Promoter structure

The Bicoid morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. According to an early model, the affinity of Bcd-binding sites in the promoters of target genes determines the posterior extent of their expression. This hypothesis, referred to as the Gene X model, predicts that genes expressed specifically in the head primordium will contain low affinity Bcd sites, so that high levels of Bcd protein are required for their activation. Higher affinity Bcd sites would permit gene expression extending into the thoracic primordium. Other parameters, such as the spacing between Bcd sites and cooperative binding have also been proposed to affect bcd target gene regulation. However, the importance of all these factors in the regulation of actual bcd target genes has not been determined. A small regulatory region upstream of the cephalic gap gene orthodenticle is shown to be sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. This element is sufficient to generate early otd-like expression. This indicates that this second fragment must contain binding sites for a different activator of early head expression. However, since lacZ expression driven by this 173 bp fragment is eliminated in embryos lacking bcd, this activator must, at least in Drosophila, be bcd-dependent. The only clue regarding the functional specificity of this activator is the reiterated sequence motif required for the activity of this regulatory element. This study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation (Gao, 1998).

To localize the control elements required for embryonic head expression, a series of lacZ reporter fusions were constructed spanning the otd genomic region. A 7.6 kb fragment extending upstream of the otd transcriptional start site is sufficient to recapitulate the endogenous pattern of otd head expression. The pattern of endogenous otd expression was compared to that driven by the 7.6 kb regulatory fragment. otd is expressed initially at relatively low levels in an anterior cap-like region of the syncytial blastoderm embryo. The posterior boundary of this early expression domain is not sharp, but is graded in intensity. Expression quickly fades from the anterior terminus, leaving a stripe extending from 75%- 92% egg length (EL) in the cellular blastoderm embryo. During this period, ventral expression also disappears. By this stage, the anterior and posterior boundaries of otd expression are sharply defined. During germ band extension, otd expression becomes more complex, appearing at the ventral midline and in other regions of the embryo. In the germ band-retracted embryo, expression can be seen in the anterior brain and in midline CNS cells. This expression persists through embryogenesis. In the blastoderm embryo, lacZ expression driven by the 7.6 kb fragment is indistinguishable from endogenous otd expression. Later in embryogenesis, lacZ expression in the anterior head and in midline cells is similar, but not identical to otd expression at equivalent developmental stage. Expression of the transgene is less localized within the head primordium, and significantly weaker in midline cells. This suggests that additional regulatory elements are required for correct late expression. The results described above indicate that the 7.6 kb fragment contains the regulatory elements that control otd expression in the blastoderm head primordium. Further dissection of the 7.6 kb fragment reveals that a 900 bp sub-fragment is the smallest contiguous regulatory region capable of driving strong head expression, and this is referred to as the Early Head Enhancer (EHE). The EHE responds to maternal cues in a similar fashion to otd expression (Gao, 1998).

To understand how the EHE functions, it was mapped at higher resolution. Progressive 5' deletions show that a 186 bp element at its 5' end is critical for maintaining the intensity of early head expression. This region contains the three putative Bcd sites in the EHE. Deletion of this element significantly decreases the intensity of lacZ expression, without significantly affecting its spatial extent. Further 5' deletions, which removed a putative Hb site, have no obvious effect on the level or position of lacZ expression. 3' deletions reveal a second important control element at the opposite end of the EHE. Removal of 173 bp from the 3' end of the 900 bp fragment also reduced the intensity of lacZ expression. Again, the spatial extent of early head expression is not significantly altered. These experiments revealed that the activity of the EHE resides primarily within two small regulatory elements, each sufficient to drive otd-like expression in the head primordium. The 186 bp element contains three candidate Bcd binding sites. Each of these sites contains 6 of the 9 nucleotides defined as a high affinity Bcd site in the hb promoter. In particular, each site contains the TAATC core critical for the recognition of purified Bcd protein in vitro. The presence of these sequences suggested that Bcd binds directly to the 186 bp fragment. As described, the removal of the single putative Hb site does not obviously affect the function of the EHE. This is consistent with previous observations that hb plays a relatively minor role in otd activation. In contrast, the loss of a putative dorsal site that lies between the 186 bp and 173 bp fragments prevents the ventral retraction of lacZ expression. This is consistent with previous findings that dorsal is required for this retraction. The EHE also contains possible binding sites for the product of the terminal gap gene huckebein, which is involved in repressing otd expression at the anterior terminus of the embryo (Gao, 1998).

One of the goals of this study was to determine whether Bcd directly activates otd in the head primordium. Purified Bcd protein indeed binds to the three Bcd consensus sites in the 186 bp fragment and these sites are required for its activity in vivo. In its original form, the Gene X model predicted that a gene expressed specifically in the head primordium would have lower affinity Bcd sites than genes expressed more posteriorly. In addition to the affinity of isolated Bcd sites, subsequent studies show that intersite spacing, the number of sites, and cooperative binding effects all contribute to the affinity of regulatory regions for Bcd. The overall affinity of the 186 bp element for Bcd was compared to that of a 250 bp enhancer from the hb promoter. The hb enhancer drives lacZ expression across both the head and thoracic primordia and binds Bcd with high affinity. Significantly higher Bcd levels are found to be required in gel retardation assays to shift the labeled 186 bp fragment than the labeled hb regulatory element. Consistent with the Gene X model, the overall affinity of the otd regulatory element for Bcd is lower than that of the hb enhancer (Gao, 1998).

The 1.8 kb regulatory fragment contains two candidate Bcd and one Hb site that lie upstream of the EHE. Deletion of the region containing these sites causes a slight decrease in the intensity of expression. To determine whether this region is sufficient to drive early head expression, more fusion constructs were generated and their functions tested in vivo. Unexpectedly, it was found that a 526 bp EcoRV- HincII fragment containing these sites drive both anterior and posterior lacZ expression. This expression resembles that of the terminal gap gene tll, suggesting that this fragment contains terminal system response elements. Consistent with this idea, the anterior and posterior expression domains specified by this fragment both expand in embryos derived from torD females, bearing a constitutively active Egf receptor, triggering excess tll activation. Since otd is not expressed at the posterior pole, it was hypothesized that additional regulatory elements exist that prevent posterior expression. To test this idea, expression driven by a larger regulatory fragment extending to the 5' end of the EHE was examined. This fragment drives expression only in the head primordium, indicating that it contains a negative regulatory element that represses posterior expression. These result strongly suggests that Bcd participates directly in the regulation of otd (Gao, 1998).

Drosophila photoreceptor cells (R cells) develop from the eye imaginal disk during the third instar larval stage and acquire their adult morphology during pupation. orthodenticle is required for R-cell morphogenesis during pupation. otdUV-insensitive (otduvi) is a hypomorphic allele of otd that only affects R-cell development. The R-cell rhabdomeres are disorganized in otduvi mutants, and there is a disruption of proximal-distal development in the eye. The otd genomic structure indicates a deletion in the third intron of otduvi. Sequences encompassing this deletion are able to direct expression of the lacZ reporter gene at all stages of the developing visual system, including the photosensitive cells of Bolwig's organ, the ocelli, and the adult eye. The third intron enhancer is the primary regulatory element controlling otd in the R cells and is not under the control of the glass gene (Vandendries, 1996).

Separable transcriptional regulatory domains within Otd control photoreceptor terminal differentiation events

Orthodenticle (Otd)-related transcription factors are essential for anterior patterning and brain morphogenesis from Cnidaria to Mammals, and genetically underlie several human retinal pathologies. Despite their key developmental functions, relatively little is known regarding the molecular basis of how these factors regulate downstream effectors in a cell- or tissue-specific manner. Many invertebrate and vertebrate species encode two to three Otd proteins, whereas Drosophila encodes a single Otd protein. In the fly retina, Otd controls rhabdomere morphogenesis of all photoreceptors and regulates distinct Rhodopsin-encoding genes in a photoreceptor subtype-specific manner. This study performed a structure-function analysis of Otd during Drosophila eye development using in vivo rescue experiments and in vitro transcriptional regulatory assays. The findings indicate that Otd requires at least three distinct transcriptional regulatory domains to control photoreceptor-specific rhodopsin gene expression and photoreceptor morphogenesis. The results also uncover a previously unknown role for Otd in preventing co-expression of sensory receptors in blue vs. green-sensitive R8 photoreceptors. Sequence analysis indicates that many of the transcriptional regulatory domains identified in this study are conserved in multiple Diptera Otd-related proteins. Thus, these studies provide a basis for identifying shared molecular pathways involved in a wide range of developmental processes (McDonald, 2011).

