the cup loaded sample (Figure 4B, left panel, braced area) and a small portion of the more acidic region is detectable within seconds (Figure 4B, left inset), while detection of the smaller isoform (Figure 4B, arrow) requires a 3-hr exposure. Although there appear to be two isoforms present, we believe this is an artifact due to overloading the first dimension gel. When the amount of actively loaded sample is reduced by 50% or more, we consistently detect multiple foci of a single isoform with a faster migrating region corresponding to the cup-loaded Western blot (Figure 4B, right panel, braced area) and a more acidic region that shows a significant shift in migration rate. We hypothesize this more acidic region corresponds to the upper band on 2-hr 1D Western blots. Interestingly, when the amount of sample loaded is reduced the signal intensity is uniform across the entire region, suggesting the more acidic modification decreases the solubility of Cnn.

View larger version (61K): In this window In a new window Download PPT slide   FIGURE 4.— The two long Cnn isoforms present in embryos are both post-translationally modified. (A) 2D Western analysis showing a 300-µg cup-loaded TEP sample (left panels) that detects a single isoform with 8 foci present after a 1-min exposure (top) and 10 foci after a 5-min exposure (lower). These modifications are stable and appear to be due to phosphorylation of Cnn as phosphatase treatment (right panel) of this fraction results in a single unresolved spot. (B) When a similar TEP fraction is actively loaded into the first dimension gel (left panel) the stable modifications are present (braced area) as well as additional modifications and the Cnn1a (Cnn-PA) isoform (arrow). When the sample amount is reduced by 50% the smaller isoform is not detectable and the Cnn-Theo (Cnn-PC-like) loads uniformly (compare inset panels). The modification in the more acidic region appears to reduce the solubility of Cnn (see text for details). (C) Two-D Western analysis of Acp70A oocyte protein extracts shows that initially (left panel) Cnn-Theo (Cnn-PC-like) is the only isoform detected and this protein has multiple modifications that are similar to the modifications shown in A. However, over time (right panel) the accumulation of Cnn1a (Cnn-PA) continues resulting in this isoform being the most abundant form of Cnn (inset). The pI for these isoforms suggests they are full-length proteins and not degradation products. The numbers in A (lower left panel) are arbitrary and do not imply any ordering system for phosphorylation. The inset panels are 5-sec exposures of the associated Western blot and the larger panels are 5-min exposures, except the left panel in B is a 3-hr exposure. All western blots were stained with rabbit anti-Cnn.Long antibody.

 
Although the cup-loading method appears to result in better focusing of Cnn, both methods resolve multiple foci suggesting sequential additions of some moiety. As stated above, we have previously shown that immunoprecipitated Cnn is present as two bands on Western blots and both bands migrate faster after phosphatase treatment (LI and KAUFMAN 1996), although we interpreted our results as a single isoform of Cnn. These earlier results suggest some of the Cnn foci on 2D Western blots represent sequential additions of phosphate groups to each isoform. Since the more acidic region of Cnn present on the actively loaded blots appears to be readily dissociated by mechanical means, we wanted to know whether the stable Cnn foci present on all the 2D Western blots was due to phosphorylation. When TEP fractions are treated with phosphatase Cnn appears as a single unresolved spot (Figure 4A, right panel), suggesting the foci present on the cup-loaded 2D Western blots represent the addition of multiple phosphates. The experimental shifts in molecular weight (M_r) and pI we observe for Cnn are also consistent with in silico predictions using the Web-based software ProteoMod (KUMAR et al. 2004) for the addition of nine phosphate groups to the Cnn-Theo (Cnn-PC-like) isoform and six phosphate groups to the Cnn-PA isoform. Taken together, these results predict a complex pattern of phosphorylation and at least one other post-translational modification is involved in the modification of Cnn during embryogenesis.

On the basis of the above analyses of TEP extracts, we next wanted to know whether the unfertilized oocyte extracts had the same isoforms and whether these proteins were post-translationally modified in the absence of development. The 2D Western blot of 6-hr unfertilized oocytes detects 8 foci of a single isoform within seconds (Figure 4C, left panel inset), but fails to detect the more acidic region described above after longer exposure times (Figure 4C, left panel). A similar blot of the 18-hr unfertilized oocyte extracts detects the smaller Cnn isoform within seconds (Figure 4C, right panel inset) and detects both isoforms after longer exposure times. Although there are minor differences in the focusing and migration of proteins between electrophoresis experiments, these results suggest both Cnn-Theo (Cnn-PC-like) and Cnn-PA isoforms are present, and these are stable isoforms and not degradation products. Additionally, both isoforms appear to have multiple post-translational modifications, even in the absence of development. Interestingly, in unfertilized oocytes egg activation appears to initiate either continuous translation of Cnn-PA or a failure to degrade it for some period of time, whereas Cnn-Theo (Cnn-PC-like) protein levels remain similar to the maternally loaded protein supply.

