Functional analysis of murine CBF1 during Drosophila development

Authors


Abstract

Transcription factors of the CSL family are the main mediators of the Notch signalling pathway. The CSL factor in Drosophila is called Suppressor of Hairless (Su(H)) and it has been shown that it acts as a transcriptional repressor in the absence of a Notch signal and as a transcriptional activator in its presence in several developmental contexts. Furthermore, recent data suggest that Su(H) can also activate and maintain transcription of some target genes in a Notch-independent manner. However, although it has been shown that the mammalian CSL ortholog, CBF1, acts as a repressor of transcription in cell culture experiments, so far in vivo evidence for such a function has been lacking. Moreover, it is not known whether CBF1 can activate transcription in a Notch-independent manner, just like Su(H). Here we have investigated these questions by introducing murine CBF1 (mCBF1) and asked whether it can functionally replace Su(H) during Drosophila development. We found that this is indeed the case. We show that mCBF1 can act as a repressor of transcription and can activate and maintain the expression of some target genes in a Notch-independent manner. Our results, therefore, indicate that CBF1 can exert these functions also in its normal context, that is during mammalian development. Developmental Dynamics 235:918–927, 2006. © 2006 Wiley-Liss, Inc.

INTRODUCTION

Transcription factors of the CSL (CBF1, Su(H), Lag-1) family are core elements of the evolutionarily conserved Notch signalling pathway and are the only known signal-transducing transcription factors of this pathway. The pathway mediates short-range cell–cell interactions via the signal-proteins of the DSL (Delta, Serrate, Lag-2) family and Notch receptors. It plays an important role in many developmental processes ranging from cell proliferation to cell fate determination. Furthermore, the pathway is linked to an increasing number of diseases, such as cancer, Algalille Syndrome, or Cadasil (reviewed in Harper et al.,2003).

Upon ligand binding, the Notch receptor is proteolytically cleaved and the resulting intracellular fragment (Nintra) acts as a co-activator for CSL proteins to activate the transcription of target genes (Kopan,2002). In Drosophila, the CSL factor is called Suppressor of Hairless (Su(H)). Similar to its counterparts in other organisms, Su(H) appears to act as a switch that represses the transcription of target genes in the absence of a DSL/Notch signal, but activates them if the pathway is activated. In its repressor state, Su(H) forms a complex with Hairless and the co-repressors Groucho (gro) and dCtBP (Barolo et al.,2002; Kopan,2002). Upon activation of the Notch pathway, the repressor complex is replaced by an activator complex containing Su(H), Mastermind/Lag-3, and other factors (Kopan,2002). Although the composition of the activator complex seems to be conserved, the composition of the repressor complex in vertebrates differs significantly from that found in Drosophila (Lai,2002). CBF1, the CSL ortholog in mammals, interacts with the co-repressors of the SMRT and N-CoR as well as CIR (Lai,2002). In addition, a Hairless ortholog has not been found in vertebrate genomes. Whereas the existence and importance of the repressor function of Su(H) could be demonstrated in vivo in several developmental processes (Morel and Schweisguth,2000; Koelzer and Klein,2003; Castro et al.,2005), such evidence is missing for CBF1.

Besides its function in Notch signal transduction, Su(H) appears to have an additional Notch-independent function: It has been shown that Su(H) can activate the expression of a subset of target genes in the absence of the Notch protein if over-expressed during Drosophila wing development (Klein et al.,2000). Furthermore, it maintains its own expression in the socket cell of the bristle sensory organ independent of the Notch pathway (Barolo et al.,2000). These results suggest that Su(H) can activate expression of target genes in the absence of Nintra, possibly in collaboration with other unidentified cofactors. For CBF1, the evidence for a Notch independent activator function is scarce. Only one recent work describes results of cell culture experiments that indicate such a function (Tang and Kadesch,2001).

In order to further understand the repressor and activator functions of CBF1, we have introduced it in the fly genome under the control of the Gal4/UAS system and assayed its ability to repress and activate expression of Su(H) target genes in the absence of Notch signalling. We show here that CBF1 can substitute for the function of Su(H) in Notch signalling in the fly. Furthermore, CBF1 can activate the expression of at least one target gene of Su(H), vestigial (vg), independent of Nintra. In addition, it is able to maintain the activity of the promoter element of Su(H), ASE, that is required for maintenance of its own expression in the socket cell. Thus, it appears that CBF1 can activate transcription of genes in the absence of the activity of Notch in Drosophila. Our results also suggest that CBF1 acts as a repressor of transcription in a complex with Hairless.

Taken together, these results show that CBF 1 can functionally replace Su(H) in possibly all functional aspects during Drosophila development.

