Identifying conserved regions within insect Otd-related proteins
The homeodomain of Otd-related factors is over 85% identical in species ranging from Cnidaria to humans. Outside of this region, however, remarkably little sequence homology exists among Otd-related factors across species, confounding attempts to identify conserved functional domains. With the recent genomic sequencing of multiple insect species, we sought to reexamine potential conservation among Otd-related proteins. Using a 550 amino acid (aa) coding sequence derived from Otd’s longer alternatively spliced mRNA (Vandendries et al., 1996
), we performed a multi-species alignment with other predicted Diptera Otd/Otx proteins. This alignment revealed that the entire coding region of Otd is largely conserved through at least ten species of Drosophilidae, and shares restricted conservation through more distant groups ().
Figure 1 Otd protein sequence and alignment with other insect orthologs. A) D. melanogaster Otd sequence used for these studies. The DNA-binding homeodomain (HD, bolded), repeated SP residues (italicized), and the GNS, ANS, and GV sequences denoted in B (underlined) (more ...)
Similar to the Drosophila melanogaster
) Otd protein, other Drosophila
Otd orthologs contain polyhomomeric stretches of glutamines (N), alanines (A), glycines (G), and serines (S) (located at aa 129–297 in D. mel
Otd), and a glycine-valine (GV) repeat sequence (aa 345-367 in D. mel
Otd). Interestingly, such repetitive sequences are commonly found in transcriptional regulators and their expansion has been associated with a number of neurodegenerative diseases (Caburet et al., 2004
; Faux et al., 2005
; Hancock and Simon, 2005
). In addition to global conservation among closely related Drosophilidae species, portions of the N-terminus and a region encompassing the C-terminus are conserved in mosquitos (Culicidae: Culex, Aedes, Anopholes
, ), and more weakly conserved in the red flour beetle (Tribolium castaneum
, Coleoptera) and honey bee (Apis mellifera,
Hymenoptera) (data not shown). This data suggests strong selective pressure for maintaining these domains. Also noteworthy is that the LDY sequence, previously proposed to represent a degenerate “Otx tail” in Otd (Acampora et al., 2005
; Freund et al., 1998
), is conserved through Culicidae.
Multiple regions within Otd are important for rhodopsin gene regulation in vitro
Currently, only three direct Otd target genes have been identified: the rhodopsins Rh3, Rh5, and Rh6. These genes are expressed in a photoreceptor subtype-specific manner (Tahayato et al., 2003
), and their expression patterns can be recapitulated using <500 bp regulatory sequence located directly upstream of the TATA box (Fortini and Rubin, 1990
; Papatsenko et al., 2001
; Tahayato et al., 2003
; Zuker et al., 1985
). Recent studies have demonstrated that many of the same factors that are necessary for controlling Rh3
gene expression in vivo
also properly regulate Rh promoter activity in cultured Drosophila
S2 cells (Xie et al., 2007
; Mishra et al., 2010
). Otd, for instance, is critical for Rh3 and Rh5 expression in vivo
, and activates the Rh3 and Rh5 promoters in vitro
() (Xie et al., 2007
). Interestingly, although Otd represses Rh6 in OPRs in vivo
, Otd weakly activates the Rh6 promoter alone and synergistically activates Rh6 with Senseless (Sens) in vitro
(Mishra et al., 2010
; Tahayato et al., 2003
; Xie et al., 2007
). Since Sens is restricted to R8 photoreceptors and is required for Rh6 expression,
but does not bind directly to the Rh6 promoter sequence, these in vitro
experiments provide evidence that Sens is involved in activating Rh6 expression by interacting with Otd, and reveal that Otd may activate or repress Rh6 in different PR subtypes. Thus, testing Rh
promoter regulation in this culture system is useful for defining and refining transcriptional regulatory processes.
Figure 2 Otd deletions differentially regulate rhodopsin promoter activity in vitro. A) Diagram of Otd deletion constructs used for rhodopsin promoter reporter assays. OtdFL = aa 1-550, OtdΔN = aa 69-500, OtdΔC” = aa 1-529 (removes the (more ...)