The pattern of Cnn short isoforms is dynamic during syncytial cleavage divisions:

The short isoforms of Cnn are much less abundant than long isoforms in early embryos and may have a limited function during development. To characterize the accumulation pattern of these isoforms during syncytial development, we immunostained early embryos with the anti-Cnn.Short antibody, which, based on our Western blotting results, appears to be specific to short isoforms. Additionally, we used the Gal4-UAS system to ectopically express a GFP::Cnn-PG fusion protein in embryos to investigate the possibility that a single isoform would mimic the short isoform pattern observed in wild-type embryos. These results show the short isoforms are more dynamic than long isoforms during the cell cycle and reveal potential functions of a new class of Cnn proteins.

The short isoform antibody initially stains the polar bodies in early embryos while the meiotic products exist in the diploid and haploid configuration (Figure 5A) and after they fuse into a triploid polar body with a starburst configuration (Figure 5B) (FOE et al. 1993). Short isoforms form a dense central core at triploid polar bodies and may be present at the cortical attachment point of the meiotic spindle (arrowhead, Figure 5A); further work will be required to understand more completely the function of these protein isoforms during meiosis.

View larger version (48K): In this window In a new window Download PPT slide   FIGURE 5.— Cnn short isoforms localize to polar bodies and mitotic centrosomes. Immunostaining with the anti-Cnn.Short antisera (red) and DNA (blue) in early embryos shows the antibody localizes to A, both haploid (1N) and diploid (2N) polar bodies. The antibody also stains one end of the haploid polar body (arrowhead), which may be the cortical attachment point. After polar bodies fuse (B) the antibody remains strongly localized at the center of the starburst of chromosomes. At mitotic nuclei, the antibody is (C) abundant at prophase centrosomes, (D) reduced at metaphase centrosomes, and (E) undetectable at anaphase B centrosomes. (F) During late telophase, the antibody is present as a haze throughout the cytoplasm and as dense foci associated with nuclei, coincident with the timing of centrosome replication. Bar for A and C–F, 20 µm; bar for B, 8 µm.

 
After development is initiated, short form staining is strongly localized at centrosomes and weakly localized around the nuclear envelope during prophase (Figure 5C) throughout syncytial development. Additionally, there is punctate staining in the cytoplasm surrounding the nuclei. As mitosis proceeds, the antibody stains small foci at the poles of elongating metaphase spindles and the cytoplasm is clear of all staining (Figure 5D). During anaphase B there is no detectable staining at the spindle poles, but there is punctate staining in the cytoplasm (Figure 5E). During telophase, when centrosome replication initiates (FOE et al. 1993), there is a single region of punctate staining associated with each nucleus, as well as an increase in cytoplasmic staining (Figure 5F). This pattern of staining suggests the short isoforms may be important during centrosome replication and/or separation from late telophase through prophase, and the protein is relocated and perhaps degraded as the cell cycle proceeds through metaphase and anaphase.

Although we cannot differentiate between specific short isoforms, Western analysis predicts at least two short isoforms in early embryos, and Northern analysis shows a transcript initiated at exon 1c would encode the most abundant form. If the function of the two short isoforms is similar, a single isoform should mimic the wild-type pattern observed in embryos. To investigate the function of a single isoform during early cleavage divisions, we ectopically expressed a GFP::Cnn-PG encoded fusion protein and immunostained embryos with an anti-GFP antibody. GFP staining localizes to two primary foci associated with nuclei and weakly surrounds the nuclear envelope during prophase (Figure 6A), similar to the pattern observed in wild-type embryos. However, the primary foci of GFP staining are more fragmented than the native protein (Figure 6B), possibly due to excess amounts of the fusion protein. During metaphase, GFP staining is localized to discrete foci at the poles of elongating metaphase spindles and at chromatin (Figure 6, C and D). The reduction in the centrosome-associated fraction suggests the localization of the fusion protein in this region is regulated by the same mechanism as the native protein, whereas the association with chromatin is unique to this fusion protein, possibly due to excess amounts, loss of regulation, or both. Interestingly, when there is strong GFP staining associated with chromatin during late anaphase and early telophase, segregation of DNA is either impaired or blocked (arrowheads, Figure 6, E–H). Although all telophase nuclei are associated with the fusion protein, many of the divisions are normal, consistent with no measurable loss of fertility in embryos expressing this fusion protein. However, there are a significant number of aberrant divisions and these must either be resolved over time, or aneuploid nuclei are eliminated from the cortex due to blocked divisions prior to cellularization (RAFF and GLOVER 1988; SULLIVAN et al. 1990; POSTNER et al. 1992).

View larger version (42K): In this window In a new window Download PPT slide   FIGURE 6.— nos::Gal4 driven ectopic expression of a GFP::Cnn-7c (Cnn-PG) fusion protein in early embryos. Immunostaining with anti GFP antibody (green), DNA staining (blue), and anti--tubulin antibody (red) in early embryos shows the fusion protein localizes to centrosomes and to DNA during mitosis. (A) During early prophase GFP staining is localized to foci associated with nuclei and (B) surrounds the nucleus but is excluded from the nuclear interior while the nuclear envelope is intact. (C) By early metaphase GFP staining is detectable at centrosomes and (D) at DNA during chromosome congression. GFP staining at DNA persists throughout the remainder of mitosis. During telophase, (E) GFP is present at all nuclei and (arrowheads, E–H) appears to interfere with DNA segregation. (arrowheads, E) The tubulin midbodies are aberrant at these nuclei and (arrowheads, G) nuclei fail to separate. These effects are either transient or rare as fertility in these flies is not significantly reduced. Bar for A–D, 40 µm; bar for E–H, 20 µm.