RESULTS AND DISCUSSION

Expression of Murine CBF1 During Drosophila Wing Development

In order to test whether CBF1 can substitute for Su(H) during Drosophila development, we carried out Gal4/UAS expression experiments using the murine CBF1 ortholog (mCBF1). mCBF1 shares high sequence homology to Su(H) (Fig. 1A) (Schweisguth and Posakony,1992): The overall identity between the two proteins is 60.9%, and the similarity 67.7%. However, a core region within both proteins stretching from aa 103 to 528 in Su(H) and from aa 55 to 481 in mCBF1 is 82% identical and 89% similar. This core makes up 71% of the Su(H) sequence. Interestingly, both proteins have low similarity outside this core region.

Figure 1.

A: Sequence comparison between Su(H) and mCBF1. For further information see text. B–E: Consequences of expression of UAS mCBF1 during Drosophila wing development. B: Wildtype wing. C: A wing in which UAS Su(H) was over-expressed with ptcGal4. The arrow points to the proximal region where additional wing pouch tissue is observable because of over-proliferation of wing pouch cells. D: Expression of UAS mCBF1 during wing development results in formation of wing notching (arrowhead). E: Expression of UAS ECN, a dominant negative acting Notch molecule that has its intracellular domain truncated, leads to a similar notching of the wing (arrowhead). In addition the veins have expanded and fused in the proximal region of the wing (arrow). Note that although expression of UAS mCBF1 causes similar wing notching as UAS ECN, no expansion of vein materials can be observed (arrow in C).

As a test system, we chose the development of the wing, a process in which the role of Notch/Su(H) signalling is well studied and several target genes have been identified (Klein,2001). During this process, Notch is strongly active in a small stripe of cells along the dorso-ventral (D/V) boundary. The activation of the pathway results in the expression of several genes, which are crucial for the development of the wing such as wingless (wg) and vestigial (vg). The stripe of Notch activity along the D/V boundary will later become the wing margin and reduction or local loss of Notch activity results in the loss of wing margin structures and gives rise to notches. We used the expression of four different target genes to monitor Notch/Su(H) activity. The vestigial boundary enhancer (vgBE) is a regulatory element of vestigial (vg) that mediates the Notch-dependent activation of vg during wing development through a single Su(H) binding site (Kim et al.,1996) (see Fig. 2B). The Gbe+Su(H)-lacZ construct consists of the ubiquitously active grainyhead promoter, which is modified by the insertion of four Su(H) binding sites (Fig. 2D) (Furriols and Bray,2001). As a consequence, lacZ-expression of this construct is suppressed in regions devoid of Notch activity, because of the repressor activity of Su(H). In regions where Notch is active and, thus, Nintra turns Su(H) into an activator of transcription, Gbe+Su(H)-lacZ is strongly expressed. In the absence of Su(H) activity, Gbe+Su(H)-lacZ is expressed at low levels throughout the wing area, because of the loss of the repressor function of Su(H) (Furriols and Bray,2001). Thus, the Gbe+Su(H) construct is an excellent detector of the repressor as well as the activator activity of Su(H) in all imaginal discs. As further markers, we used the Notch-controlled expression of wingless (wg) and cut along the D/V boundary (Fig. 2C,E).

Figure 2.

Expression of Notch target genes in wing imaginal discs upon expression of UASmCBF1 with ptcGal4. A: Expression of ptcGal4 is shown in green. It is expressed in a stripe of cells in the middle of the wing anlage, perpendicular to the expression domain of Wg (in blue) along the D/V boundary (arrow). BD: Expression of the vgBE, Wg, and Gbe+Su(H)-lacZ, respectively, in wildtype wing imaginal discs of the late third larval instar stage. Strong expression of the Notch targets can be observed in cells along the D/V Boundary (arrows). E–H: Expression of Notch targets in discs where UASmCBF1 is expressed. E: The expression of Cut is suppressed at the D/V boundary where UAS mCBF is expressed (arrowhead). F: Expression of the vgBE is ectopically activated by CBF1 (highlighted by the arrows, compare with B). In contrast, the expression of Wg (G) and Gbe+Su(H)-lacZ (H) is suppressed in the region of mCBF1 expression (arrowheads). Inset g in G shows the expression of Wg in an H null mutant wing imaginal disc upon expression of UAS mCBF1 with ptcGal4. In contrast to the wildtype, expression of Wg is not suppressed by mCBF1 in this background. I: Over-expression of UAS Su(H) with ptcGal4 results in a suppression of the Gbe+Su(H)-lacZ construct (arrow). The inset (i) shows expression of Wg in the same disc, which is not altered. J: Over-expression of UAS Su(H) with ptcGal4 induces strong ectopic expression of the vgBE (arrows). K,L: In contrast to this, the expression of Gbe+Su(H) (K) and of Wg (L) is strongly suppressed upon over-expression of UAS ECN (arrows).

