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MECHANISMS OF DEVELOPMENT

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CtBP is required for proper development of peripheral nervous system in Drosophila Mark D. Sterna, Hitoshi Aiharaa, Giorgio A. Roccaroa, Lila Cheunga, Hailan Zhangb, Dereje Negeria,1, Yutaka Nibua,* a

Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, Box 60, A308, New York, NY 10065, USA b Department of Medicine, Mount Sinai School of Medicine, Box 1079, 1425 Madison Avenue, New York, NY 10029, USA

A R T I C L E I N F O

A B S T R A C T

Article history:

C-terminal binding protein (CtBP) is an evolutionarily and functionally conserved transcrip-

Received 15 May 2008

tional corepressor known to integrate diverse signals to regulate transcription. Drosophila

Received in revised form

CtBP (dCtBP) regulates tissue specification and segmentation during early embryogenesis.

19 September 2008

Here, we investigated the roles of dCtBP during development of the peripheral nervous sys-

Accepted 8 October 2008

tem (PNS). Our study includes a detailed quantitative analysis of how altered dCtBP activity

Available online 17 October 2008

affects the formation of adult mechanosensory bristles. We found that dCtBP loss-of-function resulted in a series of phenotypes with the most prevalent being supernumerary bris-

Keywords:

tles. These dCtBP phenotypes are more complex than those caused by Hairless, a known

Drosophila

dCtBP-interacting factor that regulates bristle formation. The emergence of additional bris-

Corepressor

tles correlated with the appearance of extra sensory organ precursor (SOP) cells in earlier

CtBP

stages, suggesting that dCtBP may directly or indirectly inhibit SOP cell fates. We also found

Peripheral nervous system

that development of a subset of bristles was regulated by dCtBP associated with U-shaped

Sensory organ

through the PxDLS dCtBP-interacting motif. Furthermore, the double bristle with sockets

Transcriptional repression

phenotype induced by dCtBP mutations suggests the involvement of this corepressor in

U-shaped

additional molecular pathways independent of both Hairless and U-shaped. We therefore propose that dCtBP is part of a gene circuitry that controls the patterning and differentiation of the fly PNS via multiple mechanisms.  2008 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Proper patterns of gene expression are essential for animal morphogenesis. In many instances, broadly based signaling cues, such as morphogen gradients, initiate the formation of boundaries during development. Subsequently, the integrated function of both positive and negative regulators acts to refine previously established patterns. These reiterative mechanisms are abundantly represented in development.

Pattern formation often requires transcriptional repression, as in the case of the formation of stripes and broad bands along both the anteroposterior and dorsoventral axes in the early Drosophila embryo (Ip and Hemavathy, 1997; Mannervik et al., 1999). The transcriptional corepressor Drosophila CtBP (dCtBP) is known to interact with several DNA-binding repressors through short peptide PxDLS motifs to establish a subset of sharply defined patterns of gene expression in the early fly embryo.

* Corresponding author. Tel.: +1 212 746 6202; fax: +1 212 746 8175. E-mail address: [email protected] (Y. Nibu). 1 Present address: Max-Delbrueck Center for Molecular Medicine (MDC) Berlin-Buch, Signalling pathways, cell biology and cancer, Robert-Roessle Str. 10, D-13125- Berlin, Germany 0925-4773/$ - see front matter  2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2008.10.003

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CtBP belongs to the CtBP/BARS/RIBEYE/AN superfamily, and has been characterized as a transcriptional corepressor that is evolutionarily conserved from nematodes to humans (Chinnadurai, 2007; Stern et al., 2007). Unlike vertebrates, which contain up to three CtBP genes, Drosophila melanogaster has only a single copy of dCtBP, whose mRNA can be differentially spliced to produce at least four different isoforms (ManiTelang and Arnosti, 2007; Nibu et al., 1998a, 1998b; Poortinga et al., 1998; Stern et al., 2007; Sutrias-Grau and Arnosti, 2004). dCtBP, together with short-range DNA-binding repressors, quenches only nearby DNA-binding activators, such as Bicoid and Dorsal, to establish additive patterns of gene expression (Nibu et al., 2003, 1998a,b). The fruit fly peripheral nervous system (PNS) cell lineage is an excellent system for understanding pattern formation and cell fate specification (Bardin et al., 2004; Calleja et al., 2002; Gomez-Skarmeta et al., 2003). The formation of sensory organs, consisting of large sensory bristles (also called macrochaetes), is spatially and temporally controlled during the development of wing imaginal discs, the anlage for both the adult wings and the dorsal thorax. Specification of the PNS initiates when 20–30 cells of proneural clusters (PNCs) expressing the achaete and scute genes, which encode basic region-helix–loop–helix (bHLH) transcription factors, arise at certain positions in the monolayer of epidermal cells at the third-instar larval stage. This initial constellation of PNCs constitute a ‘‘pre-pattern’’, that is dictated by discrete enhancers that direct the spatially resolved expression of the achaete and scute genes in a position-specific manner (Gomez-Skarmeta et al., 2003; Modolell and Campuzano, 1998). Afterwards, the PNC gives rise to one or two sensory organ precursor (SOP) cells. Each SOP cell then differentiates into five cells, the bristle (macrochaete), socket, neuron, sheath, and glial cells, thereby forming mature mechanosensors. Numerous molecular players involved in this process have been identified, including two dCtBP-interacting factors, Hairless (H) and U-shaped (Ush) (Bardin et al., 2004; Gomez-Skarmeta et al., 2003; Pi and Chien, 2007; Reeves and Posakony, 2005). The H adaptor protein promotes SOP fates by recruiting the dCtBP and Groucho corepressors, thus maintaining the default state of Notch target genes (Bang et al., 1991; Bang and Posakony, 1992; Barolo et al., 2002; Castro et al., 2005). Interestingly, phenotypic descriptions of a weak dCtBP mutation have documented duplicated thoracic bristles defects (Poortinga et al., 1998; Stern et al., 2007). However, H mutations induce loss of bristles (Bang et al., 1991; Bang and Posakony, 1992). These observations would be controversial if dCtBP acted only through H. Ush, which contains a PxDLS dCtBP-interacting motif, regulates the expression of the achaete and scute genes through a dorsocentral (DC) enhancer element, thereby specifying the DC PNC (Cubadda et al., 1997; Garcia-Garcia et al., 1999; Gomez-Skarmeta et al., 2003; Haenlin et al., 1997). The vertebrate orthologs of Ush, Friend of GATA 1 (FOG-1) and FOG-2, have been shown to interact with CtBP through the PxDLS motifs in vitro and in co-immunoprecipitation assays (Fox et al., 1999; Holmes et al., 1999; Katz et al., 2002). Similarly, Ush binds to dCtBP in vitro (Waltzer et al., 2002). Expression of ush and pannier (pnr) is initiated by the Decapentaplegic morphogen gradient, but the ush expressing domain in the