 

cnn mutations revisited: the function of long and short isoforms during development:

The above analysis strongly suggests the functional complexity of cnn is greater than might be expected from a single isoform. That the loss of long Cnn isoforms in oocytes results in female sterility due to defective cleavage divisions during syncytial development has been well established (HEUER et al. 1995; MEGRAW et al. 1999; VAIZEL-OHAYON and SCHEJTER 1999). The developmental requirement for Cnn appears to be limited to early embryogenesis, as maternally supplied transcripts and proteins from heterozygous females are sufficient for adult development of mutant flies. Additionally, Cnn is not essential for mitosis in larval neuroblasts and imaginal cells, or in Drosophila S2 tissue culture cells (MEGRAW et al. 2001), demonstrating that Cnn is not required for mitosis in many cell types. Cnn is required for the mitotic and meiotic divisions during spermatogenesis, and all cnn mutant alleles result in male sterility (LI et al. 1998). Interestingly, Cnn proteins may not have an essential function during oogenesis, as mutant females produce many eggs with normal gross morphology. This has led to the general assumption that all cnn mutations are equivalent at the molecular level, but experimentally this assumption is based almost exclusively on mutant analyses of strains that have Cnn short isoforms present. The finding that excess stem cell accumulation in D. melanogaster testes, due to the loss of centrosome function in cnn mutants is more severe in cnn^hk21 homozygotes, as compared to cnn^hk21/cnn^mfs3 trans-heterozygous mutants, which are capable of producing the short isoforms from the cnn^mfs3 allele (YAMASHITA et al. 2003), suggested subtle phenotypic differences due to genotype would be detectable.

To investigate the effect of different mutations, we used the cnn^hk21, cnn^mfs7, and cnn^mfs3 nonsense mutations, which we refer to hereafter as hk21^null, mfs7, and mfs3, respectively. The hk21^null mutation eliminates all Cnn isoforms, whereas the mfs7 and mfs3 mutations result in the most and least truncated forms of Cnn long isoforms, respectively, while leaving the short isoforms unscathed and potentially functional (Figure 1). We first asked whether there were any detectable affects on gametogenesis by assaying the fecundity of males and females carrying various cnn mutant combinations as well as examining the morphology of hk21^null ovaries. Our results pertaining to spermatogenesis and oogenesis are presented in the supporting information of this publication. (See File S1; Figure S1; Figure S2; Table S1; and Table S2.)

To investigate the possibility of a range of cnn mutant phenotypes during embryogenesis, similar to those in gametogenesis (see supporting information), we examined immunostained embryos and assessed the localization of short and long isoform antibodies in metaphase spindles prior to, and at the point of failure. In control (cnn^–/cnn⁺) metaphase spindles, long isoform antibodies show strong staining at the centrosome (Figure 7, A and C), whereas the short isoform antibodies are barely detectable at the centrosome (Figure 7B), similar to what we observe in wild-type embryos. In mfs3 mutant embryos, the long form antibody is localized strongly at the metaphase plate and weakly throughout the spindle and at the poles (Figure 7D). The short form antibody localizes to the spindle poles and is more abundant than seen in control embryos (Figure 7, E and F). In mfs7 mutant embryos long form staining is barely detectable in the spindle (Figure 7G), whereas the short form staining is significantly increased at the poles (Figure 7H), and microtubule staining shows the poles in these spindles are relatively broad (Figure 7I) as compared to control and mfs3 spindles. The failure of embryogenesis in both mutants is associated with the appearance of multiple metaphase spindles fused at a common pole. In mfs3 mutant embryos showing multiple fused spindles, long form staining is weak and primarily in the spindle (Figure 8A). The short form staining is reduced but still present at all spindle poles (Figure 8B), including the fusion point of multiple spindles (arrowheads, Figure 8C). In mfs7 embryos, both long form (Figure 8D) and short form staining (Figure 8E) are barely detectable or absent at many spindle poles, although fused poles are associated with short form staining (arrowhead, Figure 8F). In hk21^null mutant embryos, both antibodies fail to detect any protein, and most nuclei are only detected with DNA staining, although spindles are occasionally present (Figure 8, G–I). These embryos fail during the first few cleavage divisions, and most of the embryos examined show no signs of development.

View larger version (50K): In this window In a new window Download PPT slide   FIGURE 7.— Long and short Cnn isoforms at early metaphase spindles in mfs3 and mfs7 mutant embryos. Immunostaining of early metaphase spindles in control and mfs3 and mfs7 mutant embryos with anti-Cnn.Long antibody (red), anti-Cnn.Short antisera (blue), and -tubulin antibody (green) reveal differences between mutant alleles and isoform function. In control embryos (A) long form staining is localized to centrosomes, (B) short form staining is barely detectable at the poles, and (C) spindles are normal. In the mfs3 mutant embryos (D) long form staining is strongest at the spindle equator and weakly detected throughout the spindle and at centrosomes. (E) Short form staining is localized to centrosomes and is more abundant than the control centrosomes, and (F) weak astral arrays are present at the centrosomes of these spindles, suggesting this allele has partial function. In the mfs7 mutant embryos (G) long form staining is weakly detected in the spindle only, (H) short form staining is relatively robust at centrosomes, and (I) the spindles have less focused poles that lack astral arrays. The increase in short form staining at centrosomes in these embryos suggests a redundant function in the absence of long forms. Bar for A–I, 15 µm.