First, we tested whether expression of mCBF1 causes a similar phenotype as over-expression of Su(H). ptcGal is a Gal4 line that is expressed in a stripe along the antero-posterior compartment boundary (see Fig. 2A). Expression of UAS Su(H) with ptcGal during wing development results in the over-proliferation of the proximal wing pouch tissue, but has no other observable defects (Fig. 1B,C) (Furriols and Bray,2000; Klein et al.,2000). Furthermore, expression of vgBE is ectopically activated, whereas expression of Gbe+Su(H)-lacZ and cut is suppressed (see Fig. 2I,J) (see Furriols and Bray,2000, and Klein et al.,2000, for data on cut). Nevertheless, expression of Wg is not affected (Fig. 2i, Klein et al.,2000; Furriols and Bray.2000), indicating that the promoters of individual Notch target genes react differently towards over-expression of Su(H).

Surprisingly, expression of UAS mCBF1 with ptcGal4 causes a different phenotype in the adult wing; the adult flies often exhibited wing notches, similar to that caused by reduction or local loss of Notch activity (arrowhead in Fig. 1D). Furthermore, expression of UASmCBF1 did not induce over-proliferation of wing pouch tissue (compare Fig. 1C and D). As reported for UAS Su(H) (Klein et al.,2000), the effects were more pronounced, but qualitatively the same, if we expressed two UASmCBF1 constructs. The results presented here were achieved by co-expression of two UAS constructs.

The wing notches suggested that mCBF1 might suppress the activity of the Notch pathway. Indeed, expression of the dominant negative ECN construct, a Notch variant that lacks the intracellular domain (Brennan et al.,1999), causes a similar notching (Fig. 1E). However, in contrast to UAS ECN, UAS mCBF1 did not cause broadening of wing veins in the proximal region of the wing (compare region highlighted with the arrow in Fig. 1D and E). Thus, mCBF1 appears to act in a different manner than ECN. This conclusion was confirmed when we looked at the expression of Notch target genes. We found that the expression of Gbe+Su(H)-lacZ as well as cut were strongly suppressed upon expression of UAS mCBF1 (Fig. 2E,G,H). In addition, expression of Wg along the D/V-boundary was reduced (Fig. 2G). However, expression of the vgBE was ectopically activated by mCBF1 (Fig. 2F, see arrows). In contrast, expression of this enhancer is suppressed by expression of UAS ECN (Gho et al., 1996). This observation confirms that CBF1 acts in a different manner compared to ECN. The ability of CBF1 to activate Notch target genes resembles that of Su(H). In both cases, expression of Cut and Gbe+Su(H)-lacZ is suppressed and that of the vgBE is activated. However, the influence of both proteins on the expression of Wg differs: it is unaffected by Su(H) over-expression (see Klein et al.,2000), but suppressed by mCBF1. The notches and lack of over-proliferation of wing tissue in mCBF1-expressing wings are probably due to the suppression of wg expression, since its activity is necessary for the formation of the wing margin and growth of the wing pouch (Couso et al.,1994; Diaz-Benjumea et al.,1994).

CBF1 Can Replace the Function of Su(H) During Adult Drosophila Development

The results above indicate that mCBF1 has functional properties that are similar, but not identical to Su(H), upon expression during normal wing development. Therefore, we next tested whether CBF1 can functionally replace Su(H) during Drosophila development by expressing mCBF1 in a Su(H) mutant background. In a first set of experiments, we expressed UAS mCBF1 with ptcGal4 in Su(H) mutant wing imaginal discs and focussed on its effects on wing development (Fig. 3). In Su(H) mutant wing imaginal discs, the expression domain of wg and of the vgBE along the D/V boundary is lost and the wing area is dramatically reduced. Consequently, the two Notch-independent ring-like expression domains of wg are strongly reduced in diameter (see Fig. 3A,C). Furthermore, Gbe+Su(H)-lacZ is ubiquitously expressed at a low level, due to the loss of the repressor function of Su(H) (Fig. 3F) (Furriols and Bray,2001). We found that CBF1 is able to re-establish the expression domains of the vgBE and wg in cells along the D/V-boundary (arrow in Fig. 3B,D,E). In addition, the vgBE is also ectopically expressed in a similar manner as during normal development (arrowheads in Fig. 3D, E, compare with Fig. 2D). ptcGal4 is expressed in a gradient that increases posteriorly in strength. Consequently, UAS CBF1 expression is highest in the posterior region of the ptc expression domain. Ectopic expression of the vgBE can only be observed in regions of high CBF1 expression. However, at the D/V boundary where Nintra is present, expression of the vgBE expands more anteriorily in regions of low CBF1 expression (highlighted by the arrow in Fig. 3D). This indicates that the presence of Nintra strongly enhances the ability of CBF1 to activate this enhancer.