Rh3 and Rh5 expression is coupled between pR7 and pR8 cells, respectively, suggesting that a 'pale photoreceptor, inner photoreceptor-specific' factor could similarly contribute to Otd's ability to activate both genes. However, increasing evidence suggests that Rh3 and Rh5 expression may rely on different regulatory processes. First, the onset of Rh3 vs. Rh5 expression is different during pupation; second, several experiments now suggest that Rh3 is the 'default'inner photoreceptore (IPR) opsin, whereas Rh5 expression requires an inductive signal from R7 cells; and third, Otd's C-terminus is essential for Rh5 activation in pR8s, whereas the N-terminus is sufficient to activate Rh3 expression, demonstrating that the activation of these two opsins are functionally separable. N-terminus-mediated activation of Rh3, however, is only observed in the dorsal third of the eye, a region recently shown to have weaker but more widespread Rh3 expression than in the ventral part of the eye. Since this dorsal region appears more 'permissive' for Rh3 expression, it is likely that the N-terminus provides weak activation potential while the C-terminus may be important for 'boosting' this expression in the remainder of the eye. One finding that is inconsistent with this model for Rh3 activation is that OtdΔAB, which maintains both the N- and C-terminus activation domains, is unable to activate Rh3. One possibility for this is that removing the AB domain creates a highly misfolded protein that prevents Rh3 activation. However, since OtdΔAB is able to activate Rh3 similar to OtdFL in vitro and is the most efficient construct tested in vivo for activating Rh5, this explanation seems unlikely. Another possibility is that Otd activates a repressor of Rh3. Indeed, an Otd-dependent yR7-restricted Rh3 repressor has been recently identified. Thus, it may be that the increased transactivation properties of OtdΔAB result in misexpression of this factor into pR7s, subsequently leading to loss of Rh3 expression. While future experiments will be required to formally test this hypothesis, this postulate underscores that much remains unknown related to how Rh3 and Rh4 are properly regulated in R7 cells, and whether Otd plays direct and/or indirect roles in this process (McDonald, 2011).

Transcriptional Regulation

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid gene is required for the development of larval head and thoracic structures, and the maternal gene torso for the development of the unsegmented region of the head (acron). otd expression responds to the activity of torso and bicoid at the anterior pole of the embryo, retracting from the most anterior region due to torso activity (Finkelstein, 1990, Eldon, 1991 and Gao, 1996). Embryos lacking both maternal and zygotic hunchback otd expression is not eliminated, but its posterior border shifts anteriorly.

Anterior repression of otd is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizes transcriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid. The relevance of runt's activity as a repressor of otd in normal Drosophila embryogenesis has been investigated. otd expression is activated in the posterior region of embryos that are mutant for runt. This posterior expression domain of otd depends on the activity of the orphan nuclear receptor protein Tailless. Repression of otd by runt does not require the conserved VVVRPY motif, which mediates interaction between Runt and the co-repressor protein Groucho. It is speculated that the genetic interactions between runt and tll involve physical interactions between the two proteins. It is interesting to note that interactions between Runt and another orphan nuclear receptor protein, Ftz-F1 have been invoked to explain runt's regulation of the pair-rule gene fushi tarazu. However, in this case runt functions to activate, rather than repress Ftz-F1 dependent transcription. It will be interesting to determine if there are binding sites for Tll that are essential for the activation of otd in the posterior region and whether these sites respond to the repressive activity of runt. It is noted that the activity of tll is necessary, but not sufficient for otd expression in the posterior region of the embryo. The observed functional interactions between runt and tailless on otd expression may indicate there are other contexts where members of these two families of transcriptional regulators interact to regulate gene expression during development (Tsai, 1998).

Epidermal growth factor receptor induces pointed P1 and inactivates Yan protein in the embryonic ventral ectoderm. Ectopic expression of secreted Spitz results in expression of orthodenticle within the entire ventral ectoderm, suggesting that ventral expression of otd is normally induced by higher levels of EGF-R activity. Of the two pointed transcripts, only pntP1 is expressed in the ventral ectoderm. It first appears prior to gastrulation in the entire neuroectoderm region. In pointed null mutants, the expression of orthodenticle, argos and tartan in these cells is abolished or significantly reduced. Since Pointed P1 is thought to be a constitutively active transcription factor, with no requirement for modulation of its activity by the EGF-R/MAP kinase signaling pathway, a direct induction of pntP1 transcription by EGF-R appears possible. Early pntP1 expression is EGF-R independent, but at stage 9/10, expression of pnt is not observed in the ventral ectoderm of Egf-R mutants. yan, which encodes a negative regulator of ETS transcriptional activators, is first detected at stage 5/6, where it is found in the dorsal ectoderm. yan expression declines in a dorsal-ventral gradient and is not found in mesoderm. Expression of yan does not depend on EGF-R, as it is unaltered in Egf-R mutants. In yan mutants, the ventralmost markers orthodenticle, argos and tartan show a clear expansion. Absence of the Yan protein may thus allow the Pointed P1 protein, which is expressed earlier in a broader domain, to efficiently induce ventralization. In the absence of Egf-R and yan, the early EGF-R-independent expression of pntP1 is capable of triggering otd expression. An activated form of Yan, which is unable to undergo phosphorylation by MAP kinase, was expressed in wild-type embryos. Indeed, the expression of orthodenticle and argos is significantly reduced or abolished, in the region where activated Yan is expressed (Gabay, 1996).

ebi (the term for 'shrimp' in Japanese) regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).

Loss of ebi affects Egfr-dependent expression of genes in the embryo. The EGFR ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggested that ebi may be a component in the EGFR signal transduction pathway (Dong, 1999).

Anterior-posterior positional information in the absence of a strong Bicoid gradient

The Bicoid (Bcd) transcription factor is distributed as a long-range concentration gradient along the anterior posterior (AP) axis of the Drosophila embryo. Bcd is required for the activation of a series of target genes, which are expressed at specific positions within the gradient. This study directly tested whether different concentration thresholds within the Bcd gradient establish the relative positions of its target genes by flattening the gradient and systematically varying expression levels. Genome-wide expression profiles were used to estimate the total number of Bcd target genes, and a general correlation was found between the Bcd concentration required for activation and the positions where target genes are expressed in wild-type embryos. However, concentrations required for target gene activation in embryos with flattened Bcd were consistently lower than those present at each target gene's position in the wild-type gradient, suggesting that Bcd is in excess at every position along the AP axis. Also, several Bcd target genes were positioned in correctly ordered stripes in embryos with flattened Bcd, and it is suggested that these stripes are normally regulated by interactions between Bcd and the terminal patterning system. These findings argue strongly against the strict interpretation of the Bcd morphogen hypothesis, and support the idea that target gene positioning involves combinatorial interactions that are mediated by the binding site architecture of each gene's cis-regulatory elements (Ochoa-Espinosa, 2009).

This study used genetic and transgenic manipulations to create pure populations of embryos with flattened Bcd gradients. These manipulations expanded specific subregions of the body plan, which reduced the complexity of cell fates in the embryo compared with wild type, and increased signal-to-noise ratios in the microarray experiments. The three levels of Bcd generated in these experiments, ≈4%, 11%, and ≈40%, cover the lower half of the full range of the Bcd gradient, and these experiments identified 13 of the 18 known Bcd target genes (Ochoa-Espinosa, 2009).

The 13 known Bcd target genes are included in a set of 242 genes that are differentially activated by increasing levels of Bcd. Ninety-seven of these genes have been tested for expression in the early embryo, and 48 are expressed differentially along the AP axis. Of these, 30 are likely to be direct targets based on known or predicted Bcd-dependent CRMs. If a linear extrapolation of this number is used to take into account the full set of 242 genes, the genome-wide estimate is ≈74 genes, and if the fact that these experiments did not identify five previously known Bcd target genes (27%), the estimate increases to ≈103 genes (Ochoa-Espinosa, 2009).

Six other genes were identified as Bcd targets based on the microarray experiments and the presence of nearby clusters of Bcd sites, but these genes are either expressed ubiquitously or in dorsal-ventral patterns, with no apparent modulation along the AP axis. It is possible that Bcd-dependent activation may partially contribute to these patterns by activating expression in anterior regions, which is consistent with recent studies that showed ChIP-chip binding of DV transcription factors to AP-expressed genes and vice versa. If these are real target genes, they would slightly increase the estimate of the total number of Bcd target genes (Ochoa-Espinosa, 2009).

