1 Developmental Biology Institute of Marseille, Laboratoire de Génétique et Physiologie du Développement, Université de la Méditerrannée, Luminy, Case 907, 13288 Marseille Cedex 09, France
2 Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA
* Present address: Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA
Present address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Present address: UMR218, Curie Institute-Section of Research, Pavillon Pasteur, 26 rue dUlm, 75 248 Paris Cedex 05, France
¶Author for correspondence (e-mail: nathalie.dostatni{at}curie.fr)
Accepted March 27, 2001
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SUMMARY |
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Key words: Bicoid morphogen, Homeodomain, Downregulation, Torso receptor tyrosine kinase, Drosophila
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INTRODUCTION |
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The terminal group genes mediate the development of the terminal structures called the acron at the anterior and the telson at the posterior (St Johnston and Nusslein-Volhard, 1992). Interestingly, in addition to the lack of anterior development, embryos from bcd females develop a telson instead of an acron at the anterior (Frohnhofer and Nusslein-Volhard, 1986). This indicates that the combined activities of both anterior and terminal determinants are required to establish the acron. Terminal activity is mediated by the torso gene, which encodes a receptor tyrosine kinase (RTK) expressed at the surface of the embryo and activated only at the poles by a locally activated ligand (Perrimon, 1993). The activation of the Torso RTK leads to the activation of the ras signal transduction cascade that results in the zygotic derepression of the tailless (tll) and huckebein (hkb) genes (Liaw et al., 1995; Paroush et al., 1997). tll and hkb are the zygotic mediators of the terminal system posteriorly but a different output of the terminal system has been postulated at the anterior, as anterior terminal structures do form in tll-/hkb- double mutant embryos (Strecker et al., 1986; Weigel et al., 1990). Interestingly, several of the Bcd target genes, including hb and otd, are first expressed during syncytial development as anterior caps that are repressed at the pole upon activation of Torso later on during cellularisation. The persistence of hb at the anterior pole alters the formation of terminal derivatives, such as the labrum and the foregut, and represses the expression of three zygotic genes, cap-n-collar (cnc), wingless (wg) and hedgehog (hh), which are involved in the formation of these structures (Janody et al., 2000a). High levels of Bcd can functionally rescue the lack of terminal activity at the anterior pole (Schaeffer et al., 2000), suggesting that Bcd and Torso can act independently on common target genes. Indeed, cnc, wg and hh are positive targets of Bcd and Torso. These genes are likely to be repressed by the persistence of hb at the anterior pole in torso mutants (Janody et al., 2000a), and over-expression of Bcd might bypass their repression by hb in this mutant background. Thus, Torso-induced repression of hb plays a key role in the interaction between the anterior and terminal systems for the formation of the acron.
The components of the ras signalling cascade, D-sor (MAP kinase kinase; Dsor1 FlyBase) (Tsuda et al., 1993) and D-raf (phl FlyBase) are required for the anterior repression of Bcd targets whereas tll and hkb are largely dispensable for this effect (Ronchi et al., 1993; Gao et al., 1996; Janody et al., 2000b). The insertion of three copies of the heterologous activation domain of the yeast GCN4 protein within Bcd renders the resulting fusion protein (Bcd-GCN4) insensitive to Torso inhibition (Ronchi et al., 1993). This indicates that modifications of Bcd could prevent the effect of Torso. Indeed, Bcd transcriptional activity is itself inhibited by the Torso pathway, as shown by Torso-induced downregulation of a Bcd-Gal4 fusion protein, in which the Gal4 DNA-binding domain replaces the homeodomain (Bellaïche et al., 1996). Most phosphorylations of Bcd are dependent on Torso activity (Ronchi et al., 1993) and occur in vivo on MAP kinase (MAPK) sites (Janody et al., 2000b). Surprisingly, however, in vivo mutational analysis indicates that these phosphorylations are not involved in the anterior repression of Bcd target genes but instead contribute to Torso-dependent strengthening of the Bcd morphogenetic gradient in the rest of the embryo (Janody et al., 2000b). This analysis suggests that the molecular mechanism responsible for the downregulation of Bcd by Torso does not rely on direct modifications of the Bcd protein but rather involves modification of a Bcd partner that interacts with Bcd. Besides the HD and the serine/threonine (ST)-rich domain that contains most of the MAPK phosphorylation sites, the Bcd protein contains three putative activation domains located at its C terminus: a glutamine-rich domain (Q), an alanine-rich domain (A) and an acidic domain (C). As a Bcd deletion variant lacking all three activation domains (Q, A and C) rescues the lack of wild-type Bcd to viability (Schaeffer et al., 1999), the N-terminal region of Bcd must contain at least one additional activation domain that mediates the activation of its target genes.