Figure 3.

mCBF1 can functionally replace Su(H) during adult development of Drosophila. A: Wg expression in a Su(H) mutant wing imaginal disc. Expression of the inner ring-like domain (IRD, arrowhead) is strongly reduced in cell diameter and expression along the D/V boundary is lost (compare with Fig. 2A). B: The diameter of the IRD (arrowhead) has increased upon expression of mCBF1 in the Su(H) mutant wing imaginal disc. In addition, the expression at the D/V boundary is re-established in the domain of mCBF1 expression (arrow in B). C: Expression of Wg (red) and the vgBE (green) in a Su(H) mutant wing imaginal disc. The expression of the vgBE is lost within the wing area. The residual expression (highlighted by the arrows) is within clusters of sensory organ precursors. D: Expression of the vgBE in a Su(H) mutant disc where UASmCBF1 is expressed with ptcGal4. Expression of this enhancer is re-established along the D/V boundary (arrow). In addition, the vgBE becomes ectopically expressed (arrowheads) within regions of high expression in the ptcGal4 domain. E: The same disc as in D showing expression of Wg in red and of vgBE in green. Arrowheads highlight the ectopic expression of vgBE and the arrow points to the re-established domain of expression of both markers along the D/V-boundary. F: Expression of Gbe+Su(H)-lacZ in Su(H) mutant wing imaginal discs. It is uniform and weak due to the loss of the repressor function of Su(H). G: Expression of Gbe+Su(H)-lacZ in a Su(H) mutant disc where UASmCBF1 is expressed with ptcGal4. The normal expression of Gbe+Su(H)-lacZ is re-established along the D/V boundary (arrowhead) and up regulated in individual spots within the ptc domain (arrows). H–L: Su(H) mutant wing imaginal discs that are rescued by ubiquitous expression of mCBF1 throughout adult development by daGal4. The expression patterns of Wg (H) and Gbe+Su(H)-lacZ (I) are very similar to their wildtype pattern (compare with Fig. 2C, D). J: Merge of the channels depicted in H, I with expression of Wg shown in red and of Gbe+Su(H)-lacZ in green. K: Expression of the SOP marker SOP-E (Culi et al., 1999) in Su(H) mutant wing imaginal discs. The expression of this SOP marker is observable in many cells of the proneural clusters, because of the loss of the Notch/Su(H) mediated selection process. L: Expression of the SOP marker SOP-E (Culi et al., 1999) in Su(H) mutant wing imaginal discs rescued by UASmCBF1/daGal4. The expression of the SOP-E is restricted to single cells as in wildtype discs (arrows), indicating a normal selection of the SOP from the proneural clusters. M–P: The adult phenotype of a Su(H) mutant fly rescued by UASmCBF1/daGal4. M,O: Notum (M) and wing (O) of wildtype flies. N,P: The phenotype of the rescued flies. The comparison of N,P with M,O reveals that the rescued flies look very similar to the wildtype flies. This suggests that UAS mCBF1 can substitute for Su(H) during all of adult Drosophila development.

Expression of Gbe+Su(H)-lacZ is activated along the D/V-boundary and in several other regions of the ptcGal4 domain (Fig. 3G). Note that expression of wg and Gbe+Su(H)-lacZ is repressed upon mCBF1 expression during normal wing development. Thus, the re-establishment of their expression by CBF1 in Su(H) mutant discs suggests that CBF1 can interact with Nintra generated in this region and forms an activator complex, if Su(H) is absent. Thus, suppression of expression of Gbe+Su(H)-lacZ by CBF1 during normal development is probably due to the competition with endogenous Su(H) for binding of co-factors that are required to form the activator and repressor complexes.

The data so far presented suggest that CBF1 can substitute for Su(H) during wing development of Drosophila. To further extend and validate the functional similarity between CBF1 and Su(H), we tested whether CBF1 can functionally replace Su(H) during all of adult Drosophila development. Su(H) is expressed ubiquitously during Drosophila development (Gho et al.,1996). Therefore, we ubiquitously expressed UAS mCBF1 during Drosophila development, using daughterlessGal4 (daGal4), which drives ubiquitous expression of UAS constructs from early embryonic stages onwards (Wodarz et al.,1995). Su(H) null mutant animals survive embryogenesis without noticeable defects because of maternally supplied Su(H) protein (Lecourtois and Schweisguth,1995). However, they die shortly after puparium formation with defects in pattern formation. We found that the expression of UAS mCBF1 with daGal4, causes a complete rescue of these defects and many Su(H) null mutant animals survive until adulthood and hatch from the pupal case. These flies looked on the whole normal (see Fig. 3M–P). Furthermore, we found that the expression patterns of wg and Gbe+Su(H)-lacZ were restored to their wildtype pattern (Fig. 3H–J). These data strongly suggest that CBF1 can functionally replace Su(H) in all developmental processes of Drosophila. Some rescued flies displayed minor defects, such as wing notches and duplication of individual bristles. We explain the minor defects with the observation that daGal4 expression is often patchy and in many wing imaginal discs there are regions in which daGal4 expression is lost (data not shown).

