Redundant function of Runt Domain binding partners, Big brother and Brother, during Drosophila development

A fundamental problem in biology is determining how different cell types are specified during the development of a multicellular organism. This process is usually initiated by extracellular cues that activate signal transduction cascades which produce a specific cellular response. Transcription factors such as the Core Binding Factor (CBF) play a vital role in enabling such signaling cascades to regulate a set of target genes (reviewed by Ito, 1999). CBF is a heterodimer composed of a DNA binding α-subunit and a non-DNA binding β-subunit. In mammals, three genes encoding the α-subunit, Runx1, Runx2 and Runx3, and a single gene encoding the β-subunit, CBFβ (also called PEBP2β), have been identified (Bae et al., 1993; Miyoshi et al., 1991; Ogawa et al., 1993a; Ogawa et al., 1993b; Wang et al., 1993). Runx1^−/− and CBFβ^−/− knock-out mice show a complete block of fetal liver blood cell development (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a; Wang et al., 1996b). The Runx2 gene is essential for osteoblast differentiation and skeletal morphogenesis, and knock-out mice are completely deficient for bone development (Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997). The Runx proteins contain within their sequence a conserved 128 amino acid motif called the Runt Domain (RD) that mediates interactions with DNA and the β-subunit (Meyers et al., 1993; Ogawa et al., 1993a; Ogawa et al., 1993b; Wang et al., 1993). The β-subunit increases the affinity of Runx proteins for DNA by altering the conformation of the Runt Domain (Meyers et al., 1993; Ogawa et al., 1993a; Ogawa et al., 1993b; Tang et al., 2000; Wang et al., 1993).

In humans, the RUNX1 and CBFβ proteins have been extensively studied in the context of oncogenic forms that cause leukemia. The most frequent translocation [t(8;21)] associated with acute myeloid leukemia (AML) encodes a fusion product between RUNX1 and the protein ETO (reviewed by Downing, 1999). The fusion protein includes the RD, interacts with CBFβ, and functions as a dominant negative transcription factor. The leukemic phenotype presumably results from the accumulation of secondary mutations. Additionally, a chromosomal inversion [Inv(16)] that generates a fusion protein, CBFβ-SMMHC, between CBFβ and the smooth muscle myosin heavy chain protein, has been associated with AML (Liu et al., 1993). This fusion protein complexes with RUNX1 but is retained in the cytoplasm, and therefore disrupts transcription (Adya et al., 1998; Kanno et al., 1998; Liu et al., 1993).

The Runt Domain was named after runt (run), best known for its role as a primary pair-rule gene in Drosophila embryonic patterning and segmentation (Gergen and Wieschaus, 1986). run also plays an important role in sex determination by directly controlling the expression of the sex lethal gene (Kramer et al., 1999). Additionally, run function is required for the development of the EL neurons in the embryonic CNS (Dormand and Brand, 1998; Duffy et al., 1991). A second protein containing the RD is Lozenge (Lz), which specifies both neuronal and non-neuronal cell types in the Drosophila eye (Daga et al., 1996; Flores et al., 1998). In the non-neuronal cone cells, Lz functions combinatorially with the transcription factors downstream of the Notch (N) and Epidermal growth factor receptor (EGFR) signaling pathways to activate the expression of D-Pax2 (also known as shaven; Flores et al., 2000). Similar mechanisms also operate in the control of the prospero (pros) gene in the eye (Xu et al., 2000). Additionally, lz functions during the development of olfactory sensory organ precursors (Gupta et al., 1998) where it regulates the expression of the proneural gene amos (Goulding et al., 2000). Finally, studies have defined a role for Lz in Drosophila hematopoiesis in establishing a sub-population of blood cells called the crystal cells (Lebestky et al., 2000; Rizki and Rizki, 1980). It is interesting to note that both vertebrate and Drosophila RD proteins are involved in the control of blood cell fate.

In Drosophila, two genes encoding β-subunits, Brother (bro) and Big brother (Bgb), were first identified through homology searches (Fujioka et al., 1996; Golling et al., 1996). These are expressed in distinct but overlapping patterns during embryogenesis (Fujioka et al., 1996; Golling et al., 1996). Furthermore, Bro and Bgb have been shown to physically interact with Run and increase its affinity for DNA (Fujioka et al., 1996; Golling et al., 1996). Overexpression experiments using either Bro or Bgb suggest that such complexes can form in vivo (Li and Gergen, 1999). A more complete functional analysis of the Drosophila partner proteins was lacking because mutations in these genes were unavailable at the time.