Bicoid has been considered as one of the best examples of a gradient morphogen. Several lines of evidence suggest that Bcd does indeed function as a morphogen, including the coordinated shifts of morphological features and target gene expression patterns in embryos with different copy numbers of the bcd gene, and the ability of bcd mRNA to establish anterior cell fates when microinjected into ectopic positions. Furthermore, manipulations of the Bcd-binding sites in the hb P2 promoter and synthetic constructs with defined Bcd sites showed that cis-regulatory elements can be designed to be more or less sensitive to Bcd-mediated transcription. These studies led to the hypothesis that differential sensitivity to Bcd binding may control the relative positioning of different target genes (Ochoa-Espinosa, 2009).

The current findings suggest that differential sensitivity to Bcd binding is not the primary mechanism that controls the relative positioning of its target genes. Though some target genes respond in an all-or-none fashion to different levels of flattened Bcd, the levels required for activation are much lower than those present in the wild-type gradient in the regions where those genes are activated. These findings suggest that Bcd concentrations are in excess of those required for activation at every position along the length of the wild-type gradient (Ochoa-Espinosa, 2009).

It was also shown that the head gap genes otd, ems, and btd are expressed in correctly ordered stripes in embryos containing flattened Bcd gradients. This is most dramatically demonstrated by the mirror-image duplication of otd, ems, and btd stripes in the posterior region of 6B (6 copies) vas exu embryos, where the Bcd gradient slopes in the opposite direction to the order of striped expression. It is proposed that these genes are patterned by the terminal system in the absence of a Bcd gradient, and though Bcd function is required for their activation, the Bcd gradient does not play a major role in establishing their relative positions along the AP axis (Ochoa-Espinosa, 2009).

Bcd seems capable of bypassing the terminal system if expressed at high levels. For example, the anterior defects in terminal-system mutants can be partially rescued by increasing bcd copy number. Also, in 6B (6 copies) vas exu embryos, higher levels of Bcd are present throughout the embryo, with a relatively weak gradient along the AP axis. This causes expansions of the anterior otd, ems, and btd expression patterns into central regions of the embryo. The posterior boundaries of these patterns are positioned correctly, suggesting that the Bcd protein gradient is sufficient to position these target genes in regions where the terminal system does not reach. This is consistent with the observation that microinjected bcd mRNA can autonomously specify anterior structures (Ochoa-Espinosa, 2009).

These data are consistent with previous studies that failed to find a strong correlation between the relative positioning of target genes and the Bcd-binding 'strength' of their associated cis-regulatory elements. They further support a model in which Bcd functions as only one component of an integrated patterning system that establishes gene expression patterns along the AP axis. A second major component is maternal Hb, which is expressed in an AP protein gradient. Hb synergizes with Bcd in the activation of several specific target genes. In vas exu embryos, the loss of vas causes ectopic translation of maternal hb in posterior regions, so Hb protein is ubiquitously expressed and available for combinatorial activation with Bcd. This combination is likely sufficient to lead to the near ubiquitous expression of zygotic hb and Kr in 1B vas exu embryos, and gt in 2B vas exu embryos (Ochoa-Espinosa, 2009).

A third major component is the terminal system, which seems to affect the expression patterns of Bcd target genes in two ways. First, it causes a repression of all known Bcd target genes at the anterior pole by a mechanism that is not clearly understood. Second, the data suggest that the terminal system functions with Bcd for the establishment of the posterior boundaries of the head gap genes. This interaction appears to be important for regulating at least two other target genes, gt and slp1, which are expressed in anterior domains that shift toward the anterior pole in terminal system mutants. Both gt and slp1 are also activated in anterior and posterior stripes in embryonic regions containing low levels of flattened Bcd. These findings suggest that interactions with the terminal system may be required for positioning most Bcd target genes. The only known target genes that may not be directly influenced by the terminal system are zygotic hb and Kr, which are expressed in middle embryonic regions, far from the source of the terminal system activity (Ochoa-Espinosa, 2009).

How synergy between Bcd and the terminal system is achieved for each target gene is not clear. One possibility is that the Torso phosphorylation cascade directly modifies the Bcd protein, increasing its potency as a transcriptional activator. Mutations in Bcd's MAP-kinase phosphorylation sites partially reduce the ability of Bcd to activate otd, consistent with this hypothesis. Alternatively, the terminal system has been shown to repress the activities of ubiquitously expressed repressor proteins. Perhaps repression by the terminal system creates posterior to anterior gradients of these proteins, which then compete with Bcd-dependent activation mechanisms to establish posterior boundaries of target gene expression (Ochoa-Espinosa, 2009).

Interactions between Bcd, maternal Hb, and the terminal system may be critical for the initial positioning of target gene expression patterns, but it is clear that other layers of regulation are required for creating the correct order of gene expression boundaries in the anterior part of the early embryo. Almost all known Bcd target genes are transcription factors, and there is evidence that they regulate each other by feed-forward activation and repression mechanisms. Each target gene contains one or more CRMs, each of which is composed of a specific combination and arrangement (code) of transcription factor binding sites. Unraveling the mechanisms that differentially position Bcd target will require the detailed dissections of CRMs that direct spatially distinct expression patterns (Ochoa-Espinosa, 2009).

Targets of Activity

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. (Mohler, 1995).

In the eye antennal disc, during larval stages, orthodenticle acts through the segment polarity gene engrailed and other target genes to specify the medial head development (Royet, 1995). engrailed expression occurs well after expression of otd (Royet, 1995).

An effect on the early stripe of Goosecoid expression is observed in sloppy-paired, orthodenticle, tailless and decapentaplegic mutants. Both slp and otd affect Gsc in a similar way: the early stripe of Gsc appears normally but at the end of the cellularization stage, there is no reinforcement of its expression and it is prematurely lost. dpp is necessary to bring aboud Gsc repression in the dorsal-most region of the embryo, while tll is required to promote Gsc expression in the lateral region, or to prevent its repression by the dorsoventral patterning system (Goriely, 1996).

Otd/Crx, a dual regulator for the specification of ommatidia subtypes in the Drosophila retina

Comparison between the inputs of photoreceptors with different spectral sensitivities is required for color vision. In Drosophila, this is achieved in each ommatidium by the inner photoreceptors R7 and R8. Two classes of ommatidia are distributed stochastically in the retina: 30% contain UV-Rh3 in R7 and blue-Rh5 in R8, while the remaining 70% contain UV-Rh4 in R7 and green-Rh6 in R8. The distinction between the rhodopsins expressed in the two classes of ommatidia depends on a series of highly conserved homeodomain binding sites present in the rhodopsin promoters. The homeoprotein Orthodenticle acts through these sites to activate rh3 and rh5 in their specific ommatidial subclass and through the same sites to prevent rh6 expression in outer photoreceptors. Therefore, Otd is a key player in the terminal differentiation of subtypes of photoreceptors by regulating rhodopsin expression, a function reminiscent of the role of one of its mammalian homologs, Crx, in eye development (Tahayato, 2003).

Six rhs are expressed in the adult fly visual systems. R1-R6 contain the wide-spectrum Rh1, encoded by rh1/ninaE, while the ocelli contain the related Rh2. Based on the Rh content of the inner PRs, three main classes of ommatidia can be distinguished. In the dorsal rim area, both R7 and R8 contain the UV-Rh3. These ommatidia form a polarizing filter that detects the polarization vector of UV light reflected by the sky. The two other classes are distributed stochastically in the rest of the retina and exhibit differences in the fluorescence of their inner PRs, appearing either yellow (y; 70% of ommatidia) or pale (p; the remaining 30%). The y ommatidia express UV-rh4 in R7 and green-rh6 in R8, whereas p ommatidia express UV-rh3 in R7 and blue-rh5 in R8. The biological significance of these two subtypes of ommatidia is not clear but presumably allows discrimination of a broader range of wavelengths, with p ommatidia better discriminating among short wavelengths, and y ommatidia discriminating colors extending to the green (Tahayato, 2003).

The expression pattern of rhs is controlled at the transcriptional level. rh promoters have a bipartite organization consisting of a conserved proximal domain and unique upstream sequences. The proximal domain (-60 bp) provides PR identity through an RCSI/P3 element present in all known insect rhs. This element is a target for the homeodomain of Pax6. Although RCSI/P3 alone is not sufficient to drive rh expression, its multimerization allows expression of a reporter gene in all PRs. Thus, RCSI/P3 must confer general PR specificity to rhs, while interaction of this subthreshold element with upstream elements (RUS; rhodopsin upstream sequences) specific to each rh is required to achieve correct subtype expression (Tahayato, 2003).