 

View larger version (47K): In this window In a new window Download PPT slide   FIGURE 8.— mfs mutant embryos fail over time; hk21 embryos fail early. Immunostaining in mfs3 and mfs7 mutant embryos is the same as in Figure 5; hk21 mutant embryos stained with anti-Cnn.Long antibody (red), -tubulin antibody (green), and DNA (blue). As development continues in mfs3 mutant embryos, (A) long form staining is less intense and is primarily localized to the spindles, (B) short form staining is reduced at the spindle poles, and (C) spindles begin to fuse (arrowheads). Short isoforms staining is present at all spindle fusion points. Failure is similar in mfs7 mutants although (D) long forms and (E) and short forms are barely detectable and (F) in general, there are less robust and fewer spindles present (arrowhead). Short form staining is present at spindle fusion point and at extremely low levels at individual poles. (G and H) In hk21 mutant embryos there are typically a few nuclei present and occasional spindles, but development fails prior to nuclear migration. We frequently find a spindle near the cortex but obvious polar bodies are not detected. This suggests that a combination of long and short isoforms is required for centrosome function during early embryogenesis. Bar for A–F, 15 µm; bar for G–I, 25 µm.

 
On the basis of these results it seems clear that hk21^null mutants result in early failure during embryogenesis and perhaps during oogenesis. However, the truncated long isoform protein encoded by the mfs3 allele actually localizes to centrosomes and weak astral microtubules are present, while the protein encoded by the mfs7 allele is much less apparent. This suggests the mfs3 allele may retain partial function as compared to the mfs7 allele. To investigate this possibility we analyzed the behavior of these two truncated proteins in control and mutant embryos on 2D Western blots. In mfs7 control embryos we detect the typical 10 foci of Cnn present in the TEP fraction, but the truncated mutant protein is undetectable (left upper panels, Figure 9). However, consistent with previous Western data (MEGRAW et al. 1999) the truncated protein is detected in mutant embryos, although the protein appears to be degraded (right upper panel, Figure 9). Surprisingly, in mfs3 control embryos the wild-type Cnn pattern is present as well as 5 foci of the truncated protein (left lower panels, Figure 9). The same truncated protein pattern is detected in mfs3 mutant embryos (right lower panel, Figure 9). The failure to detect mutant protein in the mfs7 control animals could be due to degradation of the transcript, but this seems unlikely since the protein is present in mutant TEP fractions. A more probable explanation is the truncated protein is degraded in the presence of a functional centrosome. Unlike mfs7, the mutant protein associated with the mfs3 allele is stable in control animals and appears to be modified post-translationally in control and mutant embryos, showing that mfs3 is a strong hypomorph, rather than an entire loss of long isoform function of cnn.

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 9.— The mfs3-associated long isoform is stable and post-translationally modified. 2D Western blots of TEP fractions stained with anti-Cnn.Long antibody reveal a difference in the stability of the mfs7 and mfs3 truncated long isoforms. The mfs7 protein is not detectable in control TEP fractions after long exposure times, suggesting the protein is degraded in the presence of functional centrosomes (top panels, left) and the protein fails to resolve into discrete foci in mutant embryos (top panel, right). The mfs3 protein is stable in control (lower panels, left) and mutant embryos (lower panel, right), and resolves to five foci in both protein fractions. In mutant embryos the mfs3 protein is more abundant than in control embryos, on the basis of exposure times.

 
These results demonstrate that there are functional differences among cnn mutant alleles, and the loss of all Cnn isoforms has significantly more severe phenotypic consequences during gametogenesis and embryogenesis than lesions that affect only the long isoforms. The finding that the truncated protein associated with the mfs3 allele is stable, is targeted by the PTM machinery, and prior to embryonic failure low levels of protein localize to centrosomes that are competent to nucleate astral microtubules, phenotypes not observed in mfs7 mutant embryos, provides a partial explanation for the differences between the mfs alleles. The reduced severity in mfs phenotypes as compared to the hk21^null phenotype suggests the short isoforms are necessary and may be partially redundant to the Cnn long forms, as evidenced by an increase in short forms detectable at metaphase spindle poles prior to failure in early embryos in mfs3 and mfs7 mutants. However, the loss of long form function always results in failure shortly after nuclei reach the cortex, suggesting long form function is essential for the recruitment of centrosomal components as development proceeds. Taken together, these data predict the short isoforms are not merely redundant to the long isoforms but rather have unique roles required during embryogenesis.

Rescue of cnn mutant phenotypes with Cnn1a:

The above data all strongly suggest the short isoforms have an essential function during Drosophila development. If short isoforms are required during embryogenesis, ectopic expression of a cnn1a (Cnn-RA) transgene in embryos should not be able to rescue the hk21^null mutant phenotype. To test this possibility we expressed a GFP::Cnn-PA fusion protein in embryos with the maternally driven nos::Gal4 expression vector, and crossed mutant females carrying the two transgenes to wild-type males. Wild-type males are necessary for this cross, as mutant males carrying the transgenes are semisterile. This cross allows us to test the rescuing ability of the Cnn-PA fusion protein during the early stages of embryogenesis, prior to zygotic expression.