Here we report a loss-of-function analysis of the partner proteins. A genetic screen using a sensitized background isolated a mutation in Bgb, and ds-RNA-mediated interference strategies illustrate a redundant role for Bro and Bgb during development. We further show that Bgb functions during embryonic and eye development in the context of Run and Lz and is stabilized only in the presence of these proteins.

Adult flies were fastened to metal mounts with clear fingernail polish. Images were digitally acquired using the natural SEM mode of a Hitachi S-2460N scanning electron microscope with an attached Robinson detector.

A rabbit polyclonal antibody (α-Bgb) was generated against a C-terminal peptide of the predicted Bgb sequence (RDNRQDEMEAVR) (Fujioka et al., 1996; Golling et al., 1996). The antibody was affinity purified using the peptide conjugated to a Pierce Immunopure column. The purified antibody was preadsorbed against fixed cuticle from wild-type larvae and used at a final dilution of 1:150. Additionally, the following antibodies were used: α-Run (1:250; Dormand and Brand, 1998), αPros (1:10,000; Kauffmann et al., 1996), and DSHB antibodies α2B10 (1:20), αElav (1:400), αEngrailed (1:200), and mAb22C10 (1:200). Secondary antibodies (goat α-mouse-HRP, goat α-rabbit-HRP) were obtained from Jackson ImmunoResearch and used at a final dilution of 1:200. Eye discs were stained as described previously (Rogge et al., 1995).

ds-RNA experiments were performed essentially as described previously (Kennerdell and Carthew, 1998). The results from the injections are summarized in Table 1. For each experiment, the observed segmentation phenotype is shown in three independent examples in Fig. 5. A certain fraction of the injected embryos either hatched as wild type or were grossly and non-specifically affected as a result of the injection. Their numbers relative to the total injected was independent of the nature of the ds-RNA and are included in Table 1.

EMS (methanesulfonic acid ethyl ester; 25 mM) (Lewis and Bacher, 1968) was used to mutagenize Oregon-R male flies. The mutagenized males were crossed to lz^ts1 virgin females and the progeny were reared at 25°C. The F₁ male progeny were screened for an eye phenotype. Mutations were balanced over either SM6a or TM6b chromosomes and the enhancement was mapped using standard mapping chromosomes.

Df(3L)Bgb^K4 was generated by crossing y w / Y; P[1556 ry⁺] ry e / TM3 with y w / y w; Δ2-3 Ki / Δ2-3 Ki. Male progeny were mated to lz^ts1 virgins. The progeny from this cross were reared at 25°C and the males were scored for an enhanced eye phenotype. Mosaic clones were generated by crossing w / Y; Bgb^D FRT80B / TM6b with ey-flp w / ey-flp w; P[w⁺] FRT80B / P[w⁺] FRT80B. Mutant tissue was identified by the absence of pigment.

Mosaic clones in adult eyes were fixed and sectioned as described previously (Coyle-Thompson and Banerjee, 1993). For rescue experiments, full length Bgb cDNA (Fujioka et al., 1996) was cloned into the XbaI and NotI site of hsp70-pCaSpeR and germline transformants were generated.

High resolution sequence analysis of the Bgb^D and Bgb⁹ mutants was performed using the ³³P-Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech).

Transient transfections were performed using the Drosophila Expression System Kit (Invitrogen) and antibody staining was performed as described by Fehon (Fehon et al., 1990).

A temperature-sensitive allele of lz, lz^ts1, was used in a genetic screen to isolate mutations in interacting genes. At 25°C, lz^ts1 flies have near wild-type eyes (Fig. 1B). However, when raised at 29°C these flies have an easily visible rough eye phenotype (Fig. 1C). Even at this non-permissive temperature, lz^ts1 alleles have a milder eye phenotype than that due to a lz null allele (Fig. 1D). This led us to propose that the temperature-sensitive allele of lz functions at a threshold level such that any further reduction of the functional output of the Lz pathway would generate a mutant eye phenotype. In this scenario, at 25°C, a single copy loss-of-function mutation in a gene related to the Lz pathway is expected to generate an eye phenotype resembling that of a lz^ts1 fly raised at a higher temperature.