To further confirm that CBF1 can indeed substitute for loss of Su(H) function, we looked in more detail at a second developmental process where the role of Su(H) is well understood, the development of the bristle sensory organ (Fig. 3K,L). The development of this sense organ starts with the selection of a sensory organ precursor cell (SOP) out of a group of equipotent cells called the proneural cluster (Gomez-Skarmeta et al.,2003). The Notch/Su(H) pathway is required for this selection and in the absence of its activity, all cells of a proneural cluster adopt the SOP fate (Fig. 3K) (Schweisguth and Posakony,1992). We found that the selection process is re-established in Su(H) mutants, if UAS mCBF1 is expressed with daGal4 (Fig. 3L). In these animals, the normal pattern of SOPs characteristic of the wing imaginal disc of the late third larval instar stage can be observed. Also the pattern of the later developing smaller bristles (michrochaete) that cover the notum is more or less normal in adult flies (see Fig. 3M,N).

CBF1 Can Activate Expression of Some Target Genes in the Absence of Notch-Signalling

The data presented above show that mCBF1 can ectopically activate expression of the vgBE in wildtype as well as in Su(H) mutant wing imaginal discs. We have previously shown that over-expression of Su(H) can induce ectopic expression of the vgBE in a Notch-independent manner (Klein et al.,2000). Thus, we tested whether CBF1 could also activate expression of this enhancer independent of Notch. For this purpose, we expressed UAS CBF1 with ptcGal4 and concomitantly induced Notch-mutant cell clones in the domain of expression. The clones were generated with the help of the FLP/FRT technique, using an UAS FLP construct to induce mitotic recombination. In this experiment, we obtained 6 wing imaginal discs bearing 17 clones at appropriate locations. The cells of these Notch mutant clones expressed the vgBE (see Fig. 4A–C). Thus, it appears that CBF1 is able to activate expression of the vgBE in the absence of Notch function, similar to Su(H).

Figure 4.

mCBF1 can activate and maintain expression of target genes in a Notch-independent manner. AC: Wing imaginal disc bearing Notch mutant clones and where mCBF1 is expressed by ptcGal4. The allele used is a deficiency, which removes the Notch transcription unit (see Experimental Procedures section). The expression of the vgBE in C and E is shown in red. The clones are labelled by the absence of the green GFP marker (green in C). Arrow in A–C points to a large Notch mutant territory. As revealed in B,C, the mutant cells express the vgBE, indicating that mCBF1 can activate this target in the absence of Notch function. D,E: Expression of UAS mCBF1 in a Psn mutant wing imaginal disc. D: Expression of the vgBE. E: Expression of the vgBE (red) and Wg (green). mCBF1 is able to activate the expression of the vgBE in the absence of Psn function (see arrows in D, E). F: Expression of the ASE-GFP construct in bristles of the thorax of Su(H) mutant flies rescued by UAS mCBF1/daGal4. The expression of ASE-GFP indicates that mCBF1 can maintain expression of this enhancer in adult flies in the absence of Su(H) function. For further information, see text.

In a second experiment, we tested whether mCBF1 can activate expression of the vgBE in a Psn null mutant background, where the Notch receptor cannot be activated due to the failure of S3 cleavage. We found that mCBF1 can activate this enhancer in a similar manner as during normal wing development (Fig. 4D, E).

Finally, we monitored the ability of CBF1 to maintain the expression of the Auto-regulatory Socket cell Enhancer (ASE) of Su(H) (Barolo et al.,2000). This enhancer controls expression of Su(H) in the socket cell of the bristle sensory organ. Its expression is activated by Notch/Su(H) signalling during the development of the sensory organ. However, during later development and adult stage, the ASE activity is maintained by Su(H) alone, through an auto-regulatory loop (Barolo et al.,2000). The maintenance of ASE expression is required for the physiological function of the sense organ and is the first in vivo process identified, where Su(H) regulates gene expression in a positive and Notch independent manner. To check whether CBF1 is also capable of maintaining expression of the ASE in the socket cell, we tested whether the ASE-GFP construct is active in adult Su(H) null mutant flies that were rescued by the expression of UASmCBF1 with daGal4. We found that the enhancer was active in all socket cells of the rescued flies (Fig. 4F), indicating that CBF1 can maintain its expression. Altogether, these results strongly suggest that mCBF1 can activate and maintain transcription of some target genes independent of Notch during Drosophila development.

