dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo
1998; Springer Nature; Volume: 17; Issue: 23 Linguagem: Inglês
10.1093/emboj/17.23.7009
ISSN1460-2075
AutoresYutaka Nibu, Hailan Zhang, Ewa Bajor, Scott Barolo, Stephen Small, Michael Levine,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle1 December 1998free access dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo Yutaka Nibu Yutaka Nibu Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Hailan Zhang Hailan Zhang Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Ewa Bajor Ewa Bajor Department of Biology, 1009 Main Building, 100 Washington Square East, New York University, New York, NY, 10003-6688 USA Search for more papers by this author Scott Barolo Scott Barolo Department of Biology, Bonner Hall, 9500 Gilman Drive, UCSD, La Jolla, CA, 92093-0347 USA Search for more papers by this author Stephen Small Stephen Small Department of Biology, 1009 Main Building, 100 Washington Square East, New York University, New York, NY, 10003-6688 USA Search for more papers by this author Michael Levine Corresponding Author Michael Levine Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Yutaka Nibu Yutaka Nibu Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Hailan Zhang Hailan Zhang Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Ewa Bajor Ewa Bajor Department of Biology, 1009 Main Building, 100 Washington Square East, New York University, New York, NY, 10003-6688 USA Search for more papers by this author Scott Barolo Scott Barolo Department of Biology, Bonner Hall, 9500 Gilman Drive, UCSD, La Jolla, CA, 92093-0347 USA Search for more papers by this author Stephen Small Stephen Small Department of Biology, 1009 Main Building, 100 Washington Square East, New York University, New York, NY, 10003-6688 USA Search for more papers by this author Michael Levine Corresponding Author Michael Levine Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Author Information Yutaka Nibu1, Hailan Zhang1, Ewa Bajor2, Scott Barolo3, Stephen Small2 and Michael Levine 1 1Department of Molecular and Cellular Biology, Division of Genetics, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA 2Department of Biology, 1009 Main Building, 100 Washington Square East, New York University, New York, NY, 10003-6688 USA 3Department of Biology, Bonner Hall, 9500 Gilman Drive, UCSD, La Jolla, CA, 92093-0347 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7009-7020https://doi.org/10.1093/emboj/17.23.7009 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression. Introduction Transcriptional repressors establish localized stripes, bands and tissue-specific patterns of gene expression in the pre-cellular Drosophila embryo (e.g. Rivera-Pomar and Jäckle, 1996; Dubnicoff et al., 1997; Jiminez et al., 1997; Nibu et al., 1998; Poortinga et al., 1998). Patterning of both the anteroposterior and dorsoventral axes depends on broadly distributed activators and localized sequence-specific repressors. For example, the maternal Dorsal gradient can activate rhomboid in both ventral and lateral regions of early embryos, but the Snail repressor keeps it off in the ventral mesoderm (Ip et al., 1992). Similarly, the maternal Bicoid gradient can activate the eve stripe 2 enhancer in a broad anterior domain, but the Giant and Krüppel repressors restrict the pattern within sharp stripe borders (Small et al., 1991, 1992). Recent studies have identified two putative co-repressors in the early embryo, Groucho (Paroush et al., 1994) and dCtBP (Nibu et al., 1998; Poortinga et al., 1998). Both proteins are encoded by maternally expressed genes, are ubiquitously distributed throughout the early embryo and are brought to the DNA template through interactions with sequence-specific regulatory factors. Groucho mediates transcriptional repression by Dorsal and Hairy. Dorsal is inherently an activator, but can recruit the Groucho co-repressor when it interacts with specific DNA-binding proteins located within the silencer elements of the zen and dpp genes (Dubnicoff et al., 1997). Hairy represses pair-rule genes, such as ftz and runt, in early embryos, and later is involved in neurogenesis (e.g. Jimenez et al., 1996). These functions of Hairy have been shown to depend on a specific sequence motif, WRPW, which is important for interactions with Groucho (Fisher et al., 1996). The removal of maternal Groucho products results in complex patterning defects in mutant embryos, including disruptions in both segmentation and dorsoventral patterning (Paroush et al., 1994; Dubnicoff et al., 1997). Recent studies have identified a second putative co-repressor in the early embryo, dCtBP (Nibu et al., 