Wild-type male flies were mutagenized with EMS and were mated with virgin lz^ts1 females. Approximately 100,000 male progeny were reared at 25°C and scored for an eye phenotype. 10 flies were recovered that showed a disorganized eye phenotype. The mutations were crossed inter-se to generate 5 complementation groups: en(lz)4G/I, en(lz)3C/4C, en(lz)4F/4H, en(lz)Y, and en(lz)D/9 (Fig. 1E-I). These mutations are recessive embryonic lethal with the exception of en(lz)4G, which is homozygous viable with a disorganized eye. The en(lz)4G/I mutants were determined to be alleles of D-Pax2 based on their failure to complement the eye-specific D-Pax2^spa-pol allele (Fu and Noll, 1997). The enhancer called Y maps to the left arm of the second chromosome between dp (25A2) and b(34D5), is not uncovered by any known deletions, and was not analyzed further. The en(lz)D/9 mutants were determined to be mutations in Bgb and will be discussed in detail below. The en(lz)3C/4C mutations mapped to the left arm of the third chromosome and failed to complement Df(3L)HR370. Additional complementation analysis with known mutations within the breakpoints of this deletion determined that the en(lz)3C/4C mutations are alleles of hsp83 (Hackett and Lis, 1983). The en(lz)4F/4H mutations mapped to the right arm of the third chromosome and failed to complement Df(3R)RK8-21. Complementation analysis with known mutations within the region established that en(lz)4F/4H are alleles of osa, also known as eyelid (eld) (Kennison and Tamkun, 1988; Treisman et al., 1997).

The en(lz)D/9 mutants were identified as alleles of Bgb based on several lines of evidence. First, we used a P-element excision strategy to generate a deletion belonging to the region because no chromosomal aberrations existed that eliminated this locus. A P-element located near Bgb was excised in a lz^ts1 sensitized background. About 20,000 lines were screened and one excision allele, en(lz)K4, was identified as a dominant enhancer of lz^ts1 at 25°C. This deletion completely eliminates the Bgb locus. The right breakpoint of this deletion maps within the transcribed region of an uncharacterized gene (Adams et al., 2000; Rubin et al., 2000) between Bro and Bgb. Thus, Df(3L)K4 does not eliminate any part of the Bro gene. The EMS-induced mutants, en(lz)D/9, fail to complement the lethality of Df(3L)K4 suggesting that they too carry a mutation in a gene uncovered by the deletion. Sequence analysis showed that en(lz)9 carries a mutation (A801C) in Bgb which results in a change from glutamic acid to aspartic acid in the Bgb sequence. Although non-conserved in the vertebrate protein, this amino acid is physically close to the segment that binds the α-subunit (Goger et al., 1999; Tang et al., 2000; Warren et al., 2000). More strikingly, the en(lz)D chromosome carries a mutation (G808A) in Bgb which results in a change from a conserved glycine to an arginine. This glycine in Bgb is conserved in the vertebrate CBFβ and Drosophila Bro proteins. This amino acid is also close to residues that physically interact with the Runt Domain (Goger et al., 1999; Tang et al., 2000; Warren et al., 2000). It is therefore likely that this mutation disrupts the stability of a Bgb/RD complex in vivo. Finally, flies carrying hsp70-Bgb constructs were generated using P-element mediated transformation (Rubin and Spradling, 1982) and assessed for rescue of the Bgb phenotype. Three independent hsp70-Bgb transformant lines were placed in homozygous en(lz)D and en(lz)9 genetic backgrounds. In each case, complete rescue to adult viability was obtained with the basal level of expression achieved without the application of a heat shock. Taken together, these data establish that en(lz)D and en(lz)9 carry mutations in Bgb and these lines were therefore renamed Bgb^D and Bgb⁹. The Df(3L)K4 was renamed Df(3L)Bgb^K4.

The Drosophila eye imaginal disc undergoes morphogenesis during the third larval instar. At this stage, a furrow sweeps across the disc from the posterior to the anterior. Cells entering the furrow undergo a synchronized round of cell division to give rise to a set of undifferentiated cells as well as pre-clusters of five neurons that differentiate in a stereotypical developmental order: R8, R2/R5, then R3/R4 (for a review of eye development see) (Wolff and Ready, 1993). The remaining undifferentiated cells subsequently give rise to R1/R6, R7, and the non-neuronal cone and pigment cells.