Tang and Kadesch (2001) reported results that indicate that CBF1 might act as a transcriptional activator in 293T cells in a Notch-independent manner. Our results support this possibility and suggest that CBF1 might activate transcription of genes in a Notch-independent manner also during vertebrate development. Thus, this possibility must be taken into account in any future analysis of the function of CBF1 during mammalian development.

CBF1 Can Form a Repressor Complex With Hairless

Work from several groups convincingly showed that Su(H) acts as a repressor during Drosophila development (Furriols and Bray,2000; Morel and Schweisguth,2000; Koelzer and Klein,2003; Castro et al.,2005). However, although it has been shown that CBF1 can repress gene expression in cell culture and in vitro experiments (Hsieh and Hayward,1995; see Lai, 2000, and references therein), so far such a repressor function could not be demonstrated in any developmental process. We have observed that the normal expression pattern of Gbe+Su(H)-lacZ in Su(H)-mutant discs is rescued by CBF1 expression with daGal4. During normal development, the construct is actively repressed by Su(H) in a complex with Hairless (H) to prevent its expression in regions devoid of Notch activity (Furriols and Bray,2000). Thus, the re-establishment of the pattern of Gbe+Su(H)-lacZ upon CBF1 expression indicates that CBF1 can substitute for the repressor function of Su(H).

In the repressor complex, H acts as a bridge that connects Gro and dCtBP with Su(H) (Morel et al.,2001; Barolo et al.,2002). In the absence of H function, the repressor complex cannot form and consequently Gbe+Su(H)-lacZ is expressed at basal levels throughout the wing just like in Su(H) mutants (Furriols and Bray,2001). The requirement of H for the normal expression pattern of Gbe+Su(H)-lacZ suggests that mCBF1 can form a repressor with H in the Su(H) mutant flies rescued by daGal4//UAS mCBF1. This is also supported by the observation that Wg expression is not suppressed by mCBF1 in H mutant wing imaginal discs (see Fig. 2g).

To investigate this possibility further, we performed the following genetic experiment: loss of H function in wing imaginal discs causes an enlargement of the wing region located between the inner ring-like expression domain of wg and the wing blade. This enlargement is manifested by the increase in the diameter of the inner ring-like wg expression domain and is more enhanced in the posterior part of the wing (region between arrow and arrowhead in Fig. 5A,B). This enlargement is suppressed in Su(H); H double mutant discs (Fig. 5C,D), indicating that it is caused by the activity of Su(H). We tested whether loss of H function in Su(H) mutant discs rescued by UAS mCBF1/daGal4 also results in the enlargement of the wing area between the wing pouch and the inner ring-like expression domain of wg. This was indeed to be the case (Fig. 5E). Thus, it appears that CBF1 can collaborate with H to prevent the enlargement of this wing area. We tested this further by co-expressing UAS mCBF1 and UAS H (Fig. 5F–N). Co-expression of UAS Su(H) and UAS H with ptcGal4 results in the formation of a powerful repressor that represses expression of the vgBE and wg at the D/V boundary (Furriols and Bray,2000; Fig. 5F–K). We found that the co-expression of mCBF1 with H results in a similar suppression of wg and vgBE expression (Fig. 5L–N). These results suggest that, like Su(H), mCBF1 requires H to suppress expression of target genes in Drosophila.

Figure 5.

Interactions of mCBF1 with H. A: Expression of Wg in a wildtype wing imaginal disc. The arrowhead highlights the IRD of Wg expression. B: Expression of Wg in an H null mutant wing imaginal disc. The IRD is enlarged in diameter, caused by an expansion of the region between the wing pouch (arrow) and the IRD (arrowhead). This enlargement is suppressed by loss of Su(H) function as indicated by the fact that the phenotype of Su(H); H double mutant disc (C) looks like a Su(H) mutant one (D). However, if mCBF1 is expressed in the Su(H), H double mutant, the expansion of the region between the arrow and arrowhead is re-established (E). F–N: mCBF1 can form a powerful repressor of transcription in collaboration with H. F,G: Expression of UAS H with ptcGal4. Wg expression in G is shown in red, the ptc domain is labelled in green because of the presence of an UAS GFP. The pictures reveal that expression of H weakens the expression of Wg along the D/V boundary (arrow in F, G). H: Expression of UAS Su(H) results in the ectopic activation of the vgBE (blue). However, expression of Wg, shown in red, is unaffected. IK: Co-expression of UAS H and UAS Su(H) results in the interruption of Wg (I, red in K) and the vgBE (J, blue in K) along the D/V boundary (arrow). In addition, the vgBE is not ectopically activated. The same repressing effect is observed if UAS mCBF1 is co-expressed with UAS H (see L–N). L: Expression of Wg. M: Expression of the vgBE. N: “Merge” images of the channels are shown in L,M with Wg in red and the vgBE in blue and the ptcGal4 domain in green.