In the present study, our aim was to identify the domain of Bcd that mediates its inhibition by the Torso pathway. We first aimed to understand the biological behaviour of the Bcd-GCN4 protein that is insensitive to downregulation by Torso. We show that it is the intrinsic properties of the GCN4 activation domains that render Bcd-GCN4 insensitive to downregulation by Torso. This indicates that the molecular mechanism responsible for the downregulation of Bcd by Torso probably involves an inhibition of the activation process. To better understand the activating potential of Bcd, we performed a structure/function analysis of Bcd in cell culture and in the early embryo using deletion variants (Schaeffer et al., 1999) and fusion proteins containing the Gal4 DNA-binding domain with multiple copies of the Bcd domains. This analysis established: (1) that the Q domain functions as an autonomous activation domain that is not sensitive to downregulation by Torso; (2) that the ST and C domains function as autonomous activation domains that are both downregulated by Torso; and (3) that the A domain functions as a repressor domain in vivo and its activity does not seem to be regulated by Torso at the anterior pole. We propose that the direct downregulation of Bcd transcriptional activity by the Torso pathway results from the competing activity of the Q domain insensitive to Torso with both the C and the ST domains that mediate downregulation by Torso. The contribution of the A domain in this process might be to reduce the general activity of Bcd activation domains, in particular, the activity of the Q domain that would otherwise prevent inhibition by Torso.
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MATERIALS AND METHODS |
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The transcription unit pnos-Gal4-Actdomain-Bcd3'UTR in which the sequence coding for the fusion proteins, between the Gal4 DNA-binding domain (1-94) and simple or multiple copies of the putative activation domains of Bcd, are placed under the control of the nanos (nos) promoter and the Bcd 3' untranslated region (UTR), were obtained and processed as pnos-Gal4-GCN4-Bcd3'UTR (Janody et al., 2000a). The oligonucleotides used for amplification of the sequence of each of the Bcd domains were 5'-CTATTCTAGATCGACCAATGTCAATGGCG-3' and 5'-GATAGGATCCGTAGCTAGCGGCTTGCTTTTGCTGG-3' for the Q domain; 5'-CTATTCTAGAGCCAGCGCCTGTCGCG-3' and 5'-GATAGGATCCGTAGCTAGCGTAGACCTCGGAGCCCGG-3' for the A domain; 5'CTATTCTAGAGCTGCCTCGCCGGGC-3'and 5'-GATAGGATCCGTAGCTAGCATTGAAGCAGTAGGC-3' for the C domain; and 5'-TCATCTAGAGTAGATCGCACAAG-3' and 5'-TAGGATCCTTAGCTAGCTAAGCCGCCATTGACATT-3' for the ST domain (bcd sequences underlined). The sequence coding for the B6 and B42 activation domains were amplified by PCR from genomic DNA from the T335 and T337 stocks (Tübingen collection), respectively, using the following oligonucleotides that hybridise in the bcd genomic sequence (underlined): 5'-CTATTCTAGAGGGGGGCCAGGACCTGG-3' and 5'-GATAGGATCCCTAACTTTCTACGCGTAG-3'. The three copies of the GCN4 activation domain were amplified as described above and the unique copy of this domain was amplified using the following oligonucleotides: 5'-CTATGCTAGCTCTAGAGGACCTGGGTCGACGGATCC-3' and 5'-CTATGCTAGCAGATCTCTATCGACCTGCCTTATCAGCG-3'. The amplified fragments were digested by XbaI and either NheI or BamHI (bold) for multimerisation and cloning in frame, downstream of the sequence that codes for the Gal4 DNA-binding domain (XbaI) and upstream of the Bcd 3'UTR (BamHI). The fragment coding for the VP16 activation domain was obtained by XbaI and BamHI restriction digests of the pCG-Gal4 VP16 plasmid (a gift from W. Herr (Das et al., 1995)) and inserted downstream of the Gal4 DNA-binding domain-coding sequence and upstream of the Bcd 3'UTR. The pnos-Gal4-Actdomain-Bcd3'UTR transcription units were inserted as KpnI/NotI fragments in pCasPeR4 for germline transformation and the Gal4-Actdomain-Bcd3'UTR sequences were inserted in pPAC as a NruI/NotI fragment for transfection experiments. In these latter constructs the Gal4 ATG is located approximately a hundred base pairs downstream of the actin promoter transcription start site of pPAC.