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most dorsal region of the wing disc is slightly restricted compared to pnr expression (Fromental-Ramain et al., 2008; Garcia-Garcia et al., 1999; Sato and Saigo, 2000; Tomoyasu et al., 2000). In the absence of Ush, Pnr (GATA transcription factor) directly activates achaete and scute expression by GATA sites in the DC enhancer, Ush, however, represses expression by directly interacting with enhancer-bound Pnr (Garcia-Garcia et al., 1999). Similarly, FOG-1 and FOG-2 are also known to bind GATA-family transcription factors and function as corepressors (Deconinck et al., 2000; Fox et al., 1999; Holmes et al., 1999). ush loss-of-function mutants show extra DC and scutellar (SC) bristles on the fly notum, while overexpression of this corepressor causes a reduction of the DC bristles (Cubadda et al., 1997). To date, roles mediated by the dCtBP-Ush complex in this process are largely unknown. In this study, we show that dCtBP is required for the proper patterning of the fly PNS. In the absence of dCtBP gene function, alterations in the mechanosensory bristle pattern were observed; in many cases ectopic or additional bristles formed, in agreement with the appearance of extra SOP cells. Towards a better understanding of how dCtBP functions to regulate this process, we identified ush and pnr as genes that were able to genetically interact with dCtBP. Disruption of the PxDLS motif in Ush partially impaired its function. In addition, one of the phenotypes (bald cuticle) seen in dCtBP mutants in this study was also observed in H mutants (Bang et al., 1991; Bang and Posakony, 1992). Furthermore, one particular phenotype induced by dCtBP mutations, the double bristle with sockets phenotype, suggests that additional molecular pathways involve dCtBP, independently of its interaction with both H and Ush. Taken together, we propose that dCtBP is involved in regulating different aspects of the gene network responsible for specifying the PNS via multiple mechanisms.

2.

Results

2.1.

dCtBP is involved in mechanosensory organ formation

Reduction of dCtBP activity results in a series of dorsal thoracic mechanosensory defects (Fig. 1). In this study, we used two dCtBP alleles: the hypomorphic dCtBP03463 allele and the EMS-induced dCtBP87De-10 mutation, the most severe mutant allele that is publicly available (Barolo et al., 2002; Poortinga et al., 1998; Stern et al., 2007). The most pervasive of these abnormalities are extra bristles (additional bristles attached to sockets) (Fig. 1C–E). Less frequently, double bristles (two bristles housed in the same socket structure) (Fig. 1E) and bald cuticle (complete loss of both bristles and their associated sockets) (Fig. 1F) are observed. Quantitative analysis of these bristle phenotypes shows that a subset of bristles, such as the posterior SC (PSC), anterior SC (ASC), posterior DC (PDC), anterior DC (ADC), posterior postalar (PPA), anterior PA (APA), and posterior supraalar (PSA) bristles, is affected in dCtBP03463/dCtBP03463 and dCtBP87De-10/dCtBP03463 pharate adults (Fig. 2). Other bristles, including the anterior SA (ASA), presutural (PS), posterior notopleural (PNP) and anterior NP (ANP) bristles, were relatively insensitive to reduction of dCtBP activity (Fig. 2). The ASC region most frequently displayed additional bristles. This correlates with the haplo-insufficiency observed for the ASC region in both