1998; Poortinga et al., 1998), which is the Drosophila homolog of the mammalian CtBP protein (e.g. Schaeper et al., 1995; Turner and Crossley, 1998). CtBP attenuates transcriptional activation by the adenovirus E1A protein; it binds E1A through a specific sequence motif located near the C-terminus of E1A, P-DLS-K (Schaeper et al., 1995; Sollerbrant et al., 1996). This motif is conserved in two unrelated repressors in the Drosophila embryo, Snail and Knirps (Nibu et al., 1998). Gene dosage assays are consistent with the occurrence of interactions between Knirps and dCtBP in vivo. Moreover, the P-DLS-K motif was shown to be important for the repression activity of a Gal4-Knirps fusion protein in transgenic embryos (Nibu et al., 1998). The functional significance of the P-DLS-K motif in the Snail repressor currently is unknown. The Hairy repressor contains a divergent sequence, P-SLV-K, which raises the possibility that Hairy-mediated repression depends on both Groucho and dCtBP (Poortinga et al., 1998). In the present study, we analyze the expression of a number of target genes, both authentic and synthetic, in dCtBP mutant embryos to obtain evidence that the dCtBP co-repressor is essential for Snail function. Evidence is also presented that a third sequence-specific repressor in the early embryo, Krüppel, depends on dCtBP. The C-terminal repression domain of Krüppel contains a sequence (P-DLS-H) that is related to the P-DLS-K motif in E1A, Knirps and Snail; mutations in this sequence disrupt the repression activity of a Gal4-Krüppel fusion protein in transgenic embryos. Dorsal and Hairy retain at least some repression activity in dCtBP mutants, suggesting that they do not require dCtBP as a co-repressor. It would appear that the bulk of the patterning defects observed in dCtBP mutants can be attributed to the loss of Knirps, Krüppel and Snail activity. We suggest that Groucho and dCtBP mediate separate pathways of transcriptional repression. Results Maternally encoded dCtBP products are distributed uniformly throughout early embryos (Nibu et al., 1998; Poortinga et al., 1998), and mutants derived from dCtBP germline clones exhibit altered patterns of segmentation, gene expression and severe patterning defects (Poortinga et al., 1998). These mutants also possess disruptions in dorsoventral patterning. As a first step towards identifying the repressors that might require dCtBP as a co-repressor, we have analyzed the expression of a number of authentic and synthetic marker genes in mutant embryos derived from germline clones, which are homozygous for a P-induced mutation in dCtBP (see Materials and methods). The role of dCtBP in segmentation In situ hybridization assays suggest that the mutant embryos exhibit a severe reduction in, but not complete elimination of, dCtBP expression (data not shown). dCtBP mutant embryos exhibit essentially normal patterns of Krüppel (Figure 1B; compare with A) and knirps (Figure 1D; compare with C) expression (see Poortinga et al., 1998). These results suggest that the Hunchback (Hb) repressor does not require dCtBP as a co-repressor, since Hb is important for establishing the anterior borders of both the Krüppel and knirps expression patterns (Struhl et al., 1992). Figure 1.Altered patterns of segmentation gene expression in dCtBP mutants. Embryos were obtained from dCtBP germline mosaics and hybridized with digoxigenin-labeled antisense RNA probes. The staining patterns were visualized by histochemical staining, and the embryos are oriented with anterior to the left and dorsal up (except K and L). (A and B) Krüppel expression pattern in wild-type (A) and dCtBP mutant (B) pre-cellular embryos. Strong staining is observed in central regions, and a weak pattern is detected in head regions. As shown previously (Poortinga et al., 1998), the Krüppel expression pattern is essentially normal in mutant embryos. (C and D) knirps expression pattern in wild-type (C) and dCtBP mutant (D) pre-cellular embryos. Staining is detected in both a band in the presumptive abdomen and an extended patch in antero-ventral regions. The knirps pattern is essentially normal in the mutant embryos (Poortinga et al., 1998). (E and F) giant expression pattern in wild-type (E) and dCtBP mutant (F) pre-cellular embryos. giant is expressed in both anterior and posterior regions. The posterior band is expanded in mutant embryos (see brackets). A similar alteration in giant expression is observed in Krüppel mutants (Kraut and Levine, 1991). (G and H) eve expression pattern in wild-type (G) and dCtBP mutant (H) cellularized embryos. There is a severe disruption in the eve pattern, with a loss of stripes 