The expression of the transgene in mfs7 and mfs3 mutant embryos is sufficient to rescue embryogenesis, but fails to rescue the hk21^null mutant phenotype. We find 92% of the eggs produced by mfs mutant mothers carrying the transgene hatch, as compared to 95% of the control eggs, and none of the eggs produced by hk21^null mutant mothers carrying the transgene hatch. To investigate the molecular basis of the rescue of embryogenesis in mfs embryos, but not in hk21^null embryos, we immunostained embryos produced by mutant mothers expressing the fusion protein.

Immunostaining for GFP and -tubulin in mfs3 embryos shows both GFP and tubulin antibodies localize to the centrosome during prophase (Figure 10, A and B), but the GFP antibody does not localize to the DNA (Figure 10C). The centrosomes in these embryos have astral microtubules (Figure 10A) and appear relatively normal, although the intensity of GFP staining is more variable between centrosomes than is typically seen in wild-type embryos stained with anti-Cnn antibodies. During metaphase in mfs3 embryos, spindle morphology is similar to wild-type spindles and astral microtubules are detectable at the poles, although the poles are less focused than wild-type spindles (Figure 10D). GFP antibody localizes throughout the spindle (Figure 10E), but is absent at DNA aligned along the metaphase plate (Figure 10F). The spacing of spindles in these embryos is more variable than what is observed in wild-type embryos, and the GFP antibody does not localize to a discrete region at the spindle poles. However, the high levels of GFP staining throughout the spindle, which is most likely due to the abundance of the fusion protein, may obscure detection of the centrosome. We find no detectable differences between mfs3 and mfs7 embryos, showing the Cnn-PA transgene is sufficient to rescue early syncytial cleavage divisions when the short isoforms are present.

View larger version (52K): In this window In a new window Download PPT slide   FIGURE 10.— GFP::Cnn-PA rescues mfs embryonic phenotypes but fails to rescue hk21 embryonic phenotypes. Immunostaining with anti--tubulin antibody (red), anti-GFP antibody (green), and DNA staining (blue) in early mfs3 and hk21 embryos shows the GFP::Cnn-PA fusion protein localizes to centrosomes and to spindles during mitosis in mfs3 embryos, but is only present in spindles in the hk21 embryos. In mfs3 embryos during prophase (A) tubulin and (B) GFP staining is present at centrosomes that form astral microtubules, and (C) the fusion protein is not associated with DNA. During metaphase, in mfs3 embryos, (D) tubulin staining shows the spindle poles are less focused than wild-type spindles but form astral microtubules, (E) GFP staining localizes primarily to the spindle, and (F) is not present with DNA at the metaphase plate. In hk21 embryos, metaphase spindles form during early embryogenesis, but (G) tubulin staining shows the spindles have broad unfocused poles, (H) GFP staining is weak throughout the spindles, but (I) the morphology of these spindles is improved compared to hk21 embryos that lack the fusion protein (see Figure 8). As development proceeds, the fusion protein fails to support mitotic divisions in hk21 embryos and (J) tubulin staining is weak at midbodies (arrowhead) and absent at the poles, (K) GFP staining is barely detectable at midbodies (arrowhead), and (L) many nuclei are aberrant. Bar for A–I, 15 µm.

 
Immunostaining of embryos produced by hk21^null mothers expressing the transgene shows in the absence of the short isoforms, the Cnn-PA fusion protein does not rescue embryogenesis. Although we see an increase in the number of embryos with spindles, the majority of the embryos are similar to hk21^null embryos, which lack the fusion protein. When metaphase spindles are present during the very early stages of development, the poles are extremely broad and unfocused (Figure 10G), suggesting they lack centrosomes, and GFP staining is weakly localized to the spindles and DNA (Figure 10, H and I). As development proceeds, tubulin and GFP staining is detectable at midbodies during telophase but is absent at the poles (Figure 10, J and K, arrowhead), and in most cases these antibodies form an amorphous mass around nuclei or are completely absent at nuclei (Figure 10L). The lower levels of GFP staining at metaphase spindles detected in hk21^null embryos as compared to mfs3 embryos suggest Cnn short isoforms may be necessary to transport Cnn long isoforms to the centrosome. The morphology of metaphase spindles and the absence of both GFP and tubulin at the poles of telophase spindles indicate functional centrosomes are absent in these spindles, suggesting Cnn short isoforms may be necessary for centrosome formation following fertilization. Additionally, the number of embryos that fail to initiate embryogenesis implies short isoforms may play a role in meiosis, or syngamy, or both.

The obvious test to establish a requirement for Cnn short isoforms would be to rescue the hk21^null mutant phenotype by expressing both a Cnn-PA and a Cnn short form transgene in embryos. Unfortunately the deleterious effects we observe by ectopically expressing our Cnn-PG transgene (see above) have prevented us from performing this experiment. Successful rescue of the hk21^null phenotype may require the expression of a different short isoform or some combination of short forms, which will be the subject of future studies.