Physical interactions between H and CBF1 have been reported in in vitro experiments (Brou et al.,1994). Our results suggest that these interactions can take place in vivo and lead to the formation of a functional repressor complex during Drosophila development. This functionality is surprising given the fact that no H ortholog exists in vertebrates and the CBF1 repressor complex in vertebrates consists of different co-repressors. Although the repressor function of CSL factors was originally discovered in mammalian cell culture experiments (Hsieh et al.,1996), in vivo evidence for such a function of CBF1 was lacking, although several examples have been found for Su(H) during Drosophila development (Morel and Schweisguth,2000; Koelzer and Klein,2003). The results here further suggest that it is very likely that CBF1 acts as a repressor in its normal context during vertebrate development. This also raises the possibility that target genes of the Notch pathway are de-repressed if the function of CBF1 is lost during vertebrate development. Several examples of de-repression of target genes upon loss of Su(H) function have been documented in Drosophila (Morel and Schweisguth,2000; Koelzer and Klein,2003,2005; Castro et al.,2005). Interestingly, in one example de-repression of target genes is so strong that it obscures the involvement of Su(H) in this process (Koelzer and Klein,2005). The possibility of de-repression of target genes has now to be taken into account if the consequences of loss of CBF1 function are analyzed during vertebrate development.

mCBF1 Can Interact With a Fragment That Comprises Only the CDC10 Repeats of Notch

In Drosophila, expression of a fragment containing only the CDC10/ankyrin region of Nintra (NCDC10) causes a phenotype that resembles that of expression of Nintra during bristle and neuroblast development (Struhl and Adachi,1998; Brennan et al.,1999). We found that expression UAS NCDC10 can activate ectopic expression of Wg, the vgBE and Dl in a manner similar to Nintra, during wing development (Fig. 6A). This induction is dependent on the presence of Su(H) (data not shown). These results indicate that NCDC10, together with Su(H), can assemble an activator complex that is sufficient to activate target genes during Drosophila wing development. In contrast, cell culture experiments suggest that expression of the CDC 10 region of mammalian Notch1 (N1CDC10) can activate Notch reporter constructs only in the presence of endogenous Notch1, indicating that it requires other regions of the intracellular domain of Notch1 in trans for its function (Kurooka et al.,1998). Furthermore, N1CDC10 shows only weak interactions with CBF1, and even a chimeric protein, consisting of CBF1 fused to N1CDC10, failed to activate transcription (Kurooka et al.,1998). However, a similar construct is constitutively active in Xenopus (Wettstein et al.,1997). To test whether mCBF1 can form a functional activator complex with NCDC10 in Drosophila, we first determined whether NCDC10 is able to activate target genes expression also in the absence of endogenous Notch. This has not been investigated so far. To do so, we expressed UAS NCDC10 with dppGal4 and induced Notch mutant cell clones within the dppGal4 domain with a UAS Flp construct that performs FRT mediated mitotic recombination.

Figure 6.

mCBF1 can interact with NCDC10 to activate expression of Notch target genes. A: Expression of UAS NCDC10 with dppGal4 during normal development leads to an ectopic activation of Wg expression, which is largely restricted to the dorsal wing pouch (arrow). dppGal4 is expressed very similar to ptcGal4 (see Fig. 2A). B,C: NCDCD10 can induce expression of Wg in the absence of Notch. Notch mutant clones are induced with the help of the Flp/FRT system using a UAS Flp construct. Clones can be recognized in C by the absence of GFP staining. B: Expression of Wg. C: The same disc as in B showing the expression of GFP in green and of Wg in red. Two clones are highlighted with the arrows. The Notch mutant cells still express Wg, indicating that expression of NCDC10 in these cells can compensate for the loss of Notch function. D: Expression of UAS mCBF1 with dppGal4 in Su(H) mutant wing imaginal discs leads to a similar rescue of expression of Wg along the D/V boundary (arrow) as expression with ptcGal4 does (compare with Fig. 3B). No ectopic expression can be observed. E: Co-expression of UAS mCBF1 with UAS NCDC10 results in ectopic expression of Wg in a similar manner as expression of UAS NCDC10 during normal wing development (compare with A). This result suggests that mCBF1 can collaborate with NCDC10 to activate transcription of Wg.

