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).
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).
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).
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).
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).
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).
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