Cnn is the most abundant protein in the centrosome of D. melanogaster embryos (LANGE et al. 2000) and has a homogeneous distribution throughout the PCM (LANGE et al. 2005). Although it is clear that Cnn is required for centrosome function during syncytial development, the molecular basis of this function remains unclear. Perhaps the essential function is the requirement of Cnn for the proper localization of -tubulin to the centrosome and the formation of astral microtubules (MEGRAW et al. 2001). However, in the apparent absence of Cnn associated with certain mutant alleles and the consequent failure of -tubulin to be localized to the spindle poles, the early cleavage divisions in mutant embryos nevertheless appear to be relatively normal, so this function alone fails to explain why embryogenesis fails in these mutants. To address this issue, we have revisited the cnn gene and the effect of known mutations on gametogenesis and embryogenesis in D. melanogaster. We present evidence for the presence of two unique types of Cnn protein at the centrosome and show all mutant alleles of cnn are not equivalent. This study should help to elucidate some of the questions about the function of Cnn at the Drosophila centrosome.

The Drosophila genome project and our screening of cDNA libraries and the Berkeley Drosophila Genome Project EST collection have led to the recovery of additional cnn cDNAs that comprise two families of cnn products, showing our original assessment of two protein isoforms is an underestimate of the repertoire of the gene. At present we have identified five unique transcripts and their encoded proteins similar to the Cnn proteins described in the literature denoted here as long isoforms. Moreover, there is undoubtedly a sixth member of this family (discussed below). Additionally, there is a second family of four unique transcripts and their encoded proteins, denoted here as short isoforms, which differ significantly from the long forms. Although isoforms may potentially have tissue specificity, the three promoters in cnn seem to be utilized at all stages of development. This suggests specificity may be the result of tissue-specific regulation of splicing events, similar to findings for a collection of alternatively spliced modular exons in mouse (XING and LEE 2005). The determination of transcript specificity will require further analyses, but clearly alternative splicing increases the complexity of cnn.

Our 1D Western analysis of long and short isoforms during early embryogenesis strongly correlates with the transcriptional complexity for this stage of development. There are two long and three short protein isoforms in early embryos that are present in different concentrations. However, to detect all isoforms present, an enrichment method for centrosomes or Cnn is required, as only the most abundant bands are detectable in TEP extracts. It is possible these less abundant bands represent changes in PTMs of a single protein isoform or are also unique encoded isoforms. We used 2D Western analysis to differentiate between these possibilities for the long isoforms and to verify previous results indicating Cnn is regulated by phosphorylation.

The strongly correlated Northern and Western analyses of cnn transcripts and Cnn isoforms clearly show the presence of two unique long isoforms in embryos that differ in size and pI. The size and pI of these two isoforms suggests the smaller and least abundant of the long isoforms is Cnn-PA, which has been the assumed functional isoform in most studies on Cnn. The larger, most abundant long isoform is likely to be the product of a unique and as yet unidentified transcript similar to cnn5c (cnn-RC) except at its 5' end. We predict this protein will be similar to Cnn-PC and refer to this isoform as Cnn-Theo (Cnn-PC-like) in this study. The protein product would have a predicted molecular weight of 155.7 kDa, and a predicted pI of 5.42, which is significantly different from Cnn-PA. Unfortunately, we have not recovered a full-length cDNA for this transcript, although partial fragments and Northern analysis suggest it does exist. Both isoforms have multiple PTMs that are likely to be due to phosphorylation of the proteins, and the Cnn-Theo (Cnn-PC-like) isoform has an additional unknown PTM detectable in 2-hr TEP extracts. The phosphorylation of Cnn is supported by phosphatase treatment of the larger isoform in this study, and previous work showing immunoprecipitated Cnn appears as two bands on Western blots and these bands shift after phosphatase treatment (LI and KAUFMAN 1996). The unknown PTM is less stable than phosphorylation and appears to decrease the solubility of the protein. A previous report may have identified this PTM in Drosophila tissue culture cells when mitosis was chemically blocked. The modification was hypothesized to be the result of a Polo kinase-dependent addition of a single phosphate on Cnn (DOBBELAERE et al. 2008). However, the molecular weight shift of Cnn on 1D Western blots, and the large pI shift of Cnn on 2D Western blots reported (DOBBELAERE et al. 2009) is inconsistent with a single phosphate addition. Interestingly, we apparently detect the same modification during cellularization of the syncytial embryo. At this stage of development Cnn is enriched at centrosomes but mitosis is blocked, a situation that may be mimicked in tissue culture cells when mitosis is blocked by chemical means. Future studies of this potentially Polo-dependent PTM may provide further insight into the function of Cnn during embryogenesis.