dppGal4 is expressed in a similar manner to ptcGal4 at late third larval instar stage. However, during earlier stages, dppGal4 is strongly expressed at the dorsal anterior side, but only weakly expressed at the ventral side of the wing pouch. Thus, the effects caused by expression of UAS constructs with dppGal4 are often restricted to the dorsal side of the wing (Fig. 6A). We found that NCDC10 could still ectopically activate expression of Wg within the Notch mutant cell clones (Fig. 6B,C). Thus, in contrast to N1CDC10, NCDC10 can function in the absence of Notch in Drosophila and does not require additional peptide sequences of Nintra in trans. Expression of UAS NCDC10 or UAS Nintra in Su(H) mutant wing imaginal discs does not cause any detectable effect because of the absence of Su(H) (data not shown, Klein et al.,2000). Therefore, we conclude that NCDC10 interacts with Su(H) to activate expression of Notch target genes.

In order to investigate whether CBF1 can also interact with NCDC10, we co-expressed UAS mCBF1 and UAS NCDC10 with dppGal4 in a Su(H) null mutant background (Fig. 6D,E). As a control, we expressed UAS mCBF1 with dppGal4 in the same background. As expected, expression of UAS mCBF1 leads to the re-establishment of wg expression at the D/V boundary (Fig. 6D). Co-expression of UAS NCDC10 and UAS mCBF1 extends the expression of wg into the dorsal side of the wing pouch in a similar manner as expression of UAS NCDC10 during normal wing development (compare Fig. 6E with A). Thus, co-expression of both constructs can initiate ectopic transcription of wg, indicating that mCBF1 is able to assemble a functional activator complex with only NCDC10 during Drosophila wing development. Similar results have been reported in C. elegans (Austin and Kimble,1989; Yochem and Greenwald,1989).

The failure of N1CDC10 in mammals to provide trans-activation activity can either be due to CBF1 or N1CDC10. Our results suggest that N1CDC10, but not CBF1, is responsible for the failure to provide such an activity in mammalian cells. Comparison of the CDC10 region of Notch orthologs varying from humans to Drosophila, revealed that the sequence similarity is very high (Ehebauer et al.,2005). However, a comparison of the secondary structures indicated that NCDC10 forms an additional helix at the N-terminus. Whether this difference is responsible for the contrasting behaviour of both molecules remains to be tested. Alternatively, it is possible that other factors present during Drosophila development and absent in the cell lines used for the experiments with CBF1 are responsible for the difference in the results.

EXPERIMENTAL PROCEDURES

Fly Strains

The following alleles were used in this work: Su(H)Δ47 P(B)FRT40A (Morel and Schweisguth,2000), Df(1)N81K FRT101 (Brennan et al.,1997), Su(H)SF8 and HE31 (Lecourtois and Schweisguth,1995), vgBE (Williams et al.,1994), Gbe+Su(H) (Furriols and Bray,2001), ASE-GFP (Barolo et al.,2000). UAS stocks: UAS GFP (Yeh et al.,1995), UAS Su(H) (Klein et al.,2000), UAS NCDC10 (Brennan et al.,1999). Gal4 drivers: ptcGal4 (Speicher et al.,1994); dppGal4 (Wilder and Perrimon,1995); daGal4 (Wodarz et al.,1995).

Clonal Analysis

The Df(1)N81K FRT101 cell clones were induced by dppGal4 driving an UASFlp construct. This method induces Notch mutant cells clones within the dppGal4 expression domain continuously.

Construction of UAS mCBF1

To generate the UAS-CBFI construct, we used pU1093-5, a plasmid that contains the CBFI cDNA (kindly provided by Ulrike Strobl). An EcoRI/NotI-digested 1,594-bp fragment containing the CBFI cDNA sequence was subcloned into pUAST plasmid. Cloning was verified by sequencing.

Histochemistry

Antibody stainings were performed according to standard protocols. The anti Wg antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The rabbit anti β-Gal antibody was purchased from Kappel. Fluoro-chrome conjugated antibodies were purchased from Molecular Probes.

Acknowledgements

We are grateful to S. Carroll, S. Bray, J Modolell, U. Zimber-Strobl, T. Honjo, G. Struhl, F. Schweisguth, L. Seugnet, M. Haenlin, A. Martinez-Arias, and J. Posakony for supplying stocks and reagents. We further thank Sam Mathew for comments on the manuscript. The work of M.K. and T.K. is supported by the Deutsche Forschungs-Gemeinschaft (SFB 572).

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