Drosophila stocks and transgenics
Mutant stocks were bcdE1 (Frohnhofer and Nusslein-Volhard, 1986), torPM and tor4021 (Klingler et al., 1988). P-element transformed lines were Bcd3-lacZ (Ronchi et al., 1993), UAS-placZ (Rorth, 1998), Bcd-Gal4 (Bellaïche et al., 1996), Q,
A,
C,
QA,
AC,
QC and
QAC (Schaeffer et al., 1999). Injections for transgenics were performed as described in (Bellaïche et al., 1996). For each Gal4 fusion construct at least two independent insertions were analysed and the line giving the strongest expression of the UAS-placZ was chosen for further analysis. The nature of the transgene was verified by genomic PCR amplification using primers hybridising within the sequence of the Gal4 DNA-binding domain (5'-GCATTGTTAACACTGCCAGGC-3') and the Bcd 3' UTR (5'-ACTAGACCTAACTTTCTACGCG-3'). The size of the amplified fragments was shown on agarose gel to be approximately 1007 bp for Gal4-3ST, 1421 bp for Gal4-3C, 404 bp for Gal4-2Q, 530 bp for Gal4-3A, 1889 bp for Gal4-3A3C, 1043 bp for Gal4-3Q3A, 1934 bp for Gal4-3Q3C, 347 bp for Gal4-GCN4, 911 bp for Gal4-3GCN4, 290 bp for Gal4-VP16, 287 bp for Gal4-B6 and 296 bp for Gal4-B42. The amplified fragments were sequenced on each side on 300 bases with the primers used for PCR, in order to confirm the nature of the transgenes (data not shown).
For the Bcd-GCN4 and Bcd-GCN4C lines, several P-element insertions were obtained. Some of the lines exhibited a dominant female sterile phenotype whereas others exhibited a recessive female sterile phenotype. These differences were probably due to the expression level of the transgene in each case. Indeed, decreasing the number of copy of wild-type bcd in the lines exhibiting a recessive phenotype reveal a dominant phenotype, and increasing the number of copies of bcd (using additional transgenes allowing expression of wild-type Bcd) allowed rescue of the dominant female sterile phenotype. The female sterile phenotype was, thus, only observed for a given ratio of wild-type to mutant (Bcd-GCN4 or Bcd-GCN4C) protein concentration.
Whole-mount in situ hybridisation
Diogoxigenin-labelled RNA probes and in situ hybridisation were as described (Bellaïche et al., 1996) from pKS derivatives containing the coding sequences of hb, otd and lacZ. The anti-digoxigenin antibody, coupled to alkaline phosphatase (Boehringer) was pre-adsorbed and used at 1/2000 dilution. Embryos were mounted in 80% glycerol and photographed with Nomarski optics.