The molecular players responsible for the specific expression of rhs in different inner PRs are not yet known. However, genetic experiments indicate that at least two mechanisms regulate the coordinate expression of R7/R8 rhs. For instance, in sevenless or boss mutants, both of which result in the absence of R7 cells, rh5 is drastically reduced and rh6 is expanded to almost all R8 cells. In contrast, the specific loss of R8 cells does not affect the mutually exclusive expression of rh3 and rh4 in R7. Thus, a stochastic choice is made between p and y subtypes in R7, which is then communicated to R8. This suggests that a 'horizontal' pathway sets up exclusion between rh3 and rh4 in R7 cells, and a 'vertical' mechanism coordinates the expression between R7 and R8 rhs, leading to expression of rh4 and rh6 in y ommatidia, and rh3 and rh5 in p ommatidia (Tahayato, 2003).

To explore the molecular mechanisms of p/y specification, an analysis was undertaken of the promoters of R7 and R8 rhs. Following the detailed analysis of Fortini (1990) on the rh3 and rh4 promoters, it was found that short promoters (between 137 bp and 276 bp) can mimic the complex expression pattern of all four of the inner rhs. Although most of the upstream RUS elements are unique to each rh promoter, focus was placed on a series of highly conserved binding sites for homeoproteins that bear a lysine at position 50 of their homeodomain, a residue that specifies DNA binding. These sites (TAATCC) are present in the rh3, rh5, and rh6 promoters, but absent in rh1, rh2, and rh4. Mutations of the K50 binding sites in rh3 and rh5 completely abolish their expression, while mutation of the same sites in the rh6 promoter results in the expansion of its expression to the R1-R6 outer PRs. The K50 homeoprotein Orthodenticle (Otd) acts through these sites, since rh3 and rh5 are lost in otd mutants, while rh6 is expanded to PRs. Thus, Otd plays a dual role: it is essential for establishing the expression of rh genes in the p subset as well as for repressing rh6 in outer PRs. This function of Otd is reminiscent of that of one of its vertebrate orthologs, Crx, which also plays a critical role in late aspects of PR differentiation, including the regulation of PR-specific genes (Tahayato, 2003).

otd is a highly conserved gene whose ancestral function resides in the determination of 'anterior' structures. Even in cnidarians (e.g., hydra), which demonstrate a primitive, diploblastic grade of organization, otx is expressed in the oral region. While only one otd gene exists in flies, four paralogs are expressed in mice: Otx1, Otx2, and the newly characterized Otx3, as well as Crx. From flies to mammals, otd and otx genes are involved in anterior brain development, and later in nervous system patterning. In contrast, the role of otd in PR morphogenesis appears to have been adopted by another related vertebrate factor, the Crx gene. For instance, Otx1/2 pattern the brain, while Crx affects cone and rod PR development as well as PR-specific gene expression (Tahayato, 2003 and references therein).

As for Otd, the ability of Crx to regulate PR-specific gene expression requires its binding to conserved K50 sites present within their promoters. In addition, mutations in the Crx gene are responsible for an autosomal dominant form of cone-rod dystrophy. Some of Crx functions might also be partially redundant with Otx2, which is also expressed in the retina. In Drosophila, the various roles of Otx/Crx might be represented by distinct regulatory elements that control otd anterior embryonic or eye expression (e.g., otduvi affects an eye-specific enhancer while a distinct enhancer responds to the morphogenetic gradient of Bicoid). Similarly, the data indicate that the roles of Otd in PR morphogenesis and PR-specific gene expression can be temporally separated. Whether vertebrate Crx and Otx's are able to rescue rh3, rh5, and rh6 expression in an otduvi background is currently being tested. The high homology between pathways involved in Drosophila and vertebrate eye development might reveal general principles that are applicable to the vertebrate retina (Tahayato, 2003).

The expression of the different rh genes is tightly restricted to different subsets of PRs, and this regulation is essentially transcriptional. Although the minimal promoters that were identified faithfully reproduce endogenous expression for rh4, rh5, and rh6, the rh3 transgenes exhibit weak pan-R7 expression in the dorsal compartment of the eye, consequently overlapping with Rh4. Although this could reflect the lack of a regulatory element in the reporter construct, the same weak expression is observed with several types of constructs, which included 2.4 kb of upstream sequence, the 3' UTR, and 1.2 kb of downstream genomic sequences. Furthermore, low levels of Rh3 in all R7 were detected in the dorsal region using anti-Rh3 antibodies. It is interesting to note that, in other species, several rhs have been shown to be coexpressed in the same PR. For instance, in the butterfly papillio and in bee and mouse, coexpression between Rhs is observed with dorsoventral differences. The dorsal portion of the eye is more likely to be exposed to UV-rich wavelengths and thus might have a specialized function (Tahayato, 2003).

Otd is a K50 homeoprotein required in the eye at the time of PR differentiation. otd is absolutely required, but is not sufficient, for turning on the expression of p-type rhs, rh3, and rh5. Thus, Otd is unlikely to act alone to confer spatial regulation for the following reasons: (1) it is expressed in all PRs; (2) generalized expression of Otd under heat shock control in wild-type flies does not dramatically affect the expression of rh3 and rh4, nor does it affect rh1 or rh6; (3) the ability to fully rescue the expression of rh3 in otduvi mutants by pulses of otd demonstrates that otd does not need to be restricted to p-type ommatidia; (4) otd is required in the outer PRs to repress rh6 and in all PRs for proper rhabdomere morphology, and (5) otd is required for preventing rh1 expression in most, if not all inner PRs, probably through an indirect mechanism. Indeed, no K50 sites were found within the rh1 promoter construct that shows derepression in otduvi mutants, while, rh3, rh5, and rh6 all exhibit very clear binding sites for Otd (Tahayato, 2003).

Although the sequence of the K50 sites in rh3/rh5 and rh6 is identical, they function to activate the p-type rhs and to repress rh6 in outer PRs (and in a subset of R7 cells). While conserved flanking sequences associated with the activator K50 sites in rh3 and rh5 were not detected, the rh6 KI site was found to be associated with a 21 bp sequence that is highly conserved in D. virilis and D. pseudoobscura. This sequence might represent the site of action of a corepressor binding together with Otd to transform it from an activator into a repressor. While mutation of this element in the context of the -555/+121 or -246/+121 rh6 promoter did not lead to expansion of reporter activity to outer PRs, it remains possible that this site and/or other as yet unidentified elements function redundantly in preventing rh6 expression in outer PRs (Tahayato, 2003).

Since otd does not provide the spatial specificity to rhs, other factors must do so. For instance, a coactivator might be expressed specifically in p-type ommatidia to activate rh3 and rh5, while a corepressor might be needed in outer PRs to repress rh6. These cofactors do not have to bind DNA and thus might not have a cognate site in the promoters. Alternatively, Otd could only be permissive for rh3 and rh5 expression, while spatial specificity is provided by other proteins that bind to distinct elements within their promoters. RUS3B could represent such an element for rh3, since this site is essential for expression in pR7 (Tahayato, 2003).

Although Otx family genes mostly act as activators, Otx2 has been found to repress the expression of XWnt-5a through a conserved K50 site in its promoter, suggesting that the repression activity of Otd is also conserved in vertebrates, and might depend on similar cofactors. It will be interesting to investigate whether Otx2, together with Crx, can modulate late retinal development and particularly the distribution of cone opsin genes, whose promoters contain conserved K50 sites. Finally, the observation that, in Drosophila, Otd is likely to require cofactors for its various functions in the eye, is consistent with the fact that Crx has been shown to function synergistically with a number of factors, including NRL, to activate opsin gene expression. It will be important to identify the ancestral function of Otd/Crx from which the role of these genes in regulating eye development has evolved (Tahayato, 2003).

The loss of rh3 and rh5 expression in p inner PRs is not compensated by expansion of the y inner PR rhs; rh4 and rh6 remain largely restricted to the y subset of R7 and R8. This suggests that the p ommatidia remain committed as such but fail to express their rhs. This is consistent with the direct binding of Otd to the rh3 and rh5 promoters, which are terminal differentiation markers, and also shows that otd is not the spatial determinant of p versus y fates. While the loss of p rhs should lead to a lack of proper rhabdomere formation or to their degeneration, this is not observed in otduvi flies. It is suggested that this may be due to the low levels of Rh1 that are induced by the absence of rh gene expression in inner PRs lacking otd, thus avoiding degeneration. Thus, Rh1 may serve as a 'default' rhodopsin whose expression is normally repressed in inner PRs by Otd or through an Rh-mediated exclusion process (Tahayato, 2003).

