The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish
2007; Springer Nature; Volume: 26; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7601821
ISSN1460-2075
AutoresMarco Ferg, Remo Sanges, Jochen Gehrig, János Kiss, Matthias Bauer, A Lovas, Mónika Szabó, Lixin Yang, Uwe Straehle, Michael J. Pankratz, Ferenc Olasz, Elia Stupka, Ferenc Müller,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle16 August 2007Open Access The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish Marco Ferg Marco Ferg Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Remo Sanges Remo Sanges Bioinformatics–CBM Scrl, AREA Science Park, Basovizza, Trieste, Italy CBM, AREA Science Park, Basovizza, Trieste, Italy Search for more papers by this author Jochen Gehrig Jochen Gehrig Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Janos Kiss Janos Kiss Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Matthias Bauer Matthias Bauer Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Agnes Lovas Agnes Lovas Leibniz Institute for Age Research, Jena, Germany Search for more papers by this author Monika Szabo Monika Szabo Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Lixin Yang Lixin Yang Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Uwe Straehle Uwe Straehle Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Michael J Pankratz Michael J Pankratz Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Ferenc Olasz Ferenc Olasz Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Elia Stupka Corresponding Author Elia Stupka Bioinformatics–CBM Scrl, AREA Science Park, Basovizza, Trieste, Italy CBM, AREA Science Park, Basovizza, Trieste, Italy Search for more papers by this author Ferenc Müller Corresponding Author Ferenc Müller Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Marco Ferg Marco Ferg Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Remo Sanges Remo Sanges Bioinformatics–CBM Scrl, AREA Science Park, Basovizza, Trieste, Italy CBM, AREA Science Park, Basovizza, Trieste, Italy Search for more papers by this author Jochen Gehrig Jochen Gehrig Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Janos Kiss Janos Kiss Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Matthias Bauer Matthias Bauer Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Agnes Lovas Agnes Lovas Leibniz Institute for Age Research, Jena, Germany Search for more papers by this author Monika Szabo Monika Szabo Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Lixin Yang Lixin Yang Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Uwe Straehle Uwe Straehle Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Michael J Pankratz Michael J Pankratz Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Ferenc Olasz Ferenc Olasz Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary Search for more papers by this author Elia Stupka Corresponding Author Elia Stupka Bioinformatics–CBM Scrl, AREA Science Park, Basovizza, Trieste, Italy CBM, AREA Science Park, Basovizza, Trieste, Italy Search for more papers by this author Ferenc Müller Corresponding Author Ferenc Müller Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany Search for more papers by this author Author Information Marco Ferg1,‡, Remo Sanges2,4,‡, Jochen Gehrig1, Janos Kiss3, Matthias Bauer1, Agnes Lovas5, Monika Szabo3, Lixin Yang1, Uwe Straehle1, Michael J Pankratz1, Ferenc Olasz3, Elia Stupka 2,4 and Ferenc Müller 1 1Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany 2Bioinformatics–CBM Scrl, AREA Science Park, Basovizza, Trieste, Italy 3Institute of Agricultural Biotechnology Centre, Gödöllõ, Hungary 4CBM, AREA Science Park, Basovizza, Trieste, Italy 5Leibniz Institute for Age Research, Jena, Germany ‡These authors contributed equally to this work *Corresponding authors: Bioinformatics–CBM Scrl, AREA Science Park, ss 14 km 163.5-Basovizza, Trieste 34012, Italy. E-mail: [email protected] Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Herrmann von Helmholtz Platz 1, Eggenstein-Leopoldshafen 76021, Germany. Tel.: + 49 7247 823444; Fax: + 49 7247 823354; E-mail: [email protected] The EMBO Journal (2007)26:3945-3956https://doi.org/10.1038/sj.emboj.7601821 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Early steps of embryo development are directed by maternal gene products and trace levels of zygotic gene activity in vertebrates. A major activation of zygotic transcription occurs together with degradation of maternal mRNAs during the midblastula transition in several vertebrate systems. How these processes are regulated in preparation for the onset of differentiation in the vertebrate embryo is mostly unknown. Here, we studied the function of TATA-binding protein (TBP) by knock down and DNA microarray analysis of gene expression in