Taking advantage of this reporter system, we tested whether the conserved domains in Otd described in contribute to its ability to regulate the Rh3, Rh5, or Rh6 promoters in vitro
. For this, we created deletions of the N- and/or C-terminus, as well as deletions of the middle regions of the protein (). All constructs tested retain Otd’s DNA-binding domain, a domain that is required for its ability to activate of all three promoters (Supp Fig 1
). In addition, each construct is expressed at similar levels (Supp Fig 2A
), localizes to the nucleus (data not shown), and retains at least some of Otd’s functions in vivo
Figure 6 Summary of in vivo mapping results. A) Otd deletion constructs used for otduvi rescues. “-“ represents a failure to rescue the otduvi mutant phenotype, “++” indicates that the rescued phenotype is similar to OtdFL rescues, (more ...)
We first focused on Otd-dependent activation of the Rh3 and Rh5 promoters. As shown in , a full-length Otd cDNA (OtdFL) activates Rh3 ~18-fold and Rh5 ~130-fold over basal activity. Removing Otd’s N terminus (OtdDelta;N) reduces this activity by ~60% on either promoter (), while removing the last 120 aa of Otd (OtdΔC) reduces Otd-dependent activity by 79% and 95% on Rh3 and Rh5 respectively. Removing just the last 20 aa, the region carrying the LDY motif (OtdΔC”), however, does not show a significant reduction in reporter activity compared to OtdFL, suggesting that the activation function of the C-terminus is not mediated by this “remnant” Otx tail.
We next tested internal deletions that remove different portions of Otd between its homeodomain and the C-terminal 430-550 activation domain. Removing the A region (aa 137-215) from Otd shows a significant increase in reporter activity on the Rh5 promoter (2.4 and 2.9-fold more than OtdFL with OtdΔA and OtdΔAB, respectively), while removing the B region alone (aa 215-430, OtdΔB) shows no significant change compared to Otd FL. These domains exhibit a similar trend on the Rh3 promoter, but are not statistically significant (). We also tested a construct that retains just the homeodomain and the AB domain (OtdΔNC), and this shows weak, but detectable promoter activity on both rhodopsin promoters (13% and 2% of OtdFL activity on the Rh3 and Rh5 promoters, respectively) (). Together, these data suggest that the N- and C-terminus of Otd combinatorially function as activation domains on both the Rh3 and Rh5 promoters, while the A domain suppresses Otd-dependent activation of Rh5.
Since Otd represses Rh6 in OPRs, yet weakly activates Rh6 alone and synergistically activates this promoter in the presence of Senseless in vitro, we next examined how the different Otd deletions affect Rh6 promoter activity with and without Senseless. Similar to the Rh3 and Rh5 promoters, Otd’s N- and C-termini both contribute significantly to Otd-dependent Rh6 activation in S2 cells (OtdΔN, OtdΔC) (), as removing either provides only ~25% of the activation observed with OtdFL, and removing both (OtdΔNC) leads to ~14% of OtdFL activation. In addition, removing Otd’s AB region leads to ~10-fold more activation of the Rh6 promoter than OtdFL, while individually removing the A or B region shows no statistical differences from OtdFL. Interestingly, although removing the last 20 aa of Otd shows a slight, but insignificant reduction in reporter activity on the Rh3 and Rh5 promoters, OtdΔC” shows a similar reduction in Rh6 promoter activity as OtdΔN and OtdΔC, suggesting this domain may rely on promoter context.
We next tested whether different regions of Otd contribute to its synergistic activation with Sens on the Rh6 promoter. As shown in , Sens causes a 10-fold increase in OtdFL’s ability to activate Rh6, and this same increase is maintained with OtdΔAB. OtdΔC, on the other hand, fails to activate Rh6 in the presence or absence of Sens. Surprisingly, though, OtdΔN shows even higher Sens-dependent activation (>75-fold) compared with OtdFL. These data suggest that the N-terminus suppresses Sens-dependent synergism with Otd, while the C-terminus is critical for Otd- and Otd/Sens-dependent activation of Rh6.