A general rule in many sensory systems is that expression of a sensory receptor molecule in a given cell excludes the expression of all other sensory receptors. For instance, the vertebrate or Drosophila olfactory receptors or the Drosophila rh genes are generally not coexpressed. Although the vertebrate olfactory receptor molecules themselves do not appear to play a role in the exclusion pathway, it has been argued that they might be directly involved in some step in the specification of olfactory receptor cells, in particular their projection pattern. The Rhs are, like the olfactory receptors, seven-transmembrane G-coupled receptors, and they might too play an instructive role in the exclusion pathway that is distinct from their role in phototransduction. In otd mutants, the general coexpression rule is broken, since Rh6 and Rh1 coexist in outer PRs, and Rh1 and Rh4 are present together in yR7. This suggests that otd-mediated processes are key to the exclusion process (Tahayato, 2003).

The two different inner PR subtypes (p and y) remain defined in otd mutants, since rh4 and rh6 remain restricted to 70% of ommatidia, while p ommatidia acquire rh1 or rh6. Thus, otd is likely to act downstream of other factors that determine the p subtype. Otd is present and required in all PRs and it is likely that its activation and repression roles are determined by interaction with other proteins that place Otd at the heart of the pathway that specifies the exclusion and coordination of rhs. A model is proposed to explain the multiple late roles of Otd; in pR7 and R8, Otd acts along with p-specific cofactors to direct expression of both rh3 and rh5. In pR7, Prospero represses rh5 and rh6, leaving rh3 as the only rh expressed. In pR8, both rh3 and rh5 genes are also turned on, but the exclusion mechanism might only allow rh5 to be maintained in R8. The mechanism for such regulation remains to be identified and could involve the Rh molecules themselves. In yR7, the Otd p cofactor is not present and rh3 and rh5 are not activated. rh4 does not depend on Otd and must therefore be turned on specifically in yR7 by another system. A gene that is necessary and sufficient to turn on rh4 in these cells has been identified. In yR8, rh6 is expressed by default. In all inner PRs, Otd also indirectly represses rh1. Finally, in outer PRs, Otd interacts with a corepressor to turn rh6 off, while strong activators turn on rh1 at high levels. This model suggests that otd functions downstream of the p/y decision pathway, and that specific cofactors are required to allow the spatial determination of the two classes of ommatidia (Tahayato, 2003).

Analysis of the Otd-dependent transcriptome supports the evolutionary conservation of CRX/OTX/OTD functions in flies and vertebrates

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. Global gene expression analysis in late pupal heads was used to better characterize the post-embryonic functions of Otd in Drosophila. 61 genes were identified that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. These findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria (Ranade, 2008).

The comparative analysis of gene expression in wild type and otduvi mutant heads was carried out at the late P12 stage of pupal development, at a time in the terminal differentiation of photoreceptor neurons characterized by the establishment of Rhodopsin genes expression. Genes that were found to be differentially expressed between Canton S (CS) and otduvi in the microarray analysis were further investigated by RT-PCR in two wild type strains and two otduvi fly lines. Through this analysis, of 61 genes showed at least a 2-fold change in mRNA levels by microarray, and consistently display analogous changes in gene expression by RT-PCR. This is equivalent to <0.5% of the 13,369 genes represented on the Affymetrix Drosgenome1 array (Ranade, 2008).

Among the 61 differentially expressed genes, 37 are down-regulated and 24 are up-regulated in otduvi mutant heads. Forty-six genes are presently annotated for a number of biological processes and functions, and 15 have unknown functions. Although the otduvi allele is hypomorphic, and thus, does not result in a complete loss of otd activity, the expression of 40% of the genes (24/61) was strongly affected (>4 fold). Among these, four genes that are robustly expressed in the wild type appeared to be transcriptionally inactive in the mutant (Cyp6a17, Rh3, Acyp2, and Rh5), whereas 4 genes that are normally expressed at low levels or not at all were found to be strongly induced in otduvi (CG14743, Try29F, mthl8, and Cyp4p3) (Ranade, 2008).

The absence of Rh3 and Rh5 mRNA is consistent with the direct transcriptional regulation of both genes by Otd. However, no significant change was observed in the only other known direct target in fly heads, Rh6. This is likely due to the developmental stage selected for the analysis. Rh6 is the last opsin to be expressed in the pupal retina beginning around ~79% PD and reaching 70% of the adult Rh6 mRNA levels by 82% PD. Because gene expression was sampled at ~80% PD, increased levels of Rh6 mRNA due to ectopic expression in the otduvi R1–R6 photoreceptors may not be detectable until later in pupal development or in the adult (Ranade, 2008).

Due to the time point chosen for the analysis, the use of the strong but not null otduvi allele, as well as the stringent criteria applied in the selection of differentially expressed genes, this study cannot result in the identification of all genes regulated by Otd in the head. Nonetheless, because the critical interval for Otd function in the differentiating retina extends from ~12% to ~75%-80% PD, the list of genes identified in this study should include critical downstream targets of Otd during photoreceptor morphogenesis (Ranade, 2008).

A number of genes involved in phototransduction were down-regulated in otduvi mutant tissue as compared to wild type. These include the Rhodopsins Rh3, Rh4, and Rh5, CDP diglyceride synthetase (CdsA), and Arrestin2 (Arr2). The observed down-regulation of Rh3 and Rh5 was expected, while the changes in Rh4, CdsA and Arr2 expression identify new direct or indirect targets of Otd (Ranade, 2008).

Although previous work suggested that Rh4 expression is unchanged in otduvi mutant retinas, this study found Rh4 mRNA levels to be reduced by more than 4-fold in microarray analysis. Rh4 transcript levels were confirmed to be lower at both the pupal and adult stages in two separate otduvi lines as compared to CS and OR by RT-PCR. Furthermore, a reduction in β-galactosidase activity encoded by an Rh4-lacZ transgene was detected in the otduvi mutant background. Thus, it appears that Rh4 transcript levels are in fact significantly reduced in otduvi R7 cells even though the spatial pattern of Rh4 expression remains essentially unchanged. The decrease in Rh4 expression does not reflect a general down-regulation of all opsins in mutant photoreceptors. In fact, expression of Rh1, the major rhodopsin expressed in the R1-R6 cells, is not similar affected. However, since the regulatory region included in the Rh4-lacZ transgene does not contain canonical Otd binding sites (TAATCC), the regulation of Rh4 by Otd is most likely indirect (Ranade, 2008).

Interestingly, the otduvi mutant was originally identified based on its abnormal phototactic behavior in a visible-light (VIS) versus ultraviolet-light (UV) choice test (Vandendries, 1996). Rh3 and Rh4 are the two UV-sensitive opsins expressed in the fly eye: Rh4 mediates UV detection in 70% of the R7 neurons, whereas Rh3 does so in the remaining 30%. Rh4 is therefore the predominant UV-sensitive opsin and the down-regulation observed in this study is consistent with, and likely contributes to, the abnormal phototactic behavior of otduvi mutant flies (Ranade, 2008).

The CdsA and Arr2 genes also encode critical components of the phototransduction cascade. CdsA is required to regenerate PIP2, which is the source of the intracellular signals for the visual transduction cascade. Arr2 is involved in the deactivation of the Rhodopsins, and the regulated light-dependent trafficking of the Arr2 protein is essential for light adaptation of photoreceptor cells. Both are down-regulated in otduvi mutant tissue and had not been previously identified as potential Otd targets (Ranade, 2008).

Thus, in addition to Rh3 and Rh5, one more opsin receptor, Rh4, and at least two other critical components of the visual transduction cascade, CdsA and Arr2, are positively regulated by Otd (Ranade, 2008).

Several other genes that are known to function and/or to be transcribed in the eye are also differentially expressed between otduvi and wild type, including boss, CG8889, chp, Cpn, slo, Slob, trx. Two of these, chaoptic (chp) and Calphotin (Cpn), are known to be required for the differentiation of photoreceptor neurons, and mutations in either gene result in morphological defects similar to those observed in otduvi mutants (Ranade, 2008).

The chp gene encodes an adhesion protein that is thought to mediate inter-microvillar stacking within the rhabdomere (Van Vactor, 1988). The Cpn gene encodes a Ca2+ ion-binding protein. The Cpn mutant phenotype is very similar to the chp phenotype as both display distorted, reduced and split rhabdomeres. However, the most severe Cpn alleles also lead to photoreceptor cell death, whereas chp is dispensable for photoreceptor cell viability. The aberrant rhabdomere morphology observed in otduvi flies is similar to phenotypes seen in strong chp alleles and hypomorphic Cpn alleles. Accordingly, Cpn and chp expression is not abolished in otduvi mutant flies but reduced by about 3-fold in microarray analysis (Ranade, 2008).