early embryo development. We show that a subset of polymerase II-transcribed genes with ontogenic stage-dependent regulation requires TBP for their zygotic activation. TBP is also required for limiting the activation of genes during development. We reveal that TBP plays an important role in the degradation of a specific subset of maternal mRNAs during late blastulation/early gastrulation, which involves targets of the miR-430 pathway. Hence, TBP acts as a specific regulator of the key processes underlying the transition from maternal to zygotic regulation of embryogenesis. These results implicate core promoter recognition as an additional level of differential gene regulation during development. Introduction In most animal models, including Drosophila, Caenorhabditis elegans, zebrafish and Xenopus, the onset of zygotic gene activation is delayed until the midblastula transition (MBT). (Newport and Kirschner, 1982; Kimmel et al, 1995). Whereas there is no MBT in mammals, here zygotic gene activity is also delayed after fertilisation (Thompson et al, 1998). In the zebrafish blastula, the general delay in zygotic gene activity is followed by the sudden and broad activation of a large number of genes representing all main gene ontologies (Kane and Kimmel, 1993; Mathavan et al, 2005) leading to gastrulation. The activation of the zygotic genome is parallelled by an equally significant process, the differential degradation of maternally inherited mRNAs (Giraldez et al, 2005; Mathavan et al, 2005; De Renzis et al, 2007). Whereas little is known about the mechanisms of degradation of maternal mRNA, they are known to involve both transcription-dependent and -independent pathways (Bashirullah et al, 1999; Audic et al, 2001; Giraldez et al, 2005; Schier, 2007). Dynamic changes in expression of maternally and zygotically activated genes are observed during zygotic gene activation also in the mouse (Wang et al, 2004). Not all maternally inherited mRNAs degrade during early embryogenesis and many maternal mRNAs continue to influence embryo development until later developmental stages (Wagner et al, 2004; reviewed by Pelegri (2003)). The initiation of zygotic transcription during MBT is believed to be regulated by a competition between chromatin and the assembly of the transcription machinery (Newport and Kirschner, 1982; Kimelman et al, 1987; Almouzni and Wolffe, 1995). The TATA-binding protein (TBP) has been implicated as a key regulator of transcription initiation in early embryo development in vertebrates (Veenstra et al, 2000; Müller et al, 2001; Martianov et al, 2002). TBP protein levels have been shown to be limiting for transcription before MBT and are dramatically upregulated at the initiation of zygotic transcription (Prioleau et al, 1994; Veenstra et al, 1999; Bártfai et al, 2004). TBP, together with TBP-associated factors (TAFs) are components of the TFIID complex, a key point at which activators can control transcription through the core promoter. Until recently, it was argued that TBP is required for the correct initiation of all RNA polymerase (Pol I, II and III)-mediated transcription in eukaryotes. However, recent reports have revealed the contrary: the composition of Pol II core promoter-binding complexes varies and is likely to represent a point of differential gene expression regulation (reviewed by Davidson (2003)). Consistently, whereas TBP is essential for early embryo development, it is not required for all Pol II transcription as demonstrated by studies on a small number of vertebrate genes (Veenstra et al, 2000; Müller et al, 2001; Martianov et al, 2002). The apparent redundancy of TBP in vertebrates is probably due to the function of TBP-like factors (TLF/TRF2) (Veenstra et al, 2000; Müller et al, 2001) and the recently described second set of TBP paralogue genes TBP2/TRF3 (Persengiev et al, 2003; Bártfai et al, 2004; Jallow et al, 2004). The functional requirement for different TBP family proteins in embryogenesis suggests specific nonoverlapping roles for these factors in regulating subsets of genes (Moore et al, 1999; Teichmann et al, 1999; Bártfai et al, 2004; Jallow et al, 2004). Our objective was to investigate the transcriptional regulatory mechanisms that involve core promoter recognition proteins such as TBP in the whole organism. The transition of gene activity from maternally inherited mRNAs to zygotic gene expression provides an ideal model for the analysis of the control of transcription initiation (Newport and Kirschner, 1982). By using Morpholino (MO) knockdown and microarray expression profiling, we have addressed which genes require TBP for their activity and what is the function of TBP in regulating the transition from maternal to zygotic regulation during