4-6 and a fusion of stripes 2 and 3. This altered pattern combines features seen in knirps− and Krüppel− mutants (see I and J). (I) eve expression pattern in knirps− embryo. There is a loss of stripes 4, 5 and 6. (J) eve expression pattern in Krüppel− embryo. Stripes 2 +3 and 4-6 are fused into two broad bands. (K) eve stripe 2 expression in a wild-type, gastrulating embryo. A lacZ reporter gene driven by the minimal, 480 bp stripe 2 enhancer was visualized with a lacZ antisense RNA probe. (L) Same as (K) except that the stripe 2 reporter gene was crossed into a mutant embryo derived from a dCtBP germline clone. There is a posterior expansion of the stripe 2 pattern, which suggests a loss of Krüppel-mediated repression. Download figure Download PowerPoint There is a substantial expansion of the posterior giant expression pattern in dCtBP mutants (Figure 1F; compare with E). A similar expansion was observed in Krüppel− mutants (Kraut and Levine, 1991), thereby raising the possibility that Krüppel-mediated repression depends on dCtBP. Further evidence stems from the analysis of eve. As shown previously (Poortinga et al., 1998), there is a severe disruption of the eve expression pattern in dCtBP mutants (Figure 1H; compare with G). The altered pattern combines aspects of both knirps (Figure 1I) and Krüppel (Figure 1J) mutants (Frasch et al., 1987). As in the case of knirps− embryos, dCtBP mutants exhibit a severe reduction in eve stripes 4-6. Like Krüppel− embryos, dCtBP mutants display fusions of stripes 2 and 3. This latter phenotype might result, in part, from a breakdown in the Krüppel-mediated repression of the eve stripe 2 enhancer (Stanojevic et al., 1991). To test this idea, an eve-lacZ transgene containing the 480 bp minimal stripe 2 enhancer (Small et al., 1992) was crossed into dCtBP mutant embryos (Figure 1L; compare with 1K). Expression directed by the transgene was detected by in situ hybridization using a lacZ antisense RNA probe. In dCtBP mutants, the posterior stripe 2 border expands into central regions, similar to that observed in Krüppel mutants (or when the Krüppel-binding sites in the stripe 2 enhancer are mutagenized; see Stanojevic et al., 1991). These results suggest that both Knirps and Krüppel require dCtBP to function as repressors. In contrast, neither Hb nor Giant appear to require dCtBP; the latter repressor is required for establishing the anterior stripe 2 border (Small et al., 1991, 1992), which is normal in dCtBP mutants (see Figure 1L). Dorsoventral patterning The Snail repression domain contains both a conserved copy of the P-DLS-K motif, as well as the slightly divergent sequence, P-DLS-R. Mutations in the former sequence attenuate the binding of Snail to a GST-dCtBP fusion protein (Nibu et al., 1998). As a first step towards determining whether Snail requires dCtBP to mediate transcriptional repression in vivo, the expression patterns of different Snail target genes were examined in dCtBP mutant embryos. rhomboid is expressed in lateral stripes that help specify ventral regions of the neurogenic ectoderm (Figure 2A; see Bier et al., 1990; Ip et al., 1992). It is repressed in the ventral mesoderm by Snail, and in snail− mutants there is a severe derepression of the rhomboid staining pattern (Figure 2C; compare with A). A similar disruption of the pattern is observed in dCtBP− embryos (Figure 2B). This is not due to the loss of Snail products, since snail expression appears to be essentially normal in dCtBP mutants (Figure 2D). Thus, it would appear that Snail repressor activity depends on dCtBP. Figure 2.Altered patterns of dorsoventral patterning genes in dCtBP mutants. Embryos are oriented with anterior to the left and stained after in situ hybridization with different digoxigenin-labeled antisense RNA probes. (A and B) rhomboid expression pattern in wild-type (A) and dCtBP mutant (B) pre-cellular embryos. rhomboid is normally expressed in two lateral stripes along the length of the embryo (A), but is derepressed in ventral regions in dCtBP mutants (B). This staining pattern is similar to that observed in snail− mutants (C). (C) rhomboid expression pattern in a snail−. snail− homozygote. Strong staining is observed in both lateral and ventral regions. (D) snail expression in a dCtBP mutant pre-cellular embryo. Staining is observed in ventral regions that normally invaginate to form the mesoderm. This staining pattern is similar to that observed in wild-type embryos and suggests that the derepression of the rhomboid staining pattern seen in dCtBP mutants (B) is not due to a loss in snail expression but, rather, results from a loss in Snail repressor function. (E