Tissue culture and transactivation assay
Drosophila S2 cells were grown at 25°C in M3 medium supplemented with 10% fetal calf serum. Transfections were performed at 50-70% confluence with the calcium phosphate procedure (Wigler et al., 1979). For the transactivation assay, each plate was transfected with 1 µg of the hsp83-lacZ plasmid (internal control), 1 µg of either the Bcd responder plasmid (Bcd3-CAT) (Ronchi et al., 1993) or the Gal4 responder plasmid (UAS-CAT) (Bellaïche et al., 1996) and variable amounts of the producers plasmids (pPAC and derivatives). CAT assays were performed as described (Bellaïche et al., 1996) on 20 µl or normalised quantities of cytoplasmic extracts (relative to ß-galactosidase activity). Radiolabelled and acetylated forms of chloramphenicol were detected using a Fuji Bioimaging Analyser, and quantification was made using Mac Bas V2.2 computer software. Fold activation is the ratio of CAT activity obtained with a given fusion protein to background CAT activity obtained with the pPAC plasmid producing no protein. Indicated values are the means of three independent experiments.
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RESULTS |
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(2) The Bcd protein might contain a repression domain induced by Torso. This putative repression domain might reduce the activity of the endogenous activation domains of Bcd upon Torso activation, whereas it might be unable to act on the heterologous GCN4 activation domains.
Sensitivity of heterologous activation domains to inhibition by Torso
To determine the effect of Torso activity on heterologous activation domains, we analysed the activity of fusion proteins between the Gal4 DNA-binding domain and either one or three copies of the yeast GCN4 activation domain, one copy of the VP16 activation domain (Das et al., 1995) or one copy of the B42 artificial (Ma and Ptashne, 1987) activation domain (Fig. 2A). The transcriptional activity of these proteins was first analysed in co-transfection experiments in S2 cells. These proteins were expressed under the control of the actin promoter and their activating potential, indicated by their capacities to induce the expression of a reporter for a CAT reporter gene containing five Gal4-binding sites (UAS-CAT), was determined by CAT assays performed on cytoplasmic extracts of transfected cells. As shown Fig. 2B, most of these proteins activated the expression of the UAS-CAT reporter gene in cell culture. These proteins were then expressed as maternal anterior gradients in the early embryo. As shown Fig. 2C-F, these proteins allowed expression of a lacZ reporter gene (UASp-lacZ) (Rorth, 1998) to very variable extends. Surprisingly, although the VP16 activation domain is considered as one of the most potent eucaryotic activation domain, Gal4-VP16 only very weakly directed expression of lacZ (Fig. 2E). Gal4-GCN4 and Gal4-B42 allowed expression of lacZ at the tip of the embryo (Fig. 2C,F) and Gal4-3GCN4 allowed expression of lacZ as a strong anterior cap (Fig. 2D). Despite the variable levels of activity of these proteins, none of them appeared to be inhibited by Torso at the anterior pole, indicating that the GCN4, VP16 and B42 activation domains are not downregulated by Torso in vivo.
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Sensitivity of the Bcd deletion variants to the Torso pathway
We then analysed Torso-induced anterior repression of Bcd target genes in transgenic flies bearing deletion variants of bcd rescue constructs (Schaeffer et al., 1999). These deletion variants removed either only one single domain of Bcd (Q,
A or
C) or pairs of domains (
QA,
AC or
QC). A C-terminal truncated version (
QAC) removed simultaneously the Q, A and C domains (Schaeffer et al., 1999). The transgenic lines bearing each of these Bcd deletion variants in rescue constructs were analysed in a bcd mutant background by in situ hybridisation for the expression of three Bcd target genes: hb, otd and the Bcd reporter transgene Bcd3-lacZ, which places lacZ under the control of a naive promoter containing three Bcd-binding sites (Ronchi et al., 1993).