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Fig. 1 – dCtBP is required for the development of the proper number of dorsal thoracic bristles. All panels show dorsal thoraxes which are oriented with anterior to the top. Arrowheads, arrows, and asterisks indicate ‘‘extra bristle’’ (an additional single bristle attached to a single socket), ‘‘double bristle’’ (two bristles housed in the same socket structure), and ‘‘bald’’ (complete loss of both bristles and their associated socket structures), respectively. The DC and SC regions in a dissected wild-type (WT) pupal dorsal thorax (A and B, respectively). In the same homozygous dCtBP03463 mutant pharate adult, extra bristles (C; arrowheads) were formed near the extant PDC bristle and the ASC bristle (D; arrowheads). A dCtBP87De10 /dCtBP03463 pupa exhibited lack of both bristle and socket structures at the ASC and PSC positions (E; asterisks). In another dCtBP87De-10/dCtBP03463 transheterozygous pupa, an extra bristle (F; arrowhead) near the ASC and double PDC bristles (F; arrow) were observed. RNAi against dCtBP was transgenically induced by ap-Gal4 caused the loss of PDC bristles (G; asterisks) and tufted microchaeta bristles (G; arrows). In this background, some of the microchaetes are either missing or disoriented (G). In addition, sca-Gal4-driven dCtBP RNAi caused the loss of both ADC and PDC bristles (H; asterisks). (I) A schematic drawing illustrating the positions of all dorsal thoracic 11 bristles.

heterozygous backgrounds. Further reduction of dCtBP activity (i.e., in the dCtBP87De-10/dCtBP03463 transheterozygous pharate adult) resulted in a higher frequency of the extra bristle phenotypes for the PSC and APA regions as well as a higher emergence of double bristle and bald phenotypes for the PSC, ASC, and PDC regions. In support of these data, reduc-

tion of dCtBP protein by transgenically induced RNAi against dCtBP (Supplemental Fig. 1) also produced bald and double bristle phenotypes (Fig. 1G and H, and Supplemental Fig. 2). The variability seen in the phenotypes induced by reduction of dCtBP may be due to the persistence of varying amounts of dCtBP during sensory organs development. Together, these

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were either adjacent or one cell diameter away from one another. Normally, the ADC and PDC are separated by at least 7– 10 cell diameters (Cubas et al., 1991; Skeath and Carroll, 1991). Additionally, in the dCtBP mutant DC PNCs, these duplicated PDC SOP cells occupy the same spatial plane (Fig. 3D–F) as compared to the wild-type where they reside on different planes of focus.

2.3.

Fig. 2 – Quantification of the different observed dCtBP zygotic mutant phenotypes. All the phenotypes described in Fig. 1, such as extra bristle (blue bars), double bristle (red bars), and bald (purple bars), are quantified for each bristle position indicated on the left side of the graph. 34 dCtBP87De-10/ dCtBP03463 and 120 dCtBP03463/dCtBP03463 pupae, as well as 202 and 140 adult thoraxes in dCtBP87De-10/TM6 and dCtBP03463/TM6, respectively, were analyzed. The 250 wildtype adults observed did not show any of the aforementioned phenotypes (data not shown). For all examined genotypes, neither left-right asymmetry nor sexual dimorphic-specific bias was observed. results indicate that reduced dCtBP function affects the number of mechanosensory bristles on the fly notum.

2.2. Reduction of dCtBP gene function causes duplications of specific SOP cells Since reductions in dCtBP gene activity caused alterations in the number of mechanosensory bristles, we next tested whether particular SOP cells formed in wing imaginal discs, 20–24 h before pupariation, by monitoring expression of the Senseless SOP-specific marker (Nolo et al., 2000). Normally in wild-type, only one PDC SOP forms within the DC PNC (Fig. 3B and C). However, in dCtBP03463 homozygous wing discs, which give rise to extra bristle phenotypes with significant penetrance (Fig. 2), two Senseless-positive SOP cells are observed (Fig. 3E and F). In many cases, these two SOP cells

dCtBP mutant clonal analysis in the dorsal thorax

To further eliminate the dCtBP activity, we employed clonal analyses using the embryonic lethal dCtBP87De-10 allele. Within dCtBP87De-10 mutant clones (marked yellow and Sb+), extra bristles (Fig. 4A and D), double bristles forming from the same socket (Fig. 4B and E), and bald cuticle (Fig. 4B) are observed. These phenotypes are consistent with those seen in our initial genetic characterizations of dCtBP partial loss-of-function (Figs. 1 and 2). In addition, we also observed ‘‘socket without bristle’’ phenotypes (sockets lacking bristles; Fig. 4F). This phenotype was usually associated with relatively small clones. Quantitative analysis shows that all but the PS bristle appears to be affected (Fig. 4G). The double bristle phenotypes appeared exclusively at the ASC and PDC positions (Fig. 4B, E, and G). Bald cuticle phenotypes were noted in all regions except the PPA and PS regions (Fig. 4G). Finally, ‘‘socket without bristle’’ phenotype was observed for all positions except the PS region (Fig. 4G). Of note, differences were observed in the phenotypic expressivities among the dCtBP03643 homozygotes, the dCtBP87De-10/dCtBP03463 transheterozygotes, and the dCtBP87De-10 clonal backgrounds. In dCtBP87De-10 clones, a higher percentage of bald cuticles were observed. The ‘‘socket without bristle’’ phenotype appeared exclusively in dCtBP87De-10 clones. The PSC and ASC bristles were apparently more affected in the transheterozygote than in the dCtBP87De-10 mutant clones. On the other hand, the ADC, APA, PSA, ASA, and PNP bristles are more altered in the dCtBP87De-10 mutant clones. These phenotypic differences could be accounted for by protein perdurance. During the production of the mitotic clones, it is reasonable that dCtBP protein present at the time of the FLP-mediated recombination persisted to later stages. This notion is supported by evidence that the ‘‘socket without bristle’’ phenotype also appears in particular dCtBP RNAi backgrounds (data not shown). In addition, we found a putative planar cell polarity abnormality of the microchaetes (small bristles) in dCtBP RNAi backgrounds (Fig. 1G) and dCtBP mutant clones (Fig. 4C) that establishes a first link between dCtBP and this process. This particular effect was cell-autonomous, since it was confined within the borders of dCtBP mutant clones (Fig. 4C).