early vertebrate embryo development. We show that TBP is preferentially required for genes that exhibit dynamic changes in their expression during ontogeny. Furthermore, we provide evidence for a previously undocumented negative regulatory role of TBP in zygotic gene activation. Importantly, we also describe a novel biological function of TBP: a role in the degradation of a subset of maternal mRNAs after MBT. Results TBP regulates specifically a subset of mRNAs in the dome-stage embryo In the early embryo, the steady-state levels of mRNA result from a dynamic process of gradual degradation of maternal mRNAs and the delayed initiation of zygotic gene expression at the MBT (Figure 1A). To investigate the role of TBP in regulating genes expressed in the early zebrafish embryo, we carried out a microarray analysis of 10 501 genes at the dome stage in embryos in which TBP function was blocked using MO antisense oligonucleotides as described previously (Müller et al, 2001). The dome stage occurs 1.3 h after the start of the global initiation of zygotic transcription at the MBT (Kane and Kimmel, 1993) and any TBP-dependent changes in gene activity detected at this stage are still expected to be mostly direct transcriptional effects. Loss of protein was confirmed by Western blot (Figure 1B). A total of 1927 genes from the 10 501 represented on the microarray were selected, having applied stringent but commonly used criteria (FDR cut off of 0.05) to eliminate potential false positives and false negatives from the analysis (see Materials and methods). Three distinct response groups of genes were identified: downregulated genes (⩽−2-fold change), genes with low variability in expression (values between >−2 and <+2-fold-change); upregulated genes (⩾+2-fold change) (Figure 1C and Supplementary Table I). The three groups thus identified were further validated by semiquantitative RT–PCR experiments; out of a total of 39 genes representing the above groups, 37 showed comparable activity to that observed in the microarray experiments (Supplementary Figure S1). The specificity of the effects detected was confirmed by microinjecting a second MO targeting TBP mRNA (TBP MO2), which resulted in comparable gene expression changes to the above TBP MO injection when analysed by RT–PCR of 28 genes (Supplementary Figure S2). Furthermore, the gene expression changes caused by TBP MO injection could be reverted by injecting a form of TBP mRNA that could not be targeted by the MO (Supplementary Figure S2). Figure 1.TBP is selectively required for both activation and repression of genes in the early zebrafish embryo. (A) Schematic diagram of the dynamics of mRNA degradation and zygotic gene activation during the MBT in the zebrafish embryo. Shades of blue indicate differential degradation of maternal mRNAs before the MBT. The red curve indicates the dynamics of zygotic gene activation. Time after fertilisation is indicated in hpf. Schematic drawing of respective stages of embryo development are shown below. The arrow indicates time point for collection of embryos for microarray analysis. (B) Western blot analysis of TBP protein levels in dome-stage zebrafish embryos injected with TBP (MOTBP MO) and control MO antisense oligonucleotides. (C) Pie chart diagram summary of expression profiling data of 1927 probes from microarray experiments carried out in dome-stage zebrafish embryos. (D) Schematic diagram of the protocol for identification of the intersection of genes analysed for TBP dependence among genes analysed for their expression dynamics during zebrafish development via the Unigene database. (E) Pie chart diagram of the proportion of genes found in the three response groups following TBP knockdown and overlap with the stage-dependent expression microarray. (F) Pie chart diagrams showing the distribution of constitutive and stage-specific genes among the total and the three response groups of genes in TBP morphant embryos. Numbers below the charts indicate the number of overlapping genes between the two data sets compared and χ2 analysis of the gene distributions. Download figure Download PowerPoint Within the 1927 genes that were used for this analysis, a large proportion of genes expressed in the dome-stage embryo (65.3%) showed no significant difference in signal strength between TBP MO- and control MO (c MO)-injected embryos, indicating that their steady-state mRNA levels are independent of TBP function. A smaller group of genes showed a significant reduction of expression demonstrating that these genes require TBP directly or indirectly for their activation (17.5%). A similar number of genes (17.1%) showed increased levels of transcripts in TBP MO injected embryos, suggesting that TBP is required for