In order to determine the expression pattern of Bgb during morphogenesis of the eye disc, we generated a rabbit polyclonal antibody against a C-terminal peptide of Bgb that is not homologous with any peptide stretch of Bro. Bgb expression is seen in the basal nuclei of the undifferentiated cells immediately posterior to the furrow (Fig. 2A). Among the differentiated cells, Bgb expression is first obvious in the R8 cell (Fig. 2B). Posteriorly, Bgb is seen in the R1/R6 cells (Fig. 2B), and the R7 and cone cells (Fig. 2C). The level of Bgb is much higher in the R7 cell than in any of the other cells in the eye disc.

The expression of Bgb closely corresponds to that of Lz with some notable differences. Like Bgb, Lz is also expressed in the undifferentiated cells behind the furrow as well as in the nuclei of the differentiated R1, R6, R7 and cone cells (Flores et al., 1998). Unlike Bgb, however, Lz is not expressed in R8, nor is it expressed at a higher level in R7 than in the other cells. We predicted that Bgb is likely to function with a different RD protein in R7 and R8, and therefore we studied the expression pattern of Run in the eye. Strikingly, Run is specifically expressed in the R7 and R8 cells of the eye disc (Fig. 2D,E). Taken together, the expression pattern of Bgb correlates nicely with the combined nuclear expression patterns of Run and Lz.

The correlation between Lz and Bgb staining patterns is functionally relevant. In a lz mutant background, Bgb expression is eliminated from all cells in which Lz is normally expressed (Fig. 2F,G). Additionally, when Lz is misexpressed in R3 and R4, Bgb protein is seen in the nuclei of these cells (Fig. 2H). This misexpression is not accompanied by an increase in Bgb transcripts (not shown) which suggests that Lz does not transcriptionally regulate Bgb. Instead, as Lz and Bgb are binding partners, it is likely that Lz stabilizes the Bgb protein in cells in which they are co-expressed. We also examined the relationship between Run and Lz proteins. The expression of the Run protein is genetically downstream of Lz in the eye. In a lz mutant background, Run expression is limited to the R8 cell (Fig. 2I) and is eliminated from the presumptive R7 cell (Fig. 2J). Thus, in a lz mutant background, the only cell that expresses a RD protein is R8. This is significant since in this background, R8 is also the only cell that expresses Bgb (Fig. 2F). This suggests once again that the α-subunit, in this case Run, is able to stabilize the β-protein.

A correlation of the in vivo results described above was also seen in S2 cells. The nuclear localization of Lz is constitutive and does not require Bgb protein (Fig. 2M,N). In contrast, when Bgb alone was expressed in S2 cells, nearly all of the Bgb protein remained cytoplasmic (Fig. 2K). However, when a construct expressing wild-type lz was co-transfected, a majority of the Bgb protein was seen in the nucleus (Fig. 2L). Thus, Bgb cannot be translocated to the nucleus in the absence of Lz. The cytoplasmic protein can be detected in S2 cells because of the high level of expression. Presumably, in the in vivo situation, the cytoplasmic Bgb protein is rapidly degraded in the absence of an α-subunit.

The identification of Bgb mutants as enhancers of lz^ts1 provides genetic evidence that Bgb functions during eye development. The enhanced roughness produced by the loss of one copy of Bgb in this sensitized background is rather mild (Fig. 3A,B). However, rare escaper flies of Df(3L)Bgb^K4/Bgb^D genotype can be generated in a lz^ts1 background. These flies are extremely weak and rarely seen. They can only be rescued if they are dissected from their pupal cases. Such flies have a very strong adult eye phenotype (Fig. 3D) that is identical to that of the lz null allele (Fig. 3C).

Bgb mutants also show defects in the development of the embryonic peripheral nervous system (Fig. 3E-J) as illustrated by staining with mAb22C10 which recognizes neurons in the central and peripheral nervous systems (Fujita et al., 1982). The axons of lateral chordotonal neurons present in the abdominal segments of wild-type embryos follow two distinct paths to the midline. While the axon of the lateral monoscolopidial chordotonal organ (lch1) follows the segmental nerve (SN) to the midline, the axons of the lateral pentascolopidial chordotonal organ (lch5) follow the intersegmental nerve (ISN) (Fig. 3G). In Bgb^D/Bgb^D mutant embryos the lch1 neuron turns toward the ISN and incorrectly follows this path to the midline (Fig. 3H). A similar phenotype with higher expressivity is seen in Bgb^D/Df(3L)Bgb^K4 embryos (Fig. 3I). The hsp70-Bgb; Bgb^D/Bgb^D rescued flies are completely viable and healthy flies, and as expected, do not show this PNS phenotype (not shown). PNS defects are also seen in run^YP17/run^YP17 mutant embryos (Fig. 3J), however these defects are difficult to interpret because of the additional segmentation phenotypes of run (Fig. 3M), not seen in Bgb mutants (Fig. 3L). The lack of segmentation phenotypes in Bgb could be due to a maternal contribution of the Bgb gene product. Alternatively, this could reflect a functional redundancy between the two partner proteins (see below).