and F) sim expression pattern in wild-type (E) and dCtBP mutant (F) pre-cellular embryos. sim is normally expressed in two lateral lines that coincide with the presumptive mesectoderm (E), but there is a severe derepression in the pattern in dCtBP mutants (F). This alteration in the sim pattern probably results from a loss in Snail repressor activity (see Kasai et al., 1992). Download figure Download PowerPoint Snail represses a number of neurogenic genes in the ventral mesoderm. Among these is single minded (sim), which specifies the mesectoderm at the ventral midline of advanced-stage embryos (Nambu et al., 1991; Kasai et al., 1992). sim initially is expressed in ventrolateral lines (Figure 2E) that coincide with the ventral-most cells of the presumptive neurogenic ectoderm. In dCtBP mutants, there is a severe derepression of the sim staining pattern (Figure 2F), again suggesting that the Snail repressor requires dCtBP as a co-repressor in vivo. Synthetic transgenes The preceding studies suggest that Krüppel, Knirps and Snail require dCtBP+ gene activity in the early embryo. More definitive evidence was obtained by analyzing the expression of synthetic transgenes in dCtBP mutant embryos (Figure 3). Each of the transgenes contains a modified form of the 700 bp rhomboid lateral stripe enhancer (NEE), which lacks the four native Snail repressor sites (Ip et al., 1992). The enhancer directs equally strong expression in both lateral and ventral regions due to the loss of the Snail sites. As shown previously (Gray et al., 1994), two synthetic Snail sites positioned within 50 bp of the NEE activators restore repression in ventral regions, so that the reporter gene is expressed in lateral stripes, similar to the endogenous pattern (Figure 3A). However, the same transgene exhibits a derepressed staining pattern in dCtBP mutants, indicating a loss of Snail-mediated repression (Figure 3B). In these experiments, the modified enhancer was placed between two marker genes, white and lacZ, and transgene expression was monitored with a white hybridization probe. Figure 3.Expression patterns of synthetic reporter genes in dCtBP mutants. Different lacZ-white reporter genes were introduced into dCtBP mutant embryos and stained after in situ hybridization with a white antisense RNA probe. Cellularized embryos are oriented with anterior to the left and dorsal up. (A and B) white staining patterns in a wild-type (A) and dCtBP mutant embryo (B). The reporter gene contains a modified rhomboid lateral stripe enhancer (NEE) that lacks the four native Snail-binding sites, but contains two synthetic sites flanking the four Dorsal activator sites (see diagram beneath the embryos). The synthetic sites mediate repression in ventral regions by Snail (arrowhead), so that white staining is restricted to lateral stripes (A). There is a severe derepression of the staining pattern in dCtBP mutants (B, arrowhead), suggesting the loss of Snail repressor activity. (C and D) white staining patterns in a wild-type (C) and dCtBP mutant (D) embryo. The modified NEE contains two synthetic Knirps sites in place of the Snail sites (see diagram). Normally, the enhancer is repressed in the presumptive abdomen by Knirps (C; arrowhead). This repression is lost in dCtBP mutants (D, arrowhead), which suggests a loss of Knirps repressor function. (E and F) white staining pattern in a wild-type (E) and dCtBP mutant (F) embryo. The modified NEE contains two synthetic Krüppel-binding sites in place of Knirps or Snail sites (see diagram). This enhancer directs a staining pattern with a broad gap in central regions in wild-type embryos (arrowhead, E). This gap coincides with regions containing high levels of the Krüppel repressor (see Figure 1). The gap is lost in dCtBP mutants (F, arrowhead), suggesting a loss of Krüppel repressor function. (G) GST pull-down assays. A full-length Krüppel protein was labeled with [35S]methionine by in vitro translation (lane 1). It was incubated with a full-length GST-dCtBP fusion protein produced in bacteria, and the bound protein was recovered on glutathione-Sepharose 4B beads, and fractionated on an SDS-polyacrylamide gel. Krüppel does not bind the GST moiety (lane 2), but selectively interacts with the GST-dCtBP fusion protein (lane 3). For comparison, lane 1 contains 10% of the total amount of the 35S-labeled Krüppel protein used in the binding reaction. Three amino acid substitutions in the P-DLS-H motif (PEDLSMH to AAALSMH) eliminate binding of the Krüppel protein to the GST-dCtBP fusion protein (lane 6; compare with lane 3). The binding assays were done essentially as described in Nibu et al. (1998). Download