2.4. Overexpression of dCtBP in the wing disc blocks the formation of thoracic sensory bristles Previously we reported that overexpression of a 383 amino acid dCtBP isoform, using two different Gal4 drivers, ap-Gal4 and sca-Gal4, suppressed the formation of sensory bristles and caused malformed cuticle (Stern et al., 2007). To further analyze the effects caused by dCtBP overexpression, we extended our analysis to all 11 sensory bristles (Fig. 5). ap-Gal4 driven overexpression induced bald phenotype for all but

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Fig. 3 – Additional SOP cells arise in dCtBP mutant wing imaginal discs. All panels represent a single confocal optical crosssection orientated with dorso-anterior up in (A–C) and dorsal up in (D–F). Each bar in panels indicates 10 um. Wild-type (WT) (A–C) and dCtBP03463/dCtBP03463 wing discs (D–F) were double immunostained for a SOP marker Senseless (red) and a nuclear envelope marker Lamin (green). Merged images are shown in panel (C and F). The arrows indicate the positions of the SOP cells (A and D). A single Senseless-positive PDC SOP cell is observed in a wild-type wing disc (B and C), while two are formed in the PDC region of the dCtBP03463/dCtBP03463 wing disc (E and F). the ANP bristle (Fig. 5B and E). In addition, reductions in the numbers of microchaetes (Fig. 5B) and the occasional malformation of scutellar cuticle (Fig. 5D) were observed. Similarly, overexpression using the sca-Gal4 driver disrupted the ASC, ADC, APA, and PSA bristles and their sockets (Fig. 5C and E). Given the fact that dCtBP overexpression causes only the bald phenotype, excess of dCtBP activity in these contexts might impair a single step of the sensory organ cell lineage, in contrast to the loss-of-function which results in variable phenotypes.

2.5. dCtBP shows dosage-sensitive genetic interactions with ush and pnr It has been shown that the Pnr-Ush complex, which binds to the DC enhancer, can repress achaete and scute expression, and that Ush can associate with dCtBP in vitro (Cubadda et al., 1997; Garcia-Garcia et al., 1999; Waltzer et al., 2002). To determine whether dCtBP genetically interacts with pnr and ush, the effects of dCtBP overexpression were analyzed in flies heterozygous for pnr and ush mutant alleles. Examination of the ADC, PDC, ASC, and PSC bristles showed that both ush and pnr dominantly suppressed the dCtBP gain-of-function phenotypes to different degrees (Fig. 6). Hence, these results suggest that dCtBP likely functions with ush and pnr to regulate the formation of bristles.

2.6. Mutations in the dCtBP interaction motif partially impair Ush function Our finding that dCtBP genetically interacts with ush (Fig. 6) is consistent with a previously reported in vitro Ush-dCtBP

association and an evolutionarily conserved interaction between vertebrate CtBP and the FOG proteins through the PxDLS motifs (Fox et al., 1999; Holmes et al., 1999; Katz et al., 2002; Waltzer et al., 2002). To further clarify the genetic interaction between ush and dCtBP, the necessity of the PxDLS motif in Ush was tested in vivo. Consistent with previous studies using heat-shock induced overexpression of ush (Cubadda et al., 1997), sca-Gal4-mediated expression in the wing disc caused a loss of bristles (Fig. 7D), thereby phenocopying dCtBP gain-of-function. In contrast, expression of a mutant form of Ush lacking the PxDLS motif (Ush4A) under the control of the sca-Gal4 driver resulted in a milder phenotype as compared to the wild-type ush transgene (Fig. 7E and F). The PSC, ASC, PDC, APA, PSA, PS, and ANP bristles, but not the ADC, ASA, and PNP bristles were affected by mutations in the Ush PxDLS motif (Fig. 7G). These results suggest that removal of the dCtBP interaction motif in Ush partially impairs its ability to suppress the formation of a subset of bristles. In addition, our assays suggest that there might be another DNA-binding factor(s) that interacts with Ush, since bristle formation is affected in the APA, PSA, ASA, PS, PNP, and ANP regions where pnr is not normally expressed.

3.