controlling their steady-state levels by reducing their transcription and/or by enhancing mRNA degradation. Most TBP-activated genes are dynamically regulated during zebrafish ontogeny To characterise further the genes affected by loss of TBP function, we tested whether genes in the three response groups described above showed discrete expression dynamics during zebrafish ontogeny. To this end, we compared the data set described above to an ontogenic stage-dependent expression profiling experiment on the zebrafish transcriptome (Konantz M, Otto G-W, Weller C, Saric M, Geisler R. Microarray analysis of gene expression in zebrafish development, manuscript in preparation). The two gene sets share 717 genes (Figure 1D and E) and the proportions of the three TBP morphant response groups among these 717 genes are similar to those of the total TBP microarray data set (Figure 1C and E). Meta-analysis of the ontogenic stage-dependent gene expression array was carried out to define two classes of genes. Genes showing stage specific peaks of expression activity during zebrafish ontogeny were classified as ‘stage-dependent’ and genes that showed no significant variation in gene expression during ontogeny were considered as constitutively active genes (Supplementary Figure S3, see Materials and methods). Nearly half of the 717 genes (46.9%) that overlap between the two microarray data sets were shown to be constitutively expressed genes. The remaining genes belonged to the stage-dependent class (53.1%) showing dynamic activity during zebrafish ontogeny (Figure 1F and Supplementary Figure S3). Applying the ‘constitutive versus stage-dependent’ classification to the TBP microarray gene response groups revealed that genes that require TBP for their activation were predominantly stage-dependent (77%, Figure 1F), whereas upregulated genes in TBP morphants showed the opposite tendency. The low-variable group of genes did not show a bias to either stage-dependent or constitutively expressed genes. These results indicate that TBP-dependent activation tends to be a property of genes that show dynamic activity during ontogeny. Moreover, TBP tends to negatively regulate steady-state levels of constitutively active genes. TBP dependence of transcription from isolated zebrafish promoters TBP could influence steady-state levels of mRNA in zebrafish embryos both through transcriptional as well as post-transcriptional processes. To address the former, we tested 23 GFP constructs using promoters of zebrafish genes expressed at the sphere/dome-stage and representing various gene ontology classes (O'Boyle et al, 2007). TBP-dependent promoter activation was evident for seven promoters, including the otx1 gene promoter (Figure 2A and Supplementary Figure S4 and Supplementary Table II). This result is consistent with the proposed role of TBP in activating zygotic transcription of many genes during development. On the other hand, 12 promoters, including the apoeb gene promoter, did not show significant changes of activity upon loss of TBP function (Figure 2A and Supplementary Figure S4). TBP independence of apoeb transcription was further confirmed by its mRNA levels (Supplementary Figure S1) and the utilisation of its TSS (data not shown) in TBP morphants. No correlation was found between known promoter motifs (such as TATA boxes, CpG islands, etc.) and TBP response (data not shown). Figure 2.TBP is required for both activation as well as repression of zebrafish promoters. (A) Representative samples of whole-mount immunochemical staining of embryos injected with promoter:gfp constructs (view on animal pole) with brown staining indicating mosaic pattern of GFP activity. (B–F) Rescue of the TBP morphant phenotype by overexpression of recombinant TBP. (B) Noninjected embryos, (C–G) Injection of tbp:yfp reporter construct carried out together with MO oligonucleotides as indicated above the images. Injected embryos were split into separate batches and exposed to a subsequent injection of water, tbp or is30 mRNA, as indicated below the horizontal line (D–F). Lateral views of 7 hpf embryos in bright field (top) or fluorescence views under YFP (bottom). Download figure Download PowerPoint Several promoters (4 out of 23) showed a clear increase of promoter activity upon loss of TBP, including the 1.4-kb promoter of the tbp gene (Figure 2A and Supplementary Figure S4). This finding suggests negative regulatory role of TBP on the tbp gene promoter and is in line with the inverse correlation between tbp mRNA and TBP protein levels at the late blastula and early gastrula stages (Bártfai et al, 2004; Supplementary Figure S4D). Co-injection of a synthetic TBP MO-resistant Xenopus (x) tbp mRNA, but not of bacterial IS30 transposase control