In addition to the complex pattern of PTMs present on Cnn long isoforms, our Western analyses reveal a dynamic translational complexity for these two isoforms. As a maternally supplied protein, Cnn-Theo (Cnn-PC-like) is post-translationally modified at multiple sites during oogenesis or immediately after egg activation and is the predominant form of Cnn in unfertilized oocytes, ovaries, and in developing embryos. The translation of Cnn-PA initiates following egg activation and this protein continues to accumulate even in unfertilized eggs, whereas translation or accumulation of Cnn-Theo (Cnn-PC-like) appears to require development. Therefore, during normal embryogenesis the relative abundance of the Cnn-Theo (Cnn-PC-like) isoform is always much greater than Cnn-PA and is typically the only isoform detected with Western analysis of total protein extracts. However, since the Cnn-PA transgene can rescue the mfs mutant phenotypes, this isoform may be the one seen at the meiosis II spindle (LLAMAZARES et al. 1999) and may be required for proper positioning of the female pronucleus prior to fertilization (RIPARBELLI and CALLAINI 2005). Additionally, Cnn-PA may be required for initiation of the centrosome cycle during each cleavage division and Cnn-Theo (Cnn-PC-like) isoforms may represent the stable protein that remains at the centrosome throughout syncytial development. The molecular dissection of the exact function of these isoforms clearly requires deeper investigation but on the basis of our rescue results Cnn-PA provides the essential functions required during early embryogenesis. The presence of multiple long and short isoforms in early embryos potentially adds a level of complexity that has to date not been considered, and may alter earlier interpretations of the function of cnn during embryogenesis in D. melanogaster.

The requirement of long Cnn isoforms during syncytial development is well established, but what is the function, if any, of the short isoforms? Immunostaining of syncytial embryos demonstrates these proteins begin to concentrate at the centrosome during late telophase, coincident with centrosome replication (FOE et al. 1993) and the timing of localization of long Cnn isoforms to centrosomes (EISMAN et al. 2006). This suggests short Cnn isoforms may be functionally significant during these processes. Staining is the most intense at centrosomes throughout prophase, decreases during metaphase, and is absent in anaphase centrosomes. These short isoforms may be inactivated as the cell cycle proceeds, although this seems unlikely, since we readily detect cytoplasmic staining during telophase, whereas the cytoplasm of anaphase embryos has very little staining. This suggests the short isoform proteins do not function during chromosome segregation and that these proteins are degraded, consistent with the low abundance of these proteins on Western blots as compared to the long isoforms.

In addition to the centrosomal staining, the antibody also localizes at haploid, diploid, and triploid polar bodies. In wild-type embryos, haploid and diploid polar bodies enter mitosis coincident with the first zygotic mitotic division, but arrest at metaphase and fuse together with the chromosomes arranged in a starburst configuration (FOE et al. 1993). The persistent staining of short isoforms at polar bodies raises the possibility that a second function of these isoforms may be to block nuclear divisions. Consistent with this possibility, we find that the ectopic expression of a GFP::Cnn-PG encoded fusion protein in early embryos either interferes with or completely blocks chromosome segregation during anaphase. If all short isoforms have a high affinity for chromatin it would be important to degrade or inactivate these proteins at actively dividing nuclei while maintaining high levels at polar bodies. Alternatively, since the fusion protein is not a perfect mimic of native protein at mitotic centrosomes, Cnn-PG may function at polar bodies, while a different isoform functions at the centrosomes. Additional studies will be necessary to determine functional differences among specific isoforms.

While the above suggests a function for short isoforms, is the function essential for development? To test for function we reexamined the phenotypes of the hk21^null allele, which lacks all isoforms, and the mfs7 and mfs3 alleles, which both potentially express wild-type short form transcripts but differ with respect to the severity of the truncation of long isoforms. We assessed reproductive fitness on the basis of male fertility of mutant control strains and female egg production in mutant and control strains, in addition to mutant embryonic phenotypes. If short isoforms are essential there should be a detectable phenotypic difference between hk21^null and the mfs alleles.

Male fertility is significantly reduced compared to wild-type males in the three mutant heterozygous control strains, and sterility is increased in mfs7 and males and hk21^null control males. Although all heterozygous control and mutant females lay a large number of morphologically normal oocytes, egg production was significantly reduced in hk21^null/+ control and hk21^null mutant females, as well as mfs7 mutant females compared to wild type. Surprisingly, the hk21^null mutant females stop laying eggs after 10–14 days and ovaries from these females have various defects that are not found associated with any of the mutant mfs alleles. Our preliminary characterization of the underlying cause of these defects, provides the first indication that Cnn may have an essential function during oogenesis related to the loss of both long and short isoforms. It is also of interest that the mfs7 allele reduces both male and female reproductive fitness. Why the mfs7 phenotype is more severe than the mfs3 phenotype is not apparent at the morphological level, but it implies a subtle functional difference between these two alleles.

Since all oocytes produced by mutant mothers fail to develop, it has been assumed all mutant alleles result in embryonic failure due to an equivalent loss of centrosome function. Our reexamination of embryogenesis in mfs3, mfs7, and hk21^null mutant embryos with respect to the Cnn short isoforms, shows this is not the case. In mfs3 and mfs7 mutant embryos, the short isoforms are present at the poles of all spindles during early cleavage divisions. Perhaps more importantly, we find an increase in the amount of protein at the poles of mfs7 mutants compared to mfs3 mutants, and short isoforms are increased at the poles in both mutants compared to wild-type centrosomes. This suggests short isoforms may have a partially redundant long isoform function in the absence of long forms at the centrosome. As development proceeds, the amount of both long and short forms decreases, spindles begin to fuse or fail to form, and development stops. The progressive failure is likely due to accumulation of direct and indirect effects resulting from loss of centrosome function.