Two other genes, trithorax or trx (transcription regulation) and bride of sevenless or boss (cell-cell signaling) are required in the early stages of photoreceptor cell development, primarily at the time of cell fate acquisition. Although no changes in cell fate have been reported in otduvi mutants, these genes may continue to be expressed and function during later stages of retinal development. Indeed, boss expression has been detected in multiple retinal cell types during pupation, including in all photoreceptors and the neurons associated with the bristles of the eye, consistent with a potential role for boss in later aspects of photoreceptor morphogenesis (Ranade, 2008).

Lastly, several other genes are associated, either experimentally and/or through electronic annotation, with biological processes that could be relevant to the otduvi mutant phenotype, including three factors involved cytoskeleton organization, six factors involved in protein processing or seven signaling/cell-adhesion factors, in addition to boss (Ranade, 2008).

In summary, the regulation of chp and Cpn directly ties Otd to the control of R-cell morphology and several other otd-dependent loci identified in this study may contribute to specific aspects of R-cell development and function (Ranade, 2008).

The Otd homologue CRX/OTX5 has been linked to circadian-regulated processes in vertebrates, including photic entrainment and circadian gene expression in the pineal gland. To explore whether Otd may also contribute to the regulation of metabolic, physiological and/or behavioral processes under the control of the circadian clock, the set of differentially expressed genes was compared with a list of loci previously identified as cycling in fly heads (Ranade, 2008).

It was found that 13 of the 61 genes differentially expressed in the otduvi mutant background are included in this circadian gene list. All but one is down-regulated in mutant tissue and, therefore, would be positively regulated by Otd in wild-type flies. Twelve of the genes are reported to show altered expression in circadian mutants. Moreover, some of these genes may mediate the circadian regulation of visual sensitivity (Rh3, Rh4 and Rh5), detoxification (Cytochrome P450-6a2 or Cyp6a2, Cyp6a17, Glutathione S transferase E1 or GstE1), and locomotor behavior (slowpoke or slo and Slowpoke binding protein or Slob) (Ranade, 2008).

In the case of the calcium-activated potassium channel Slo and its modulator Slob, analyses of mutant phenotypes more directly implicate these factors in the circadian control of locomotor activity. Wild-type flies entrained to a 24 h light-dark (LD) cycle are more active at dawn and dusk and are quiescent during the day. Once entrained, they maintain this behavioral rhythm even if moved to constant darkness (DD). Flies mutant for slo exhibit an arrhythmic locomotion phenotype lacking clear peaks of activity but displaying overall activity levels similar to wild type. Similarly, flies with neuron-targeted expression of UAS-Slob (under the control of the pan-neural driver elav-Gal4) exhibit a loss of photic entrainment when shifted from LD to DD as suggested by the breakdown of rest/activity patterns over time (Ranade, 2008).

The contribution of the other cycling genes to circadian rhythms has not been investigated, and in all but two cases (Cyp6a17 and Rh3), gene expression is reduced rather than abolished in the otduvi hypomorphic background. Because stronger otd mutant alleles are embryonic lethal and therefore less easily analyzed, it is currently difficult to evaluate the role of otd in regulating biological rhythms. However, whether the Otd transcription factor would exercise its influence exclusively at the level of the retina, where it is known to be broadly expressed, was investigated, or whether it may also function elsewhere in the head, particularly in the other circadian centers of the fly (specifically in the Hofbauer-Buchner eyelet and/or in pacemaker cells of the central brain) (Ranade, 2008).

As previously shown, Rh6 is expressed in the eyelet, and the enhancer-trap line R32-lacZ in all pacemaker neurons. Using these molecular markers, it was found that Otd is expressed in all four cells of the eyelet and in group 3 of the dorsal pacemaker neurons (DN3). It was estimated that about half of the ca. 40 DN3 cells expressed Otd. Interestingly, the DN3 neurons can synchronize molecular rhythms in the absence of external photoreceptors and appear to be non-homogeneous based on variations in cellular size and in R32-lacZ expression level. The presence of Otd in only a subset of these neurons confirms this observation and provides the first endogenous molecular marker for a distinct DN3 subtype (Ranade, 2008).

Because the retina, eyelet, and pacemaker neurons contribute somewhat redundantly to the entrainment of circadian rhythms, understanding the consequences of the loss of otd function in the various specific cell types will require extensive analyses. Nonetheless, the expression of Otd in cells of all three circadian centers as well as the potentially direct control of slo and slob expression suggests that otd contributes to the regulation of circadian-related gene networks (Ranade, 2008).

The Otd/OTX/CRX transcription factors belong to a subgroup of homeodomain proteins known as the K50-type based on the presence of a lysine at the critical amino acid 50 of the homeodomain. In the case of the only known direct targets of Otd in the fly (Rh3, Rh5 and Rh6), gene transcription is regulated through TAATCC (GGATTA) sites located within the first few hundred base pairs upstream of the start of transcription. Although Otd binding characteristics have not been extensively studied, the availability of these sites and their variable conservation in other Drosophila species (D. pseudoobscura and D. virilis) permit the generation of an Otd-binding-site position weighted matrix (PWM). Based on this PWM, Otd-binding sites were sought within each of the differentially expressed genes, and their evolutionary conservation was investigated in the distantly related Drosophila species, D. pseudoobscura (ca. 55+ million years). Because of the limited characterization of Otd-binding specificity and the short nature of the consensus sequence (6 bp; TAATCC), four additional constraints were introduced to the search: (1) the analysis was limited to the 1000 base pairs (bp) of genomic DNA immediately upstream of each start of transcription (5′-flank) reasoning that many functional promoters (including for the known Otd targets Rh3, Rh5, and Rh6) are present in this region; (2) a PWM score cutoff of 4.5 was selected in order to exclude any sites with more than one mismatch from the TAATCC sequence; (3) only perfectly matching sites between D. melanogaster and D. pseudoobscura were considered conserved; and, (4) whenever the 5′-flank contained another gene, the DNA within this upstream transcription unit was excluded from consideration because of the potential for additional evolutionary constraints on sequence variation (Ranade, 2008).

Using these criteria, it was possible to investigate 60 of the 61 genes. A total of 129 PWM matching sites were identified within the ~54 kb of DNA analyzed. This constitutes more than twice the site frequency expected based on random occurrence (~53 sites at 1 in 1024 bp). Among the 60 genes, 19 (31%) have one or more putative Otd-binding sites that are perfectly conserved between D. melanogaster and D. pseudoobscura. The presence of up-regulated (6) and down-regulated (13) loci among the 19 putative direct targets is consistent with the ability of Otd to function as a repressor (Rh6) as well as an activator (Rh3 and Rh5). Ten genes have one conserved site, four genes contain two conserved sites (CG10924, CG8942, Cpn, Rh5), four genes have three (CG30492, CG5391, Dyb, Slob) and one gene, Rh3, contains four conserved sites. Lastly, it was possible to further investigate 13 of these 19 loci in the more distantly related species D. virilis (ca. 63+ million years), and evidence of conservation was found in 10 cases (10/13) (Ranade, 2008).

The identification of conserved target sites in the Arr2, Cpn, slo and Slob genes supports the direct involvement of Otd in phototransduction and photoreceptor cell morphogenesis and strongly suggests that Otd is involved in aspects of circadian rhythmicity as well (Ranade, 2008).

Otd not only is important for photoreceptor neuron differentiation but also plays a critical role during embryonic development. At this stage, Otd functions in patterning rather than terminal differentiation. The transcriptome regulated by Otd in the Drosophila embryo has been investigated through genome-wide microarray analysis by Montalta-He (2002). In this study, the expression level of 287 annotated genes was found to be significantly changed in response to Otd overexpression (Ranade, 2008).

A comparison of the Otd-regulated transcriptome characterized in this study with data from Montalta-He (2002) has allowed investigation of whether similarities exist between Otd function during embryonic development and R-cell morphogenesis. Whereas differences in the Otd-regulated transcriptome at embryonic and pupal stages was expected, it was surprising to find a complete lack of overlap between the 'Montalta-He set' of 287 putative Otd targets and the current list of 61 loci. The difference in experimental design between the two studies may contribute to this result, as Montalta-He relied on a gain-of-function study in whole embryos, whereas this study analyzed the consequences of a tissue-specific loss of otd function. However, the observation that none of the 61 genes identified in this study appears to respond to heat shock induced expression of Otd at the embryonic stage is nonetheless surprising, and suggests that the Otd transcription factor regulates gene expression in profoundly distinct ways as a patterning factor during embryogenesis and as a differentiation factor in the pupal head. Thus, it will be interesting to investigate how transcriptional regulation by Otd is modified at these different stages at the level of chromatin structure and through interactions with specific cofactors (Ranade, 2008).

Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function

The function and integrity of photoreceptor cells are dependent upon the creation and maintenance of specialized apical structures: membrane discs/outer segments in vertebrates and rhabdomeres in insects. A molecular and morphological comparison was performed of Drosophila Pph13 and orthodenticle (otd) mutants to investigate the transcriptional network controlling the late stages of rhabdomeric photoreceptor cell development and function. Although Otd and Pph13 have been implicated in rhabdomere morphogenesis, this study demonstrate that it is necessary to remove both factors to completely eliminate rhabdomere formation. Rhabdomere absence is not the result of degeneration or a failure of initiation, but rather the inability of the apical membrane to transform and elaborate into a rhabdomere. Transcriptional profiling revealed that Pph13 plays an integral role in promoting rhabdomeric photoreceptor cell function. Pph13 regulates Rh2 and Rh6, and other phototransduction genes, demonstrating that Pph13 and Otd control a distinct subset of Rhodopsin-encoding genes in adult visual systems. Bioinformatic, DNA binding and transcriptional reporter assays showed that Pph13 can bind and activate transcription via a perfect Pax6 homeodomain palindromic binding site and the Rhodopsin core sequence I (RCSI) found upstream of Drosophila Rhodopsin genes. In vivo studies indicate that Pph13 is necessary and sufficient to mediate the expression of a multimerized RCSI reporter, a marker of photoreceptor cell specificity previously suggested to be regulated by Pax6. These studies define a key transcriptional regulatory pathway that is necessary for late Drosophila photoreceptor development and will serve as a basis for better understanding rhabdomeric photoreceptor cell development and function (Mishra, 2010).

To date, little is known about the transcriptional network required for establishing a rhabdomere. Significantly, these findings emphasize that there are at least two homeodomain transcription factors, Pph13 and Otd, that are required for photoreceptor cell morphogenesis and function. First and foremost, Pph13 and Otd cooperate for rhabdomere elaboration. The loss of either results in poorly formed rhabdomeres, although the rhabdomeres are still present and phototransduction proteins still accumulate within. Only upon the removal of both factors do the rhabdomeres fail to materialize. Moreover, the morphological analyses demonstrate that the failure of rhabdomeres to develop is due neither to degeneration nor to an inability to initiate the process. The data suggest that the roles of Pph13 and Otd are to coordinate and direct the morphological changes of the actin cytoskeleton and apical membrane into the specific and stereotypic structure of a rhabdomere. This dependency on two homeodomain transcription factors is in contrast to vertebrate photoreceptor cells, in which the identical process of expanding the membrane to house the phototransduction machinery is relegated to one protein, Crx, a vertebrate homolog of Otd (Mishra, 2010).

Another intriguing observation from these studies is the difference between the morphological role of Otd and Pph13 in ocelli versus eye photoreceptor cells. The loss of either factor in ocelli does not result in noticeable defects in rhabdomere formation, in stark contrast with the situation for the eye. Why the difference? One possibility is that the lack of an inter-rhabdomeral space decreases the pressure on the microvilli to create a cohesive structure. For example, a defined target of Otd is chaoptin, which encodes a protein that is crucial for proper microvilli adhesion and which is essential to keep the microvilli together during the formation of the inter-rhabdomeral space. As a result, the lack of an extracellular matrix does not interfere with the ability of the apical membrane to form microvilli. Nevertheless, determining exactly how Pph13 and Otd coordinate rhabdomere morphogenesis or why expansion of the photoreceptor membrane in rhabdomeres has been partitioned to two homeodomain transcription factors will require further characterization of many of the Pph13-, Otd- and Pph13-Otd-dependent transcription targets (Mishra, 2010).

The expression of Rhodopsin or other eye-specific phototransduction proteins is a key indicator of when a ciliated or rhabdomeric cell has been designated to act as a photoreceptor cell. With respect to Drosophila, the characterization of Rhodopsin promoters has indicated a bipartite structure (Fortini, 1990) for directing photoreceptor cell expression and subtype specificity. To ensure photoreceptor cell expression there is a common essential element that is found in all Drosophila Rhodopsin promoters: RCSI. RCSI represents one half of the bipartite structure, and mutation of this element eliminates photoreceptor cell expression. Although this element alone is not sufficient for photoreceptor cell expression, when multimerized, such as in the 3XP3 reporter, it is sufficient to drive and limit expression to all photoreceptor cells. Thus, the presence of the RCSI suggests that there is a common factor(s) required in all photoreceptor cells that ensures eye-specific expression of Rhodopsin genes. Based on these observations, it would be expected that an RCSI regulatory factor would have the following characteristics: (1) it should be expressed in all Drosophila photoreceptor cells; (2) it would be a homeodomain transcription factor that is able to form a hetero- or homodimer due to the presence of the palindromic sequence TAAT; and (3) it should be capable of binding the various RCSI elements and, most importantly, be sufficient and necessary for the expression of 3XP3 in vivo. The data presented in this study indicate that Pph13 satisfies all the above criteria. Furthermore, the microarray profiling has identified other known, and yet to be characterized, photoreceptor proteins (Rhodopsins, Gβ, NinaC, Arr2, Osi18, PIP82) that also share this RCSI sequence in their promoter region and are dependent on Pph13 for expression. Overall, Pph13 is not merely a factor ensuring Rhodopsin expression, but has a greater role in photoreceptor cell function (Mishra, 2010).

One important and conflicting question is, if Pph13 is the general transcription factor binding to the RCSI elements then why is there selective downregulation of specific Rhodopsin promoters in a Pph13 mutant? A model is proposed in which the dependency for expression of Rhodopsins on either the RCSI site or additional elements responsible for subtype-specific expression has shifted between the different Rhodopsin promoters. In other words, Pph13 does bind to every RCSI site but, owing to the bipartite structure of the Rhodopsin promoters, sequence differences in the individual RCSI elements and resultant differences in Pph13 affinity contribute to a situation in which the presence of Pph13 alone is not a limiting factor for expression. There are several observations that support such a model. First, whereas Pph13 is necessary and sufficient for expression of 3XP3, no ectopic expression of any Rhodopsin promoter reporters is observed in tissues outside of photoreceptor cells, confirming the idea that Rhodopsin promoters are coordinately regulated by other factors. Second, each RCSI element is not created equally. Swapping of RCSI domains between the different promoters does not dramatically affect spatial or temporal specificity (Papatsenko, 2001), but does reflect predictable changes in the level of expression based on Pph13 affinities that were observe in this study. For example, when the Rh6 RCSI site is placed into an Rh3 or Rh5 minimal promoter, there is an increase in expression level, and a reciprocal downregulation of expression is observed when the Rh6 RCSI is replaced by the RCSI of Rh3. These results directly correlate with the relative affinity of Pph13 for these RCSI elements, as described in this study. Furthermore, the lower affinity of Pph13 for the Rh3 or Rh5 RCSI element would predict a greater dependency on other elements in these promoters for expression - hence the dependency on Otd. Indeed, the expression of both Rh3 and Rh5 is dependent on Otd binding to, and activating transcription outside the region of, their respective RCSI sequences (Tahayato, 2003). By contrast, as observed in Drosophila, the perfect palindrome/higher affinity binding site contained within the Rh6 RCSI element requires promoter regions outside of the RCSI element to repress expression in every photoreceptor cell (Tahayato, 2003: Mishra, 2010 and references therein). Altogether, these data correlate well with a model in which there is a shifting of dependency between the RCSI element and other upstream photoreceptor subtype-specific elements among the different Rhodopsin promoters. As for the residual Rh1 expression in the otd; Pph13 double mutant, the model predicts that a second element outside of the RCSI site contributes to the expression of Rh1. There are two binding sites for Glass, one inside the minimal promoter of Rh1 and one outside. Glass is expressed in all photoreceptor cells and is required for Rh1 expression. Moreover, like the RCSI element, Glass binding sites when multimerized are sufficient to drive and limit expression to photoreceptor cells (Mishra, 2010).