As mentioned by Schaeffer et al. (Schaeffer et al., 1999), the position of the posterior border of expression of hb, otd and Bcd3-lacZ was variable in different Bcd deletion variants. Despite these quantitative differences, which probably reflected the different level of expression of the various proteins, anterior repression of hb and otd at the anterior pole was observed in each of the lines tested (data not shown). Interestingly, although the expression of Bcd3-lacZ was repressed in the QAC (Fig. 6B), the
A (Fig. 6C), the
C (Fig. 6D) and the other deletion variants tested (not shown), it persisted at the pole during cellularisation in the
AC deletion variant line (Fig. 6A). To confirm that the
AC protein was insensitive to downregulation by Torso, the expression of Bcd3-lacZ was also analysed in embryos from tor4021 females where the Torso cascade is activated throughout the embryo. As mentioned previously (Janody et al., 2000b; Ronchi et al., 1993), Bcd3-lacZ expression is totally repressed in a tor4021 mutant background (Fig. 6G). As Bcd3-lacZ contains only Bcd-binding sites upstream of a naive promoter, its expression probably reflects the intrinsic transcriptional activity of the Bcd protein (Bellaïche et al., 1996) and its extinction in tor4021 mutants indicates a complete inhibition of Bcd transcriptional activity by the constitutively activated Torso pathway (Janody et al., 2000b; Ronchi et al., 1993). In contrast, in this same tor4021 background, Bcd3-lacZ remained expressed when activated by
AC (compare Fig. 6F with 6H). Torso-induced anterior repression of Bcd target genes is generally observed during the first half of cellularisation, when the activation of the Torso pathway is optimal (Gabay et al., 1997). In Fig. 6E-H, slightly younger embryos were stained. At this stage, anterior repression of Bcd3-lacZ in wild type background is observed (Fig. 6E) and the effect of the constitutive allele of torso (torso4021) on Bcd3-lacZ expression is fully visible (Fig. 6G). This observation is consistent with the results of Gabay et al. (Gabay et al., 1997) who detected Torso induced ERK activation in tor4021 background at cycle 12. Altogether, these observations indicate that the
AC protein is not downregulated by Torso and that the deletion of both the A and C domains is sufficient to prevent the direct downregulation of Bcd transcriptional activity by the Torso pathway.
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DISCUSSION |
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The molecular mechanism that leads to the downregulation of the ST and C domains by the Torso pathway remains to be uncovered. As ST and C are large domains, composed of 100 and 149 amino acids, respectively, they might be bipartite and might contain, in addition to an activation domain, a repression domain stimulated by Torso. Alternatively, the ST and C domains might function with positive co-factors whose activities are directly downregulated by activation of the Torso pathway. A simple scenario might be that both the ST and C domains of Bcd function through the same co-factor. Alternatively, there might be different co-factors for each of these domains. The sensitivity to Torso of the Gal4-3C and the Gal4-3ST proteins was slightly different: although the Gal4-3ST protein was clearly repressed by Torso at the beginning of cellularisation, it became insensitive to Torso during the second half of this process. The reason for this unique dynamic sensitivity of Gal4-3ST to Torso is not clear. One trivial explanation might be that Gal4-3ST is an unusually stable protein compared with the other Gal4 fusion proteins that we have analysed. If the Gal4-3ST protein persists in the embryo during the second half of cellularisation, it might escape the Torso pathway activity that decays notably during this period (Gabay et al., 1997). In contrast, other Torso-sensitive proteins, such as Gal4-3C or Bcd, might be degraded during the second part of cellularisation and the detection of lacZ, in these cases, might only reflect the persistence and the stability of the lacZ message.
Is the alanine-rich domain of Bcd a repression domain?
We have accumulated evidence that points to the A domain of Bcd as having a repressive function. These results are surprising as several studies have indicated that the A domain of Bcd had features of an activation domain, interacting in vitro with TAF60 and mediating transcriptional activation and synergism in a cell free system (Sauer et al., 1995). We do not fully understand the reasons for these discrepancies but several observations enlighten them. First, a role for the TAFIIs in transcriptional activation by Bcd in vivo has been recently questioned (Schaeffer et al., 1999) as the QAC deletion variant line, lacking TAF110 and TAF60 interaction domains, can rescue the lack of bcd to viability. Second, although the alanine-rich domain (A) of Bcd binds TAF60, this type of domain is also frequently implicated in transcriptional repression in vivo (Hanna-Rose and Hansen, 1996). Finally, expression of Bcd3-lacZ in the
A deletion variant line was not only expanded towards the posterior but the intensity of the lacZ message was also much stronger than in the other lines tested (Fig. 6C). The shift of the posterior border of Bcd3-lacZ expression observed in this line is consistent with a two to three time increase in Bcd concentration when compared with wild type (Schaeffer et al., 2000). The increase in the intensity of the lacZ message indicates that the
A protein is probably a stronger activator than the wild-type Bcd and confirms that the A domain might function as a repression domain.