Discussion

This study provides evidence that dCtBP is required for different aspects of PNS development. In addition, our extensive genetic characterization demonstrates how altered dCtBP activity can influence the formation of the adult dorsal thoracic mechanosensory organs. Our data show that overexpression of dCtBP impairs mechanosensory formation. In contrast, reduction of dCtBP activity leads to variable bristle

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Fig. 4 – Clonal analysis of dCtBP in the dorsal thorax region. All adult dorsal thoraxes are oriented anterior up. dCtBP mutant bristles are observed as non-Sb and yellow (black arrows and arrowheads). The boundaries of the mutant clones are traced with a white line. (A and D) Unaffected dCtBP mutant bristles (A; white arrowheads) and extra bristles within dCtBP clones (D; black arrowheads in higher magnification view of a boxed region panel A) were seen within the confines of the mutant clones. Typically these bristles were in close proximity to the extant bristles. Additionally, extra bristles at the ADC region were observed outside the mutant clones (red arrowheads label extra black and Sb bristles in A). This phenotype, however, dose not seems to be due to non cell-autonomous effect, since penetrance of this effect was similar to that observed in the heterozygous background (dCtBP87De-10/TM6 shown in Fig. 2). (B and E) At a smaller frequency, double bristles form at ASC positions (E; arrow in higher magnification view) and a complete loss of both bristle and sockets at the PDC position in the dCtBP mutant clone (B; asterisk). (F) In a small percentage of cases, ‘‘socket without bristle’’ phenotypes were observed within the mutant clones (indicated by a circle). (C) When relatively large clones were recovered, the orientations of the microchaetes were cell-autonomously disrupted. Typically, these smaller bristles point posteriorly but in dCtBP clones the directional orientation of these bristles is affected. (G) Quantification of the dCtBP clonal phenotypes. Phenotypes in dCtBP clones, such as the ‘‘extra bristle’’ (blue bars), ‘‘double bristle’’ (red bars), ‘‘socket without bristle’’ (socket structure remains without bristle; green bars), and ‘‘bald’’ (purple bars), are systematically quantified on the dorsal thorax. 52, 56, 50, 45, 35, 15, 18, 11,13, 14 and 16, mutant clones were analyzed, respectively, that overlapped the PSC, ASC, PDC, ADC, PPA, APA, PSA, ASA, PS, PNP and ANP bristle positions on the mosaic thorax. phenotypes, suggesting that dCtBP is likely operating in different molecular complexes. Namely, the mechanisms by which dCtBP regulates cell fate specification within the PNS may involve protein–protein interactions between dCtBP and at least two factors: Ush and possibly H.

Our data strongly suggest that dCtBP associates with the Ush-Pnr repressor complex through the Ush PxDLS motif to inhibit the expression of achaete and scute in particular PNCs. This model is supported by the following evidence. First, the ush loss-of-function and gain-of-function phenotypes (Fig. 7)

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Fig. 5 – dCtBP overexpression causes a loss of bristles on the adult dorsal thorax. All adult thoraxes are oriented anterior up. (A) Wild-type (WT) thorax. Overexpression of a 383 amino acid dCtBP isoform using ap-Gal4 (B) and sca-Gal4 (C) strains. Missing bristles are marked by asterisks. (D) Higher magnification view of the SC region of an ap-Gal4+UAST-dCtBP fly showing a malformed cuticle that occasionally develops. (E) Missing bristle phenotypes were quantified for each of the different overexpression backgrounds. 24 and 54 flies for dCtBP overexpression by the ap-Gal4 (blue bars) and sca-Gal4 (red bars) strains, respectively, were analyzed. In all cases, dCtBP overexpression resulted in a loss of both the bristle and socket structures of the sensory organ. Neither left-right asymmetry nor sexual dimorphic bias was observed in these dCtBP overexpression backgrounds.

Fig. 6 – The bristle loss phenotype caused by dCtBP overexpression is dominantly suppressed by halving the gene dosage either of ush or pnr. ap-Gal4 and UAST-dCtBP/ CyO females were crossed to yw (WT) flies, ush2 and pnrVX6 heterozygotes. Bristles corresponding to the ADC, PDC, ASC, and PSC positions were counted and tallied as a percentage of missing bristles. At least 50 flies were scored for each of the different genotypes indicated by blue, red, and green bars in WT, ush2/+ and pnrVX6/+ heterozygotes, respectively.

(Cubadda et al., 1997) were phenocopied by the corresponding genetic alterations to dCtBP activity (Figs. 1, 2, 4 and 5). Second, Ush interacts with Pnr and the Ush-Pnr complex inhibits expression of the achaete and scute genes through GATA sites located within the DC enhancer (Cubadda et al., 1997; GarciaGarcia et al., 1999; Haenlin et al., 1997). Third, the additional SOP cells were formed in both the dCtBP (Fig. 3) and ush mutant imaginal discs (Cubadda et al., 1997). Fourth, both ush and pnr alleles exhibited dominant genetic interactions with dCtBP (Fig. 6). Finally, disruption of the PxDLS motif of Ush partially mitigated the effects of ush overexpression on particular bristles (Fig. 7). The evolutionarily conserved physical interaction of dCtBP with Ush is essential for the propagation of certain cell lineages, such as blood cells (crystal cells) of the fruit fly, but not for heart development, processes known to be regulated by Ush and the GATA factors, Pnr and Serpent (Fossett et al., 2001; Waltzer et al., 2002). Surprisingly, the interaction between CtBP and FOG-1 is not required for erythroid development in mice, despite the fact that this interaction was found to be important in tissue culture experiments and in frog embryos (Deconinck et al., 2000; Fox et al., 1999; Katz et al., 2002). Our results from the ush overexpression assay suggest that Ush may utilize both the PxDLS motif and