mRNA rescued the epiboly movements of the animal cap (Figure 2C–F, bright field view) and tbp:yfp activity (Figure 2C–F, fluorescence views). Finally, the injection of TBP MO2 resulted in comparable effects to TBP MO both in blocking epiboly movements and in the increased activity of the tbp:yfp promoter construct (Figure 2G). These results demonstrate that the specific loss of TBP protein is the reason for the observed upregulation of the tbp promoter in TBP MO-injected embryos. TBP is required for degradation of a large number of maternal mRNAs It is known that degradation of many maternal mRNAs involves zygotic transcription-dependent mechanisms, which may be specifically regulated by TBP. Thus, the steady-state levels of maternal mRNAs may appear increased in TBP morphants. To test if the inhibition of the degradation of maternal mRNA occurs in TBP morphants, we searched for maternally expressed genes in the TBP morphant microarray gene sets. We classified genes as being maternal or zygotic through another microarray experiment utilizing mRNA pre- and post-MBT; those showing a decrease of mRNA levels from pre- to post-MBT (MBT down) were classified as prevalently maternal and vice versa (MBT up) for prevalently zygotic ones (Supplementary Tables III and IV). We then compared this experiment with the TBP morphants data set, which resulted in an overlap of 131 genes (Supplementary Tables III and IV). The overlap showed that maternal mRNAs were enriched among the upregulated genes of TBP morphants (Figure 3A, MBT down) and the inverse was observed for zygotic mRNAs (Figure 3A, MBT up). A side by side hierarchical clustering analysis of gene activity fold changes in the MBT experiment versus the TBP MO experiment demonstrates further the inverse correlation between the levels of mRNAs before or after MBT as compared to mRNA levels in TBP morphants versus controls (Figure 3B). Figure 3.A programme of maternal mRNA degradation requires TBP function. (A) Distribution of TBP MO response genes given as percentage of the genes expressed primarily at pre- or post-MBT stages. The number of overlapping genes between the microarray data sets is shown in the columns. Genes showing a higher abundance in pre-MBT stages (primarily maternal) are termed MBT down, genes showing a higher abundance in post-MBT stages (primarily zygotic) are termed MBT up. (B) Hierarchical clustering of genes based on their fold change values derived from the intersection of two microarray experiments. In the left column, the change of gene activities at post-MBT stages is shown in comparison to pre MBT (MBT). On the right, the response of the same genes is shown upon TBP knockdown (TBP). The fold change values for each gene are demonstrated as lines with colour intensities where red indicates higher expression. (C) Distribution of maternally expressed genes showing highest accumulation in the unfertilised egg (Mathavan et al, 2005) among significantly regulated genes present on the TBP-knockdown microarray given as percentages of the respective TBP response groups. The number of overlapping genes are given in the respective bars. (D) The expression patterns and levels of the maternally expressed zorba gene before (64 cells) and after MBT (sphere, dome). WISH of randomly oriented groups of embryos with DIG-labelled riboprobes against zorba are shown in MO injected embryos. Dark blue/purple staining in the animal pole (arrows) indicate specific gene activity in individual embryos. Arrowheads indicate loss of zorba mRNA signal. (E) RT–PCR analysis of selected maternally expressed genes before MBT and after MBT. Injection of MOs xtbp or is30 transposase mRNAs are indicated by ‘+’ symbol. Abbreviations, hpf, hours post fertilisation; up, upregulated; down, downregulated; low var, low variable expression in TBP MO injected embryos; c, cycle number of PCR reactions; s, synthetic mRNA; E, early embryos before MBT; L, late embryos after MBT. Download figure Download PowerPoint We further verified our findings by intersecting the TBP MO microarray experiment with an independent set of 622 maternal mRNAs (Mathavan et al, 2005), which resulted in an overlap of 143 genes (Supplementary Table V). As shown in Figure 3C, maternally inherited transcripts were significantly (P-value 1.043e−11) enriched among mRNAs upregulated in TBP morphants and underrepresented in the downregulated gene set (P-value 3.483e−5). Together, these results suggest that the upregulation of genes observed in TBP morphants could be in large part due to the specific loss of degradation of many maternal mRNAs. Identification of TBP-dependent maternal transcripts To validate the predicted involvement of TBP in the degradation of maternal mRNAs, we investigated the