The results described in Fig. 3A-D establish a role for Bgb in eye development. However, when Bgb^D/Bgb^D clones were generated in an otherwise wild-type (lz⁺) eye, the external phenotype of the facets (Fig. 4A) as well as the morphology of all retinal cells (Fig. 4B,C) appeared completely normal. Clones of Bgb^D were also examined in larval eye discs that were stained with antibodies directed against Elav, Cut and Pros. The expression pattern of these markers of neuronal and non-neuronal cells was wild type in all discs examined (not shown). Thus, all neuronal and non-neuronal cells in the eye are able to develop normally in the absence of the Bgb protein. The presence of a gene encoding the second partner protein, Bro, in the Drosophila genome, suggested to us the possibility that Bro and Bgb may function redundantly during development. This was investigated during embryonic development using the ds-RNA-mediated genetic interference method (Kennerdell and Carthew, 1998).

We injected embryos with ds-Bgb, ds-bro, or a mixture of ds-Bgb and ds-bro, and compared the resulting cuticular phenotypes with those resulting from loss of run function. run mutants are characterized by defects in segmental patterns which are visualized as deletions of denticle belts. run alleles can be ordered according to their strength by examining the denticles of the second and third abdominal segments (Gergen and Wieschaus, 1986). Weaker alleles of run tend to have only small deletions between these denticles (Fig. 5B), while stronger alleles have large deletions that are often accompanied by mirror-image duplications of denticle belts (Fig. 5C). As expected, injection of ds-run results in a cuticular phenotype (Fig. 5D-F) that is identical to the one seen with run null alleles (Fig. 5C).

Injection of ds-Bgb does not have any significant effect on the pattern of segmentation (Fig. 5G-I). In comparison, ds-Bro causes a segmentation phenotype (Fig. 5J-L) reminiscent of hypomorphic alleles of run (compare with Fig. 5B). Only a partial deletion of the naked cuticle between the second and third abdominal segments is apparent. Doubling the concentration of ds-Bro did not affect the severity of the phenotype (not shown). Strikingly, simultaneous injection of both ds-Bgb and ds-Bro resulted in embryos that have a much stronger segmentation phenotype (Fig. 5M-O), which is identical to that seen in a null allele of run (Fig. 5C), or in embryos injected with ds-run (Fig. 5D-F). These data show that although the disruption of Bgb does not generate a phenotype on its own, a role for Bgb in segmentation is revealed upon the disruption of both partner genes.

Sensitized genetic screens have proved to be powerful tools in identifying interacting proteins that participate in many different developmental pathways. A particularly impressive use of this technique in the Drosophila eye led to the identification of the mutations in the components of the RTK pathway (reviewed by Zipursky and Rubin, 1994). We have used such a screening technique to generate mutations in genes that function with lz during eye development. The identification of mutations in a direct transcriptional target of Lz, D-Pax2 (Flores et al., 2000), and the gene encoding a binding partner of Lz, Bgb, suggests that this screen is able to detect proteins whose function is directly related to that of Lz.

In our screen, two alleles of hsp83 were isolated as dominant enhancers of lz^ts1. Drosophila Hsp83 is a chaperone protein that has been shown to physically interact with Raf (van der Straten et al., 1997). Mutations in hsp83 were identified as downstream modifiers of the sevenless and EGFR RTK pathways (Simon et al., 1991; van der Straten et al., 1997). Recent studies have indicated an extensive collaboration between RTK pathways and Lz in the regulation of direct target genes such as D-Pax2 and pros (Flores et al., 2000; Xu et al., 2000). It is therefore likely that hsp83 strengthens the RTK signal transduction cascade that functions with Lz in the regulation of target genes. In addition, HSP90, the mammalian homolog of hsp83, has been shown to associate with a variety of different transcription factors and has also been proposed to function in nuclear transport (reviewed by Helmbrecht et al., 2000). An analysis of the relationship between Hsp83 and Lz/Bgb might provide insight into the mechanism by which this transcription factor complex is translocated to the nucleus.