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Fig. 7 – The effects caused by ush overexpression can be partially suppressed by removal of its dCtBP interaction motif. (A) Schematic representation of wild-type Ush. (B) Mutant form of Ush lacking the consensus PXDLS motif (Ush4A), that was changed to ALAAA to disrupt association with dCtBP. In all panels anterior is situated to the top. (C) Wild-type (WT) thorax. (D) Overexpression of wild-type ush using sca-Gal4 results in the loss of both bristles and sockets (asterisks), except for the PPA bristle. (E and F) When the mutant Ush lacking its dCtBP interaction motif was expressed under the same conditions as D, fewer bristles were missing. (G) Quantification of the loss of bristle phenotype caused by wild-type and mutant ush overexpression at each bristle position. 52 and 120 flies for overexpression of wild-type Ush (blue bars) and Ush4A (red bars), respectively, were analyzed. Removal of the dCtBPinteracting motif affects formation of all of bristles, except the ADC, ASA, and PNP bristles.

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another repression domain(s) to fully function, since particular bristles are affected by disruption of the PxDLS motif of Ush (Fig. 7G). A putative corepressor that interacts with the additional repression domain may act additively or cooperatively with dCtBP or function in different tissue/cell-type contexts. In fact, recently other repression domains in Ush, required for repression of the D-mef2 cardiac gene, were identified and these seemed to cooperatively work with the dCtBPdependent motif (Tokusumi et al., 2007). Consistent with this hypothesis, some dCtBP-interacting factors contain multiple repression domains. Knirps (nuclear receptor), Snail (zinc-finger protein), and H all have two repression domains, dCtBPdependent and -independent, which can function additively in transgenic flies and/or in tissue culture (Barolo et al., 2002; Castro et al., 2005; Keller et al., 2000; Qi et al., 2008; Struffi et al., 2004). It has been also demonstrated that H has an additional repression activity independent of Groucho and dCtBP-binding (Nagel et al., 2005). Kru¨ppel (zinc-finger protein) has two evolutionarily conserved repression domains (Hanna-Rose et al., 1997). The dCtBP-dependent domain is functional in tissue culture and in transgenic embryos, while the other repression domain is only active in tissue culture but not in transgenic embryos, suggesting a cell-type specific effect (Hanna-Rose et al., 1997; Licht et al., 1994; Nibu et al., 2003). Finally, Brinker (a helix-turn-helix protein) contains at least three repression domains (dCtBP-dependent, Grouchodependent, and the third repression domain) that are important for repression of different target genes (Hasson et al., 2001; Winter and Campbell, 2004). The physical interaction of dCtBP with H is implicated in sensory organ formation, wing formation, and embryonic patterning (Barolo et al., 2002; Castro et al., 2005; Morel et al., 2001; Nagel et al., 2005, 2007). H acts as an adaptor protein to bridge the Groucho and dCtBP corepressors to the DNA-binding factor Su(H), to ultimately inhibit Notch target genes. Vertebrate Notch target genes are similarly repressed by a complex consisting of CtBP with RBP-Jkappa (the mammalian counterpart to Su(H)) and the SHARP/CtIP corepressors (Oswald et al., 2005). Our study demonstrates that the bristles that are affected in dCtBP mutants also show defects in H loss-of-function mutants, although the effect of H is stronger than that of dCtBP (Figs. 2 and 4G) (Bang et al., 1991). H mutations induce two distinct phenotypes associated with loss of bristles; one is the bald phenotype (a complete loss of both sockets and bristles) due to lack of SOP cells, and the other is the double-socket phenotype (also lack of bristles) (Bang et al., 1995, 1991; Bang and Posakony, 1992). A similar bald phenotype was observed in dCtBP mutant backgrounds, such as dCtBP RNAi, the dCtBP87De-10/dCtBP03463 transheterozygote, the dCtBP87De-10 clonal backgrounds (Figs. 1, 2 and 4). Although compared to what is seen in dCtBP mutants, reduction of H activity interferes more uniformly with the formation of all 11 bristles that we analyzed (Bang et al., 1991), the bald phenotype further supports previous observations that dCtBP is involved in H-mediated repression (Barolo et al., 2002; Castro et al., 2005; Nagel et al., 2005, 2007). The double-socket phenotype seen in H loss-of-function mutants was never observed in dCtBP mutants. This distinct phenotype suggests that H may play a role independent of dCtBP, possibly by interacting with another