fate of individual maternal mRNAs. Zorba is a maternally expressed gene (Bally-Cuif et al, 1998), which is upregulated 2.51-fold in TBP MO embryos. We analysed the expression of zorba in wild-type and TBP-morphant embryos at regular intervals for the first 6 h of development by whole-mount in situ hybridisation (WISH). We found high levels of zorba expression in fertilised wild-type eggs and early embryos before MBT, followed by a sharp decrease soon after the MBT, followed by a slight increase at the dome stage (Figure 3D). In contrast, in TBP morphant embryos, zorba mRNA levels showed similar levels throughout early development, consistent with the assumption that degradation of maternal mRNA was impaired. We verified that the lack of degradation of zorba mRNA in TBP morphants was not due to a general delay in embryo development by observing the expression of two zygotically expressed genes: the TBP-independent gene no tail (Schulte-Merker et al, 1994), which correctly initiated transcription after the sphere stage in TBP morphants (Supplementary Figure S5A); and the TBP-dependent goosecoid (Schulte-Merker et al, 1994), whose activity was lost in TBP morphants (Supplementary Figure S5B). These results suggest efficient depletion of TBP at dome stage. To further verify the defect in maternal mRNA degradation, RT–PCR analysis was carried out on several maternally expressed genes that showed upregulation in TBP morphants. Zorba and smad2 (expressed both maternally and zygotically; Muller et al, 1999) showed elevated levels at dome stage in comparison to c MO-injected embryos, suggesting loss of degradation of maternal mRNA, as opposed to the control gene β-actin, which did not show a change in its steady-state levels (Figure 3E, lanes 1–4). RT–PCR analysis of zygotic genes was also carried out; the TBP-independent ntl showed no change in its mRNA levels, whereas zygotic activity of gsc dropped (data not shown) as shown previously by WISH. To test directly the fate of mRNAs deposited in the egg, we utilised a synthetic smad2 mRNA microinjected into the fertilised eggs (Figure 3E, smad2 (s)). This mRNA could be readily distinguished from endogenous smad2 by reducing the cycles in the RT–PCR reaction (Figure 3E, compare lanes 1–2 to 5–6 of smad2 (s)). Microinjected smad2 mRNA was more efficiently degraded in c MO- than in TBP MO-injected embryos (Figure 3E, compare lanes 6 and 8) and similar results were obtained by WISH (data not shown). Thus, the apparent increase of smad2 mRNA levels in TBP morphants is not due to premature activation of zygotic smad2 expression, but due to the loss of degradation of smad2 mRNAs. To verify the specificity of the maternal mRNA degradation phenotype to loss of TBP protein function, the ability of a MO-insensitive TBP mRNA to rescue the phenotype in TBP MO-injected embryos was tested. TBP MO and smad2 (s) co-injected embryos were split after injection and separate batches were injected for a second time either by xtbp mRNA or is30 mRNA. Expression of recombinant TBP resulted in increase of degradation of zorba (Figure 3E, compare lanes 7, 8 with 9, 10) as well as that of microinjected synthetic smad2 mRNA. In contrast, is30 tpase control mRNA did not result in rescue of the degradation phenotype of TBP morphants (Figure 3E, compare lanes 8 and 10). These results demonstrate that the effect of TBP MO on maternal mRNA degradation is directly attributable to the loss of TBP protein function. TBP regulates a zygotic transcription-dependent mRNA degradation process Little is known about the mechanisms of maternal mRNA degradation in zebrafish, however, it is likely to involve several maternal as well as zygotic transcription-dependent mechanisms. Not all maternal mRNAs were degraded in TBP morphants (Figure 3A and C). This may be due to different regulatory mechanisms acting in parallel during maternal mRNA degradation. To investigate this further, we verified the kinetics of mRNA degradation by exploiting a published microarray data set on maternal mRNAs (Mathavan et al, 2005) and compared it to our TBP morphant data set. We established three classes of mRNAs based on the time of their degradation (see Figure 4A and B and Supplementary Table X): a ‘fast’ group of mRNAs, which degrade transcription independently or in a transcription dependent manner immediately after initiation of zygotic transcription; a ‘medium’ group which is mostly degraded after MBT by early gastrula stage; and a ‘late’ group degraded during neurulation and somitogenesis. The comparison of these groups with the TBP-morphant experiment (Figure 4C) showed that maternal mRNAs upregulated in TBP morphants follow the pattern of expression dy
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