In addition to the effect of short isoforms in mutant embryos, we observe significant differences between the truncated long isoforms associated with the mfs3 and mfs7 alleles. The mfs3 protein is much more abundant than the mfs7 protein and low levels are present in the centrosomes. Additionally, the centrosomes are competent to nucleate weak astral microtubules, and the spindle poles are more focused than mfs7 spindles. This suggests the mfs3 long form protein is partially functional, and much lower levels of protein than are present in wild-type centrosomes will temporarily support mitotic divisions. Consistent with the mfs3 protein having some function, we find the protein is stable and post-translationally modified in embryos, on the basis of 2D Western blotting results. The mfs7 protein does not have this characteristic and embryogenesis appears to fail earlier in these mutant embryos, although accurately assigning a specific number of mitotic divisions to either mutant is difficult. Taken together, these results suggest the mfs3 allele is hypomorphic, while the mfs7 allele is a loss of long isoform function allele.

Unlike the mfs mutant alleles, we do not observe any significant development in hk21^null mutant embryos. Embryos typically have multiple foci of DNA and occasional spindles, but these nuclei are deep within the egg cytoplasm and do not appear to migrate toward the cortex. Additionally, we do not find normal polar bodies, suggesting the multiple nuclei may be meiotic products that have undergone multiple rounds of division. In wild-type embryos, short isoforms remain associated with polar bodies during early embryogenesis, raising the possibility that loss of these isoforms in hk21^null mutant embryos releases the normal mitotic block. Short isoforms may directly block polar body division, or they may be required to recruit other factors to the polar bodies, such as Polo kinase. In Polo mutant embryos meiotic products undergo multiple rounds of division (RIPARBELLI et al. 2000), similar to our observations in hk21^null mutants, but Polo mutant pronuclei divide more frequently than observed in hk21^null mutants. The failure of hk21^null mutant embryos to produce a significant number of spindles and the absence of fused spindles typically found in mfs mutant embryos, strongly suggests short isoforms have a required function during very early embryogenesis in D. melanogaster.

The requirement of short isoforms is demonstrated by the failure of the Cnn-PA protein to rescue the hk21^null embryonic phenotype. In hk21^null embryos the presence of the GFP::Cnn-PA fusion protein resulted in a minor improvement in acentrosomal spindle formation in some embryos, whereas the Cnn-PA isoform in mfs embryos is sufficient to rescue embryogenesis in the presence of Cnn short isoforms. While spindle morphology and the organization of spindles at the cortex are not as precise in mfs rescued embryos as that observed in wild-type embryos, these embryos complete embryogenesis and hatch. Additionally, the level of the fusion protein at spindles in hk21^null embryos was much lower than what was observed for the fusion protein in mfs embryos. This implies Cnn short isoforms may be required for normal localization of Cnn to the centrosome, or for the formation of centrosomes. Interestingly, the Cnn-PA isoform is sufficient for the rescue of the mfs mutant phenotypes, suggesting the larger, more abundant Cnn-Theo long isoform is not absolutely required during early embryogenesis. However, we cannot rule out the possibility that this isoform is required after nuclei reach the cortex at cell cycle 10. Since mutant females were crossed to wild-type males, the embryos carry a wild-type copy of the cnn gene, which could be expressed following cycle 10 (FOE et al. 1993). Additionally, we found no improvement in most of the hk21^null embryos, suggesting syngamy failed, similar to what is observed in hk21^null embryos that lack the fusion protein. This same fusion protein rescued 92% of the embryos produced by mfs mothers, demonstrating Cnn short isoforms are required for syncytial development. These results show Cnn short isoform function is necessary for syngamy, centrosome formation, and maintenance, and possibly for localization of Cnn to the centrosome. These data also show that wild-type function of Cnn during syncytial development involves a complex mix of isoforms, which are likely to be important during meiosis and syngamy, and are required for centrosome function and maintenance during the rapid syncytial cleavage divisions. The molecular basis of these functions, the specific isoforms required for each function, and the interactions between long and short Cnn isoforms during syncytial development in D. melanogaster will be the subject of future studies.

The requirement for Cnn function at mitotic centrosomes during syncytial development is well established, but this function has been largely attributed to a single protein. In this study, we present new evidence on the transcriptional and protein complexity of cnn. Rather than a single isoform, Cnn function can be attributed to two families of protein, and both families have essential functions during gametogenesis and embryogenesis. Additionally, mutations in cnn are not all equal, as different mutant genotypes can be correlated with phenotypic severity. While this study raises many new questions about the function and regulation of Cnn at the centrosome, it should provide a more comprehensive background for all experiments with Cnn as an indicator of centrosome function in D. melanogaster.

We thank Jeff Cecil, Brad Elmore, Lei Gong, Stacy Holtzman, Dave Miller, and Dave Waning for excellent technical assistance; Josef Heuer, Ling-Rong Kao, Kaijun Li, Tim Megraw, and Eugene Xu for their seminal contributions to our dissection of cnn; and Kevin Cook for sage advice, unwavering support, and careful reading of the manuscript. Any remaining deficiencies in the presentation are caused by our failure to take his advice. This work was supported by Indiana Genomics Initiative and in its early stages by Howard Hughes Medical Institute.

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.103887/DC1.

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