DEVELOPMENT 1845 RESEARCH ARTICLE INTRODUCTION Precise temporal and spatial control of gene transcription is crucial for development. Sequence-specific DNA-binding factors and their association with a variety of modulator proteins, the co-factors, achieve this control. Co-factors do not bind DNA but act as adaptors between DNA-binding factors and other proteins. A number of transcription factors have been characterized, many of which act by recruiting multiprotein complexes with chromatin-modifying activities (Knoepfler and Eisenman, 1999). By recruiting co-factors, a DNA-binding protein can act as co-activator or as co-repressor depending on the context (Mannervik et al., 1999; Chinnadurai, 2002). An example of a co-repressor is the retinoblastoma protein that converts the E2F transcription factor into a repressor of cell- cycle genes (Weintraub et al., 1995). The identification of co-factors and the determination of their precise roles are crucial for understanding the mechanisms that govern development. Nab (NGFI-A–binding protein) proteins form an evolutionarily conserved family of transcriptional regulators. Nab was originally identified in mouse as a strong co-repressor by virtue of its capacity to interact directly with the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and inhibit its activity. Two Nab genes, Nab1 and Nab2, have been identified in vertebrates. Nab proteins do not bind DNA but they can repress (Svaren et al., 1998) or activate (Sevetson et al., 2000) gene expression by interacting with Egr transcription factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The NCD1 domain interacts with the R1 domain of Egr1 (Svaren et al., 1998). The NCD2 domain is required for transcriptional regulation (Swirnoff et al., 1998). Mice harboring targeted deletions of Nab1 and Nab2 have phenotypes very similar to Egr2 (Krox20)-deficient mice, suggesting that they act as co-activators of this gene (Le et al., 2005). In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain (Mechta- Grigoriou et al., 2000). Egr2 has been implicated in determining the segmental identities of r3 and r5 by controlling the expression of several target genes as well as cell proliferation. Misexpression experiments suggest that Nab1/Nab2 antagonize Egr2 transcriptional activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins might have additional functions as these experiments also led to alterations of the neural tube not found in Egr2-deficient embryos (Mechta-Grigoriou et al., 2000). Conversely, Egr2-deficient mice have a severe hindbrain segmentation defect that is not found in mice deficient in Nab1 and Nab2. Nab might also have Egr-independent functions in mice because, although epidermal hyperplasia has been observed in Nab1 Nab2 double mutant mice, this phenotype has not been observed in mice lacking any of the Egr proteins (Le et al., 2005). In Drosophila, only one Nab gene has been identified; it is highly homologous to vertebrate Nab genes in the NCD1 and NCD2 domains. Drosophila nab mutants are early larval lethal. Detection of nab transcripts by in situ hybridization indicates expression in a subset of neuroblasts of the embryonic and larval CNS and weak expression in imaginal discs (Clements et al., 2003). The role of Nab in Drosophila development is not known and so far no binding partner has been identified. In this report we show that nab is a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and that nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons. In early larval development, the wing fate is established in the distal-most region of the wing disc by a combination of two factors: activation of the gene vestigial (vg) (Williams et al., 1991) and repression of the gene teashirt (tsh) (Ng et al., 1996). Later, in early third instar larvae, wingless (wg) is activated in a ring of cells (the inner ring, IR) that borders the vg expression domain in the presumptive wing region (Fig. 1A). It has been suggested that activation of the IR involves a signal from the vg-expressing cells to the adjacent cells (del Álamo Rodríguez et al., 2002). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development Javier Terriente Félix 1 , Marta Magariños 2 and Fernando J. Díaz-Benjumea 1, * Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. Here we analyze the role of nab in Drosophila development. By employing genetic approaches we found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. We identified two partners of Nab: the zinc-finger transcription factors Rotund and Squeeze. Nab is co- expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. Our results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. We also showed by pull-down assays that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. We propose two mechanisms by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing. KEY WORDS: Drosophila, nab, squeeze, rotund, Transcriptional co-factors, Proximodistal development, CNS Development 134, 1845-1852 (2007) doi:10.1242/dev.003830 1 Centro de Biología Molecular-C.S.I.C. and 2 Dpto. Fisiología Animal, Facultad de Biología, Universidad Autónoma-Cantoblanco, 28049 Madrid, Spain. *Author for correspondence (e-mail: diazbenjumea@cbm.uam.es) Accepted 11 March 2007