figure Download PowerPoint To assess the importance of dCtBP in Knirps-mediated repression, a different version of the enhancer was analyzed that contains synthetic Knirps-binding sites in place of the Snail sites (see diagram below, Figure 3C and D). In wild-type embryos, Knirps represses the modified NEE so that the white expression pattern includes a gap in the presumptive abdomen where there are high levels of the Knirps repressor (arrowhead, Figure 3C). This gap is lost in dCtBP mutants (Figure 3D), similar to the situation observed in knirps− embryos (Arnosti et al., 1996). These results suggest that dCtBP is required for Knirps-mediated repression of the modified NEE. Insertion of Krüppel-binding sites in the NEE results in a central gap in the white expression pattern (Figure 3E). This gap is lost in dCtBP mutants (Figure 3F), which suggests that dCtBP is also required for Krüppel-mediated repression. Further evidence for this possibility stems from in vitro binding assays (Figure 3G). In these experiments, a full-length Krüppel protein was labeled with [35S]methionine by in vitro translation, and mixed with a GST-dCtBP fusion protein. The wild-type protein binds to GST-dCtBP, but not to a GST control protein. Amino acid substitutions in the Krüppel P-DLS-H motif abolish dCtBP binding (Figure 3G). dCtBP is not essential for Dorsal or Hairy repression Dorsal and Hairy require Groucho to mediate transcriptional repression (Paroush et al., 1994; Dubnicoff et al., 1997). Hairy also contains a weak dCtBP interaction motif, P-SLV-K, and it has been suggested that Hairy-mediated repression depends on both Groucho and dCtBP (Poortinga et al., 1998). To investigate this possibility, a synthetic Hairy target gene was analyzed in dCtBP mutants (Figure 4). The target gene contains a modified NEE with two synthetic Hairy repressor sites (Barolo and Levine, 1997). In wild-type embryos, the enhancer directs a pair-rule pattern of expression (Figure 4C) due to interstripe repression by Hairy (Figure 4A). The same synthetic transgene directs an altered pattern of expression in dCtBP mutants (Figure 4D), whereby there are only three sites of repression rather than seven. These sites coincide with the abnormal hairy pattern observed in dCtBP mutants (Figure 4B), which results from disruptions in Krüppel and knirps activity. Instead of seven hairy stripes, there are only two stripes and a broad band, similar to the abnormal eve pattern (see Figure 1H). It would appear that the residual Hairy products continue to repress the modified NEE, although the repression may not be as robust as that observed in wild-type embryos. Figure 4.Hairy and Dorsal mediate repression in dCtBP mutants. Embryos were hybridized with various digoxigenin-labeled antisense RNA probes and are oriented with anterior to the left and dorsal up. (A and B) hairy expression pattern in wild-type (A) and dCtBP mutant (B) cellularized embryos. hairy is normally expressed in seven stripes and a small patch near the head (A). There is a severe disruption in the pattern in dCtBP mutants (B), similar to the altered eve pattern (see Figure 1H). There is a loss of hairy stripes 4-6 and a fusion of stripes 2 and 3 (B). (C and D) white staining pattern of a reporter gene containing a modified NEE. The enhancer contains two synthetic Hairy repressor sites in place of the Snail, Knirps and Krüppel sites used in Figure 3. The Hairy sites result in the periodic repression of the white staining pattern; the sites of repression coincide with the hairy stripes (C; compare with A). The white staining pattern is altered in dCtBP mutants (D), although there is repression in regions of residual hairy expression (arrowheads, D; compare with B). This suggests that Hairy can continue to function as a repressor in dCtBP mutants. (E and F) zen expression pattern in a wild-type (E) and dCtBP mutant (F) pre-cellular embryo. zen can be activated throughout early embryos, but is normally repressed in ventral and lateral regions by the maternal Dorsal nuclear gradient. This repression depends on Dorsal-Groucho interactions (Dubnicoff et al., 1997). The broad dorsal on/ventral off zen expression pattern is not altered in dCtBP mutants (F), suggesting that Dorsal-mediated repression does not depend on dCtBP. Download figure Download PowerPoint The Dorsal protein requires groucho+ gene activity to repress the expression of dpp and zen (Dubnicoff et al., 1997). To determine whether Dorsal-mediated repression also depends on dCtBP+ activity, zen expression was analyzed in dCtBP mutants (Figure 4F). There is no obvious change in the zen pattern as compared with wild-type embryos (Figure 4E). In both cases, zen exhibits a broad dorsal on/ventral off pattern, suggesting that the maternal Dorsal gradient can repress zen in ventral and lateral regions in both wild-type and mutant embryos. In summary, the genetic analysis of dCtBP mutants suggests that Krüppel, Knirps and Snail depend on dCtBP+ activity, while Hairy and Dorsal continue to function as repressors in the absence of the dCtBP co-repressor. However, it is possible that the full repression activity of Hairy depends on both Groucho and dCtBP (see Discussion). The P-DLS-K/H motif is essential for repression by Snail, Knirps and Krüppel Previous studies have shown that a Gal4-Knirps fusion protein containing the C-terminal third of the Knirps protein (amino acid residues 255-429) can repress a modified eve stripe 2-lacZ reporter gene in transgenic embryos (Nibu et al., 1998). The fusion protein contains the Knirps P-DLS-K motif, and mutations in this sequence (PMDLSMK to AAAASMK) inactivate its repression activity. These results suggest that dCtBP is an important component of Knirps-mediated repression, but do not exclude the possibility that additional sequences in Knirps are also important for repression. To address this issue of sufficiency, the function of the P-DLS-K motif was examined in the context of the full-length, wild-type protein (Figure 5). Knirps is normally expressed in two domains, one anterior to eve stripe 1 (Figure 5A) and the other in the presumptive abdomen, spanning eve stripes 4, 5 and 6. The posterior border of stripe 3 is thought to depend on repression by Knirps (Small et al., 1996). As shown previously, ectopic expression of knirps with the eve stripe 2 enhancer results in the loss of stripe 3 expression (Figure 5B) and dominant lethality (Kosman and Small, 1997). It has been suggested that the endogenous stripe 3 pattern is repressed by the diffusion of ectopic Knirps products from stripe 2 (Kosman and Small, 1997). A mutant form of Knirps that lacks the P-DLS-K motif does not repress stripe 3 expression (Figure 5C). The mutant protein is identical to native Knirps except for four changes in the P-DLS-K motif (PMDLSMK to AAAASMK). The mutant protein is expressed at the same levels as the wild-type protein (Figure 5C; compare with B, and data not shown), but does not mediate efficient repression. Moreover, while the ectopic expression of the wild-type Knirps protein results in embryonic lethality, transgenic strains that misexpress similar levels of the mutant protein are fully viable (E.Bajor and S.Small, unpublished observations). These results suggest that P-DLS-K represents the primary repression motif in the Knirps protein, although high levels of the mutant protein cause weak and variable disruptions in the stripe 3 pattern (data not shown). Figure 5.The P-DLS-K motif is essential for Knirps-mediated repression. Cellularizing embryos were hybridized with mixtures of a digoxigenin-labeled knirps antisense RNA (red) and a fluorescein-labeled eve antisense RNA (black). They are oriented with anterior to the left and dorsal up. (A) Double staining pattern in a wild-type embryo. eve is expressed in a series of seven stripes, while knirps is expressed at the anterior pole and antero-ventral regions, as well as in a broad posterior band which encompasses eve stripes 4 and 5. (B) Same as (A) except that the embryo contains a transgene with the full-length knirps coding region placed under the control of the eve stripe 2 enhancer. As shown previously (Kosman and Small, 1997), the ectopic knirps stripe leads to the repression of eve stripe 3. (C) Same as (B) except that the knirps coding region was mutagenized to disrupt the P-DLS-K motif (PMDLSMK to AAAASMK). The ectopic knirps stripe does not cause an obvious change in the eve pattern; in particular, stripe 3 expression is normal. Transgenic strains that express higher levels of the mutagenized protein exhibit weak alterations in the stripe 3 pattern, suggesting that the mutant Knirps protein retains weak repressor activity (data not shown). Download figure Download PowerPoint Similar assays were used to assess the significance of the P-DLS-K and P-DLS-R motifs in the Snail repressor (Figure 6). The eve stripe 2 enhancer was used to misexpress snail in transgenic embryos (Figure 6A). snail is normally expressed in ventral regions where it helps establish the limits of the presumptive mesoderm by repressing various target genes such as rhomboid (see Figure 2). The ectopic snail stripe results in an abnormal rhomboid pattern (Figure 6B) that contains a gap in the vicinity of eve stripe 2 (ar
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