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MECHANISMS OF DEVELOPMENT

corepressor Groucho (Barolo et al., 2002; Castro et al., 2005; Nagel et al.,2005, 2007). Interestingly, the bald phenotype was also induced by overexpression of dCtBP (Fig. 5). The mechanism by which overexpression causes the bald phenotype in all regions except the DC region remains unclear, although one simple explanation could be that overproduction of dCtBP may disrupt the stoichiometric balance of the H/dCtBP/Groucho repression complex. The double bristle phenotype observed in dCtBP mutants suggests that dCtBP may be required to execute cell fate decisions within the SOP lineage. A similar phenotype seen in the H gain-of-function background was the result of a socket-tobristle cell fate transformation (Bang and Posakony, 1992; Barolo et al., 2002; Castro et al., 2005). Of note, this phenotype is clearly distinct from the double bristle phenotype observed in dCtBP mutants, which is always associated with a socket(s) (Figs. 1 and 4E). This dCtBP phenotype implies that cousinto-cousin cell fate conversions may be occurring within the sensory organ lineage. This type of cell fate switch could be similar to the conversion of sheath to bristle observed in hamlet mutants (Moore et al., 2004). Hamlet is a zinc-finger transcription factor and interestingly contains a PLDLS peptide sequence located between amino acid 747 and 751, identical to the CtBP-interacting motif (Chinnadurai, 2002; Moore et al., 2002). Future experiments will address whether dCtBP and Hamlet can physically interact and function together within the same biological process. Based on our results, we conclude that dCtBP regulates the development of the mechanosensory organs likely via multiple mechanisms. This highlights the centrality of this transcriptional corepressor in integrating multiple inputs to define boundaries and thereby control pattern formation during development. Note added in proof: dCtBP has been shown to regulate sensory organ prepattern by binding to both Pnr and Ush (Biryukova and Heitzler, in press).

4.

Experimental procedures

4.1. Fly stocks, crosses, and P-element mediated germ-line transformation All crosses, unless otherwise specified, were carried out at 25 C. In all experiments, comparison to yw was used as a baseline wild-type control. The following Drosophila stocks were used: ush2, pnrVX6, md544 (ap-Gal4), sca-Gal4, and yw hs-FLP; Dr/TM3, Sb, w; ap FRT82B P[w+, ubi-EGFP] (all obtained from the Bloomington stock center), yw; FRT82B P[w+, hs-pmyc] Sb P[ry+, y+]/TM3, Ser, and HE31 (both kind gifts from Dr. Eric Lai). Three fly strains carrying dCtBP alleles were used: yw; FRT82B dCtBP03463/TM6, Tb and yw; FRT82B dCtBP87De-10/TM6, Tb for Fig. 1 and yw; FRT82B dCtBP87De-10/TM3, Sb for the clonal analyses (Fig. 4). To generate dCtBP03463/dCtBP87De-10 flies, the yw; FRT82B dCtBP03463/TM6, Tb strain was mated to yw; FRT82B dCtBP03463/TM6, Tb flies and the non-Tubby progeny were selected and dissected at the pharate adult stage and visualized to reveal their dorsal thorax phenotypes. dCtBP03463 homozygous pupae were selected against Tb.

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Transgenic strains were obtained by injecting yw embryos with various P-element transformation vectors as previously described (Rubin and Spradling, 1982). For Figs. 5 and 7, the ap-Gal4 and sca-Gal4 fly strains were crossed into transgenic strains carrying UAST-dCtBP encoding 383 amino acid isoform (transgenic #1, #10, and #14) (Stern et al., 2007), UAST-ush (transgenic #5-2, #5-4, and #5-6), or UAST-ush4A (transgenic #1 and #2), and bristle formation was scored. Individual lines gave a very similar penetrance. Phenotype obtained from UAST-dCtBP (transgenic #1) was shown in Fig. 5. Results obtained from UAST-ush (transgenic #5-4) and UAST-ush4A (transgenic #2) were shown in Fig. 7. For Fig. 6, both ap-Gal4 and UAST-dCtBP (transgenic #1) transgenes, located on the second chromosome, were recombined together and balanced over CyO. ap-Gal4 UAST-dCtBP/ CyO females were then crossed to yw, ush2 and pnrVX6 heterozygotes. At least 50 flies carrying both the dCtBP overexpression cassette and the candidate mutation were selected and analyzed. Each fly was scored for the presence of the ADC, PDC, ASC, and PSC bristles, respectively. dCtBP expression was silenced by crossing fly strains carrying the pWIZ-dCtBP RNAi (transgenic #7-2 and #7-3) and pHIZdCtBP RNAi (transgenic #20-5) transgenes with both the scaGal4 and ap-Gal4 drivers, respectively. RNAi against dCtBP and overexpression of dCtBP using these transgenes above were confirmed by immunostaining for dCtBP in wing imaginal discs (Supplemental Fig. 1).

4.2.

dCtBP mutant clonal analysis

In order to induce FLP-dependent mitotic recombination (Xu and Rubin, 1993), the larval progeny derived from the cross: yw hs-FLP/Y; FRT82B P[w+, hs-pmyc] Sb P[ry+, y+]/TM3, Sb · yw/ yw; FRT82B dCtBP87De-10/TM3, Sb (staged at 36–48 h AEL) was heat-shocked twice at 37 C for 2 h with an intervening 24 h rest period at 25 C. Subsequently, these stocks were incubated at 25 C to allow development to proceed. Adults were selected based on the appearance of cuticle-marked dCtBP mutant clones (identified as yellow, non-Sb bristles). dCtBP87De-10 clonal production was verified by immunostaining for dCtBP and GFP, when yw hs-FLP/Y; FRT82B P[w+, Ubc-EGFP]/FRT82B P[w+, UbcEGFP] flies were used (data not shown).