The screen also uncovered two alleles of osa/eld (Kennison and Tamkun, 1988; Treisman et al., 1997), a member of the brahma (brm) complex, involved in chromatin remodeling (Collins et al., 1999; Kennison and Tamkun, 1988; Vazquez et al., 1999). The identification of osa as a dominant enhancer suggests that Lz may have a function related to chromatin remodeling. This is not surprising as other Runx family members are thought to function in this manner. For example, Runx2 binding has been implicated in the remodeling of the rat osteocalcin promoter (Javed et al., 1999). Additionally, during myeloid differentiation, Runx1 has been shown to interact with p300/CBP (Kitabayashi et al., 1998), a protein involved in histone acetylation. Further, Drosophila Run has been shown to bend DNA and is likely involved in modifying the architecture of target enhancers (Golling et al., 1996). In the eye, Lz is essential for pre-patterning an undifferentiated population of cells and preparing them to activate different target genes in response to signal transduction cascades. It is possible that this process involves remodeling of the individual enhancers through the mediation of an Osa/Lz complex. The identification of osa as a genetic modifier of lz suggests the need for future biochemical experiments to establish if such protein complexes are indeed formed during development.

In this paper we have focused on the function of the partner proteins since mutations in Bgb were identified as modifiers of lz. The similarity in the phenotype of lz^ts1; Bgb^D/Df(3L)Bgb^K4 mutants to the null allele of lz suggests an absolute functional requirement of the partner protein during eye development. Similarly, the ds-RNA interference results suggest that both partner proteins are able to function with Run during embryonic pattern formation.

It remains to be proved if the disorganization seen in the PNS of Bgb is attributable to Bgb function with the known RD proteins. Similar PNS defects are seen in run mutants, but these phenotypes are difficult to interpret because of the additional segmentation phenotypes that could indirectly affect PNS development. It remains possible that Bgb functions with an as yet uncharacterized RD protein in the PNS. Consistent with this explanation, a survey of the sequence of the Drosophila genome (Adams et al., 2000) reveals two additional RD proteins.

Our S2 cell expression data show that Bgb is only translocated to the nucleus in the presence of Lz. Although Bgb has a nuclear localization signal (NLS; Fujioka et al., 1996), these data suggest an additional requirement of Lz binding for its transport to the nucleus. Similar regulation of nuclear transport has been reported with Single-minded (Sim) and Tango (Tgo) heterodimers (Ward et al., 1998) as well as with Homothorax (Htx) and Extradenticle (Exd) heterodimers (Pai et al., 1998). In these examples, the localization to the nucleus of either Tgo or Exd, depends on the presence of Sim or Hth, respectively (Pai et al., 1998; Ward et al., 1998). Recent work has shown that Hth binding allows nuclear transport of Exd by simultaneously inhibiting its nuclear export signal (NES) while activating its NLS (Abu-Shaar et al., 1999; Berthelsen et al., 1999). Bgb does not have a leucine-rich sequence typically associated with an NES (Fischer et al., 1995; Wen et al., 1995); co-localization into the nucleus in this case is likely to involve an unmasking of the NLS causing its exposure to the transport machinery. Obviously, nuclear localization of both the α- and the β-subunit is a prerequisite for activation of transcription. In fact, in human AML caused by Inv(16), the CBFβ fusion protein is exclusively retained within the cytoplasm (Adya et al., 1998; Kanno et al., 1998; Liu et al., 1993).

The Lz/Bgb complex provides an interesting example of post-translational stabilization of proteins through the formation of heterodimeric complexes. While we cannot rule out the possibility that low levels of Bgb protein remain in the cytoplasm of the cell in a lz mutant background, the likely explanation for the Bgb protein not being detectable in the absence of Lz or Run is that the β-subunit is degraded in the absence of the α-partner. Similar mechanisms involving degredation of a subunit operate in creating stable Exd/Hth and Sim/Tgo complexes. Tissue lacking Hth or Sim will cause degradation of Exd and Tgo, respectively (Pai et al., 1998; Ward et al., 1998). As an interesting contrast to our results, in mammalian systems it is the α-subunit, Runx1, that is stabilized by CBFβ. In this case, the absence of the β-partner causes a proteosome-mediated degradation of the α-subunit (Huang et al., 2001).