There are several possible molecular mechanisms to explain the repressive activity of the A domain. It is possible that the presence of the A domain modifies targeting of the protein to a cellular compartment or to degradation and indirectly prevents it from activating transcription. However, we favour the possibility that the repressive effect of the A domain directly involves the transcriptional process and that a specific co-repressor interacts with it. Indeed, it has recently been shown that the Bcd protein interacts in a two-hybrid screen with dSAP18, a member of the Sin3/Rpd3 deacetylation complex involved in repression mechanisms (Zhu and Hanes, 2000); it is tempting to speculate that dSAP18 might be this co-repressor.
Torso-induced repression of hb and otd involves two parallel processes
Our analysis indicates that hb and otd are repressed at the anterior in the AC deletion variant line, whereas Bcd3-lacZ is not. Similarly, whereas expression of Bcd3-lacZ is totally repressed by the constitutive activation of Torso in tor4021 background (Ronchi et al., 1993; Janody et al., 2000b), expression of hb and otd is only slightly affected in this mutant (Gao et al., 1996; Janody et al., 2000b). This indicates that the repression of hb and otd by Torso at the anterior pole occurs through a slightly different mechanism from that mediating the regulation of Bcd3-lacZ. The Bcd3-lacZ reporter transgene contains only three Bcd-binding sites upstream of a naive promoter. In contrast, hb and otd promoters are more complex and probably involve, in addition to Bcd binding sites, response elements for other regulators. The observation that hb and otd are still repressed in the
AC deletion variant line suggests two hypotheses. As
AC still carries the ST domain sensitive to Torso, ST might contribute to hb and otd repression but not to Bcd3-lacZ repression. Alternatively, anterior repression of hb and otd might also occur indirectly by Torso-induced zygotic expression of a repressor that will bind to the regulatory sequences of these genes. Such a mechanism has been proposed for otd by Gao et al. (Gao et al., 1996) who have observed that otd anterior repression is reduced in hkb mutants although not eliminated. This process would be parallel and partially redundant with Torso-induced direct inhibition of Bcd transcriptional activity, mediated by the ST and C domains.
Finally, the AC deletion variant line does not rescue the lack of bcd to viability leading to small head defects (Schaeffer et al., 1999). These defects might be due to a low expression level of the
AC protein in this line. We believe that this is unlikely as the morphogenetic activity of this line (as indicated by the position of the posterior border of Bcd3-lacZ expression) is comparable with that of the
QAC deletion variant line (compare Fig. 6A with 6B) that rescues the lack of bcd to viability (Schaeffer et al., 1999). The head phenotype observed with
AC is more probably due to the absence of the A and C domains of Bcd, and to the lack of Torso-induced anterior repression of Bcd transcriptional activity. As hb and otd are normally repressed by Torso in
AC, this indicates that another Bcd target gene might exist that is not properly regulated by Torso in this mutant background.
Transcriptional activation by Bcd in the early embryo
We have dissected the activating potential of the Bcd protein in vivo and have shown that the morphogen possesses three autonomous activation domains with different sensitivity to Torso, as well as a repression domain. These functional domains probably regulate transcription through interaction with different and specific co-factors. The reason for this complexity is not evident. It has been proposed that the Bcd morphogenetic activity (as measured by the position of the posterior border of a given Bcd target gene) is dependent on Bcd concentration and on the affinity of Bcd-binding sites found in the control region of each target gene (Driever and Nusslein-Volhard, 1988a; Struhl et al., 1989). In contrast, the strength of expression of Bcd target genes might be modulated by their promoter context. It is probable that the transcription factors binding in the proximity of the Bcd protein in these control regions might have synergistic or antagonistic interactions with some of the Bcd activation and repression domains or co-factors bound to these domains. These interactions might selectively affect the levels of expression of Bcd target genes. Our results therefore suggest a new level of complexity in the mechanism of action of the Bcd morphogen that resides in the multiple and diverse trans-acting potential of the Bcd protein.
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ACKNOWLEDGMENTS |
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