The results form the basis for a better understanding of all rhabdomeric photoreceptor cell development and function. Interestingly, the activity of the 3XP3 reporter is a common marker for transgenic constructs in many invertebrate species and thus its activity is not limited to Drosophila. Given the relationship between Pph13 and Otd in determining rhabdomere morphogenesis and the ability of both factors to ensure photoreceptor cell function, it will be crucial to determine whether these same relationships exists in other invertebrate rhabdomeric photoreceptor cells (Mishra, 2010).

Defective proventriculus specifies the ocellar region in the Drosophila head

A pair of the Drosophila eye-antennal disc gives rise to four distinct organs (eyes, antennae, maxillary palps, and ocelli) and surrounding head cuticle. Developmental processes of this imaginal disc provide an excellent model system to study the mechanism of regional specification and subsequent organogenesis. The dorsal head capsule (vertex) of adult Drosophila is divided into three morphologically distinct subdomains: ocellar, frons, and orbital. The homeobox gene orthodenticle (otd) is required for head vertex development, and mutations that reduce or abolish otd expression in the vertex primordium lead to ocelliless flies. The homeodomain-containing transcriptional repressor Engrailed (En) is also involved in ocellar specification, and the En expression is completely lost in otd mutants. However, the molecular mechanism of ocellar specification remains elusive. This study provides evidence that the homeobox gene defective proventriculus (dve) is a downstream effector of Otd, and also that the repressor activity of Dve is required for en activation through a relief-of-repression mechanism. Furthermore, the Dve activity is involved in repression of the frons identity in an incoherent feedforward loop of Otd and Dve (Yorimitsu, 2011).

This study presents evidence that Dve is a new member involved in ocellar specification and acts as a downstream effector of Otd. The results also revealed a complicated pathway of transcriptional regulators, Otd-Dve-Ara-Ci-En, for ocellar specification (Yorimitsu, 2011).

Transcription networks contain a small set of recurring regulation patterns called network motifs. A feedforward loop (FFL) consists of three genes, two input transcription factors and a target gene, and their regulatory interactions generate eight possible structures of feedforward loop (FFL). When a target gene is suppressed by a repressor 1 (Rep1), relief of this repression by another repressor 2 (Rep2) can induce the target gene expression. When Rep2 also acts as an activator of the target gene, this relief of repression mechanism is classified as a coherent type-4 feedforward loop (c-FFL). During vertex development, Ara is involved in hh repression, and the Dve-mediated ara repression is crucial for hh expression and subsequent ocellar specification. However, the cascade of dve-ara-hh seems to be a relief of repression rather than a cFFL, because Dve is not a direct activator of the hh gene. Furthermore, dve RNAi phenotypes were rescued in the ara mutant background, suggesting that a linear relief of repression mechanism is crucial for hh maintenance (Yorimitsu, 2011).

In photoreceptor R7, Dve acts as a key molecule in a cFFL. Dve (as a Rep1) represses rh3, and the transcription factor Spalt (Sal) (as a Rep2) represses dve and also activates rh3 in parallel to induce rh3 expression. Interestingly, Notch signaling is closely associated with the relief of Dve-mediated transcriptional repression in wing and leg disks. These regulatory networks may also be cFFLs in which Dve acts as a Rep1, although repressors involved in dve repression are not yet identified. In wing disks, expression of wg and ct are repressed by Dve, and Notch signaling represses dve to induce these genes at the dorso-ventral boundary. The Dve activity adjacent to the dorso-ventral boundary still represses wg to refine the source of morphogen. In leg disks, Dve represses expression of dAP-2, and Notch signaling represses dve to induce dAP-2 at the presumptive joint region. The Dve activity distal to the segment boundary still represses dAP-2 to prevent ectopic joint formation. Taken together, these results suggest that Dve plays a critical role as a Rep1 in cFFLs in different tissues. In the head vertex region, it is likely that the repressor activity of Dve is repressed in a cFFL to induce frons identity (Yorimitsu, 2011).

The homeodomain protein Otd is the most upstream transcription factor required for establishment of the head vertex. During second larval instar, Otd is ubiquitously expressed in the eye-antennal disk and it is gradually restricted in the vertex primordium until early third larval instar. Expression of an Otd-target gene, dve, is also detected in the same vertex region at early third larval instar. Otd is required for Dve expression, and the Otd-induced Dve is required for repression of frons identity through the Hh signaling pathway in the medial region. However, Otd is also required for the frons identity in both the medial and mediolateral regions (Yorimitsu, 2011).

This regulatory network is quite similar to the incoherent type-1 feedforward loop (iFFL) in photoreceptor R7. Otd-induced Dve is involved in rh3 repression, whereas Otd is also required for rh3 activation. iFFLs have been known to generate pulse-like dynamics and response acceleration if Rep1 does not completely represses its target gene expression. However, the repressor activity of Dve supersedes the Otd-dependent rh3 activation, resulting in complete rh3 repression in yR7. In pR7, Dve is repressed by Sal, resulting in rh3 expression through the Otd- and Sal-dependent rh3 activation. Thus, Dve serves as a common node that integrates the two loops, the Otd-Dve-Rh3 iFFL and the Sal-Dve-Rh3 cFFL (Yorimitsu, 2011).

In the head vertex region, Otd and Dve are expressed in a graded fashion along the mediolateral axis with highest concentration in the medial region. It is assumed that Otd determines the default state for frons development through restricting the source of morphogens Hh and Wg, and also that high level of Dve expression in the medial ocellar region represses the frons identity through an iFFL. It is likely that repression of dve by an unknown repressor X occurs in a cFFL and induces the frons identity in the mediolateral region (Yorimitsu, 2011).

Interlocked FFLs including Otd and Dve appear to be a common feature in the eye and the head vertex. However, other factors are not shared between two tissues. In R7, a default state is the Otd-dependent Rh3 activation, an acquired state is (1) Rh3 repression through the Otd-Dve iFFL and (2) Spineless-dependent Rh4 expression. In the vertex, a default state is Otd-dependent frons formation, an acquired state is (1) frons repression through the Otd-Dve iFFL and (2) Hh-dependent ocellar specification associated with En and Eya activation (Yorimitsu, 2011).

Both Otd and Dve are K50-type homeodomain transcription factors, and they bind to the rh3 promoter via canonical K50 binding sites (TAATCC). The Otd-Dve iFFL in the eye depends on direct binding activities to these K50 binding sites, but the iFFL in the vertex seems to be more complex. Although target genes for frons determination are not identified, the iFFL in the vertex includes some additional network motifs. For instance, in the downstream of Dve, Hh signaling is critically required for repression of the frons identity (Yorimitsu, 2011).

Since iFFLs also act as fold-change detection to normalize noise in inputs, interlocked FFLs of Dve-mediated transcriptional repression may contribute to robustness of gene expression by preventing aberrant activation. It is an intriguing possibility that, in wing and leg disks, Dve also serves as a common node that integrates the two loops as observed in the eye and the vertex. Further characterization of regulatory networks including Dve will clarify molecular mechanisms of cell specification (Yorimitsu, 2011).

Regulation of twin of eyeless during Drosophila development

The Pax-6 protein is vital for eye development in all seeing animals, from sea urchins to humans. Either of the Pax6 genes in Drosophila (twin of eyeless and eyeless) can induce a gene cascade leading to formation of the entire eye when expressed ectopically. The twin of eyeless (toy) gene in Drosophila is expressed in the anterior region of the early fly embryo. At later stages it is expressed in the brain, ventral nerve cord and (eventually) the visual primordium that gives rise to the eye-antennal imaginal discs of the larvae. These discs subsequently form the major part of the adult head, including compound eyes. This study has sought genes that are required for normal toy expression in the early embryo to elucidate initiating events of eye organogenesis. Candidate genes identified by mutation analyses were subjected to further knock-out and misexpression tests to investigate their interactions with toy. The results indicate that the head-specific gap gene empty spiracles can act as a repressor of Toy, while ocelliless (oc) and spalt major (salm) appear to act as positive regulators of toy gene expression (Skottheim Honn, 2016).

Protein Interactions

Comparison of the DNA targets of Bicoid, Fushi tarazu and Orthodenticle reveal the importance of the amino acid at position 50 of the homeodomain in discriminating between bases that lie adjacent to the TAAT core of homeodomain binding sites. FTZ has a preference for TAATG due to the presence of glutamine at position 50, while Bicoid prefers the consensus sequence TAATCC, specified by lysine at position 50. OTD also has a lysine at position 50 and the consensus sequence recognized is similar to that of BCD. Structural studies suggest that water-mediated hydrogen bonds and van der Walls contacts underlie the preferences for bases adjacent to the TAAT core (Wilson, 1996).

orthodenticle: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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