The initial cloning of Bro and Bgb raised the possibility that these genes might function redundantly during development. Although there is a stretch of 156 amino acids at the N terminus of Bgb that is not present in Bro (Fujioka et al., 1996), these proteins are 59% identical throughout the remainder of their sequence. Furthermore, Bro and Bgb have overlapping expression domains during embryogenesis (Fujioka et al., 1996; Golling et al., 1996). ds-RNA-mediated genetic interference experiments used here clearly show that Bro and Bgb function redundantly during development as heterodimeric partners of Run. A loss-of-function phenotype equivalent to a complete run null allele is only revealed in the absence of both Bro and Bgb.

The two partner proteins do not function redundantly in all tissues. This is highlighted by the fact that Bgb mutants have a PNS defect on their own. Thus, at least in this tissue, Bgb function is not redundant with that of Bro. This is different from redundant gene pairs such as BarH1 and BarH2 which are co-regulated in all tissues and always function together (Higashijima et al., 1992). It is also interesting to note that injection of ds-Bro generates a fairly strong segmentation phenotype, while injection of ds-Bgb does not affect segmentation patterning at all. Therefore, it is possible that in the wild-type fly, when both partners are present, Run preferentially functions with Bro. However, only in the absence of Bro, can compensation of Run function be achieved through its binding Bgb. A comparable situation exists in mice. The paralogs Hoxa3 and Hoxd3 are expressed in the same tissue, but clearly have distinct functional requirements (Chisaka and Capecchi, 1991; Condie and Capecchi, 1993). Yet, a compensating mechanism can be created in a background when one of the two genes is eliminated (Greer et al., 2000).

Redundancy is a classical and important problem in genetics. As vertebrate genomes are analyzed in increasing detail, it is becoming evident that single mutant phenotypes may be masked due to the function of an alternate redundant gene (Müller, 1999). The completion of the sequence of the Drosophila genome (Adams et al., 2000) has revealed many gene families that function redundantly. Indeed, the identification of the Rhomboid family of genes led to the observation that Rho1 and Rho3 function redundantly during EGFR signaling (Wasserman et al., 2000). Another example is sloppy paired1 (slp1) and sloppy paired2 (slp2) which were identified as functionally redundant genes based on the fact that they are expressed in the same tissue but at slightly different levels (Bellen et al., 1989; Grossniklaus et al., 1992; Wilson et al., 1989). In spite of these advances, detection of mutations in genes that function redundantly poses a difficult challenge to genetic analysis. Our data show that at least for the case in study, dosage-sensitive screens involving sensitized genetic backgrounds can be used for the purpose of identifying redundant genes. Bro and Bgb together can be considered to contribute 4 copies of the partner gene. Loss of 1 out of these 4 copies in a sensitized background (lz^ts1; Bgb⁻ Bro⁺/Bgb⁺ Bro⁺) gives rise to a detectable eye phenotype. Yet, loss of 2 copies in a wild-type background (lz⁺; Bgb⁻ Bro⁺/Bgb⁻ Bro⁺) does not generate a mutant phenotype. This remarkable sensitivity to dosage suggests that properly sensitized genetic screens could be used in the detection of redundant gene function.

We are grateful to Y. Ito for sharing results prior to publication. We appreciate the helpful comments from P. Gergen. We thank A. Brand, R. Carthew, I. Duncan, P. Gergen, K. Matthews, N. Perrimon, G. Rubin, J. Treisman, and the Bloomington stock center, for fly stocks and antibodies. We thank V. Hartenstein for help in analyzing the PNS phenotype. We thank the members of the Banerjee laboratory for thoughtful discussions and comments on the manuscript. We thank Rahul Warrior for helpful suggestions about ³³P sequencing. We acknowledge the work performed by Karin Dumstrei in the isolation of the Df(3L)Bgb^K4 mutant and we are grateful for help from Hui Wang in generating the germline transformants. The 2B10, ELAV, 4D9 anti-engrailed/invected, and 22C10 antibodies were obtained from the University of Iowa Developmental Studies Hybridoma Bank developed under the auspices of the NICHD. This work was supported by an NIH grant to U. B. (2RO1EY08152) and a US Public Health Service National Research Service Award to J. K. (GM07185) and T. L. (GM07185).