Faculty Bibliography

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Goosecoid (Gsc) is a homeodomain protein expressed in the organizer region of vertebrate embryos. Its Drosophila homologue, D-Gsc, has been implicated in the formation of the Stomatogastric Nervous System. Although there are no apparent similarities between the phenotypes of mutations in the gsc gene in flies and mice, all known Gsc proteins can rescue dorsoanterior structures in ventralized Xenopus embryos. We describe how D-Gsc behaves as a transcriptional repressor in Drosophila cells, acting through specific palindromic HD binding sites (P3K). D-Gsc is a 'passive repressor' of activator homeoproteins binding to the same sites and an 'active repressor' of activators binding to distinct sites. In addition, D-Gsc is able to strongly repress transcription activated by Paired-class homeoproteins through P3K, via specific protein-protein interactions in what we define as 'interactive repression'. This form of repression requires the short conserved GEH/eh-1 domain, also present in the Engrailed repressor. Although the GEH/eh-1 domain is necessary for rescue of UV-ventralized Xenopus embryos, it is dispensable for ectopic induction of Xlim-1 expression, demonstrating that this domain is not required for all Gsc functions in vivo. Interactive repression may represent specific interactions among Prd-class homeoproteins, several of which act early during development of invertebrate and vertebrate embryos.

Specificity in transcriptional regulation lies in a large part in the specificity of DNA binding by transcription factors. One group of transcription factors which are of great interest for studying transcriptional specificity is the Pax/Homeodomain (Pax/HD) proteins which contain two conserved DNA binding domains, a paired domain (PD) and a Paired-class homeodomain (HD). The Pax/HD proteins can bind to at least three types of specific DNA sequences: the PD binding sites, the dimeric HD binding sites and a composite HD and PD binding site. We propose that Pax/HD proteins regulate different subsets of their target genes through modular binding to one of these three specific sequences. We show that, in a tissue culture system, a member of the Pax/HD family, Paired, is able to activate transcription after binding through either its PD or its HD. The transactivation mediated by one domain does not require DNA binding of the other domain. Furthermore, binding sites specific for the PD of Paired are sufficient to mediate embryonic expression of a reporter gene in a paired-like pattern. The expression of the reporter gene is dependent on wild type paired function and, in a prd mutant background, it can be rescued by an exogenous paired gene encoding a protein whose HD is not able to bind to DNA. Finally, we show that the Paired protein uses differently its C-terminal activation domain when transactivation is mediated through its PD or its HD. These results and recent evidence from other Pax/HD proteins strongly suggest that this class of proteins is able to achieve specific and modular transcriptional regulation through its multiple DNA binding domains.

Pax-6 is a transcription factor containing both a homeodomain (HD) and a Paired domain (PD). It functions as an essential regulator of eye development in both Drosophila and vertebrates, suggesting an evolutionarily conserved origin for different types of metazoan eyes. Classical morphological and phylogenetic studies, however, have concluded that metazoan eyes have evolved many times independently. These apparently contradictory findings may be reconciled if the evolutionarily ancient role of Pax-6 was to regulate structural genes (e.g., rhodopsin) in primitive photoreceptors, and only later did it expand its function to regulate the morphogenesis of divergent and complex eye structures. In support of this, we present evidence that eyeless (ey), which encodes the Drosophila homolog of Pax-6, directly regulates rhodopsin 1 (rh1) expression in the photoreceptor cells. We detect ey expression in both larval and adult terminally differentiated photoreceptor cells. We show that the HD of Ey binds to a palindromic HD binding site P3/RCS1 in the rh1 promoter, which is essential for rh1 expression. We further demonstrate that, in vivo, P3/RCS1 can be replaced by binding sites specific for the PD of Ey. P3/RCS1 is conserved in the promoters of all Drosophila rhodopsin genes as well as in many opsin genes in vertebrates. Mutimerized P3 sites in front of a basal promoter are able to drive the expression of a reporter gene in all photoreceptors. These results suggest that Pax-6/Ey directly regulates rhodopsin 1 gene expression by binding to the conserved P3/RCS1 element in the promoter.

The photoreceptor cells of the Drosophila compound eye are precisely organized in elementary units called ommatidia. The outer (R1-R6) and inner (R7, R8) photoreceptors represent two physiologically distinct systems with two different projection targets in the brain (for review see Hardie, 1985). All cells of the primary system, R1-R6, express the same rhodopsin and are functionally identical. In contrast, the R7 and R8 photoreceptors are different from each other. They occupy anatomically precise positions, with R7 on top of R8. In fact, there are several classes of R7/R8 pairs, which differ morphologically and functionally and are characterized by the expression of one of two R7-specific opsins, rh3 or rh4. Here, we describe the identification of a new opsin gene, rhodopsin 5, expressed in one subclass of R8 cells. Interestingly, this subclass represents R8 cells that are directly underneath the R7 photoreceptors expressing rh3, but are never under those expressing rh4. These results confirm the existence of two subpopulations of R7 and R8 cells, which coordinate the expression of their respective rh genes. Thus, developmental signaling pathways between R7 and R8 lead to the exclusive expression of a single rhodopsin gene per cell and to the coordinate expression of another one in the neighboring cell. Consistent with this, rh5 expression in R8 disappears when R7 cells are absent (in sevenless mutant). We propose a model for the concerted evolution of opsin genes and the elaboration of the architecture of the retina.

The Drosophila gap-like segmentation genes orthodenticle, empty spiracles and buttonhead (btd) are expressed and required in overlapping domains in the head region of the blastoderm stage embryo. Their expression domains correspond to two or three segment anlagen that fail to develop in each mutant. It has been proposed that these overlapping expression domains mediate head metamerization and could generate a combinatorial code to specify segment identity. To test this model, we developed a system for targeted gene expression in the early embryo, based on region specific promoters and the flp-out system. Misexpression of btd in the anterior half of the blastoderm embryo directed by the hunchback proximal promoter rescues the btd mutant head phenotype to wild-type. This indicates that, while btd activity is required for the formation of specific head segments, its ectopic expression does not disturb head development. We conclude that the spatial limits of btd expression are not instructive for metamerization of the head region and that btd activity does not contribute to a combinatorial code for specification of segment identity.

Bicoid (Bcd) is a maternal morphogen responsible for patterning the head and thorax of the Drosophila embryo. Correct specification of head structure, however, requires the activity of the Torso receptor tyrosine kinase cascade, which also represses expression of Bcd targets at the most anterior tip of the embryo. Here, we investigate the role of both the homeodomain (HD) and the activation domain of Bcd in the anterior repression of its targets. When a Bcd mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of Bcd is also not specifically required for its down-regulation by Torso. We propose that Torso does not affect the ability of Bcd to bind DNA, but instead directs modification of Bcd or of a potential Bcd co-factor, which renders the Bcd protein unable to activate transcription.

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.