4.3.

Standardization of larval ages

To collect larvae of specific ages, flies were allowed to lay eggs on a yeasted Apple Juice (AJ) plate for 4 h in a standard collection cage. Afterwards, the AJ plate was removed and aged for 72 h or more at 25 C, and larvae were selected against the Tb marker for dissection and fixation. The remainder of the larva was permitted to develop at 25 C until pupation. The age of dissected larvae were estimated in terms of hours Before Pupa Formation (BPF) by comparing the length of time their siblings needed to develop into pupae.

4.4.

Transgene construction

The wild-type ush cDNA (clone LD12631, BDGP) was subcloned into a modified Bluescript SK+ vector containing two

MECHANISMS OF DEVELOPMENT

AscI sites. The dCtBP-interacting motif, 540-PLDLS-544 in the Ush polypeptide, was disrupted by oligonucleotide-directed mutagenesis using the following mutagenic oligonucleotide: 5 0 -CTAGAGAATCTGCGGCTCTCGCTGCGGCGCTGCGTCGATCG CC-3 0 . The underlined nucleotides in the primer indicate substitutions that create four alanines, converting the 540PLDLS-544 motif into 540-ALAAA-544. The alanine substitutions were verified by DNA sequencing. The wild-type ush and the altered ush cDNAs were subcloned as AscI–AscI fragments into a modified pUAST vector containing a unique AscI site in the multiple cloning site. Gal4-regulatable dCtBP RNAi transgenes were constructed using the pWIZ vector (Lee and Carthew, 2003) and its derivative. An inverted-repeat of dCtBP gene was created to drive RNA hairpin by the Gal4-UAS system (Brand and Perrimon, 1993) as follows. A 360-bp dCtBP cDNA fragment, flanked by synthetic XbaI sites, was amplified by PCR from the dCtBP cDNA using the following primers: 5 0 -TAATCTAGACGAACA TGGTGCGCGAGGG-3 0 and 5 0 -ATTTCTAGACGCCGCGTGCAGTG TTCACC-3 0 ; underlined sequences correspond to the synthetic XbaI sites. After restriction enzyme digestion, the 360-bp XbaI fragment were sequentially inserted into unique AvrII and NheI cloning sites of the pWIZ vector to generate an inverted-repeat separated by the 74-bp intron of the white gene. The resulting plasmid was transformed into Sure-2 Escherichia coli strain (Stratagene). To further facilitate the subcloning, a longer intron sequence, the 1-kb first intron of hairy, was also used. The 1-kb hairy intron containing both 5 0 and 3 0 consensus splice sites was isolated by PCR from genomic DNA using the following primers: 5 0 -AATCCTAGGTAAGTTTCTTT CGAGAAAAATAAGAC-3 0 and 5 0 -AATGCTAGCTGCAAGAGGCA AGAAATGG-3 0 ; underlined sequences correspond to the synthetic AvrII and NheI restriction sites, respectively. After restriction enzyme digestion, the 1 kb AvrII-NheI fragment containing the hairy intron was used to replace the white intron of the pWIZ vector, thus creating the pHIZ vector. In the pHIZ vector, the inverted-repeat of the 360-bp dCtBP cDNA fragment was constructed in a manner similar to that described above. For both pWIZ-dCtBP RNAi and pHIZ-dCtBP RNAi plasmids, orientations of each repeat were confirmed by DNA sequencing.

4.5.

Immunohistochemistry

Dissected larvae in 1 · PBS were fixed and processed according to Cubas et al. (1991) except for the blocking reaction which was either performed for 2 h at RT or overnight at 4 C in 5% FBS in PBT. Incubation with affinitypurified guinea pig a-dCtBP (1:250), Mouse a-Lamin (ascites, 1:1000; Developmenal Hybridoma Studies Bank) and guinea pig a-Senseless (1:500; gift from Hugo Bellen) antibodies was carried out overnight at 4 C with rotation. Rhodamine (Jackson Labs), Alexa488- or Alexa546-conjugated secondary antibodies (Invitrogen) was added for 2 h at RT with rotation. Immunostains were imaged on a Zeiss LSM510 laser scanning confocal microscope (Rockefeller University) or a Leica DMR microscope attached to a Leica DC500 CCD camera.

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Acknowledgements We thank Anna Di Gregorio, Janice Imai, and Jamie E. Kugler for their critical reading of this manuscript, Makoto Sato for discussion, and Sofiya Chernyak and Darya Khazanova for technical help. We also thank Eric Lai, Hugo Bellen and the Bloomington stock center for providing fly strains and antibodies. H.A. is supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science (JSPS). This work was supported by grants from the Charles A. Frueauff Foundation and from the Speaker’s Fund for Biomedical Research awarded by the City of New York, and by a Research Scholar Grant (RSG-08-042-01-DDC) from the American Cancer Society to Y.N.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2008.10.003.

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