Combined, these experiments indicate that the C-terminus functions as an essential activation domain on all three Otd Rhodopsin targets, while other domains appear to function in a context-dependent manner. For instance, removing the N-terminus from Otd leads to reduced activation on all three promoters, but in the presence of Senseless, even higher synergistic activation of the Rh6 promoter is observed. We also find that while removing the A region enhances Rh5 activation, removing the entire AB region is required to cause a significant increase in Rh6 activation. Finally, the 20 aa “tail” domain of Otd only weakly contributes to Rh3 and Rh5 activity, yet functions similarly to the entire 120 aa C-terminus activation domain on Rh6 promoter activity.
Regions important for Otd-dependent rhodopsin expression are independent transcriptional regulatory domains
We next examined whether the domains that, when removed from Otd, affect Rh target gene expression in vitro,
are also sufficient to regulate transcription of a heterologous promoter. For this, we fused various portions of Otd () to the DNA-binding domain (DBD) of the yeast transcription factor GAL4 and measured the ability of these fusion proteins to regulate a UAS-luciferase reporter. Western blot analysis indicates that these constructs are expressed at similar levels (Supp Fig 2B
The GAL4 DBD lacks its own transcriptional activation domain, and only minimal basal luciferase activity is observed with this control (). In contrast, fusing the region that is downstream of Otd’s homeodomain (aa 129-550) to the DBD causes approximately 500-fold activation of the reporter (). Constructs containing aa 215-550 () or 304-550 (data not shown) also activate the report strongly, by ~250-fold, while the C-terminal domain (aa 430-550) that is necessary for Otd to activate rhodopsin gene expression, activates reporter expression by ~150-fold (). Further reducing the C-terminus to include only the last 90 aa () shows weaker, but significant, activation of reporter gene expression (~20X), while fusing the last 20 aa of Otd to the DBD shows no significant difference in activity compare to the DBD alone (inset in ). Like the C-terminus, the N-terminus alone (aa 1-70, N) is able to activate heterologous gene expression, but only by ~40-fold. In contrast, the A, B, and AB regions do not cause a significant increase in basal DBD activity, but rather, the entire AB region is sufficient to mediate weak repression (~30%, inset in ). Together, these data support our rhodopsin reporter assays that a potent transcriptional activation domain resides between residues 430-530, that a weaker activation domain is present within the N-terminus, and that the AB region may function as a repression domain. However, since the ABC region shows higher activation than the C region alone, the AB region may participate in activation and/or repression based on protein and/or promoter context.
Establishing a GAL4-based rescue paradigm for measuring Otd functions in vivo
To test whether the transcriptional regulatory domains identified in vitro
contribute to Otd’s functions during photoreceptor differentiation in vivo
, we developed an eye-specific Otd rescue paradigm. Previous studies have shown that transient heat shock-inducible expression of a full-length Otd cDNA is sufficient to rescue otd
mutants (Leuzinger et al., 1998
; Nagao et al., 1998
; Tahayato et al., 2003
; Vandendries et al., 1996
). However, this approach causes mosaic misexpression of Otd throughout the organism, making cell- and tissue-specific functions difficult to dissect. Thus, we developed a GAL4/UAS-based system that takes advantage of a 1.6 kb enhancer present within otd
’s third intron. This region is deleted in the eye-specific otduvi
mutant and is sufficient to drive reporter gene expression in all photoreceptors (Vandendries et al., 1996
). As shown in , this enhancer cloned upstream of the yeast GAL4 transcription factor (otd1.6
-GAL4) activates a UAS reporter similar to endogenous Otd expression: its expression initiates soon after the completion of photoreceptor specification in late third instar eye imaginal discs (), and is maintained throughout photoreceptor development ( and data not shown). We also observe limited reporter expression in a subset of neurons within the medulla neuropil of the optic lobe with this driver (data not shown, and (Morante and Desplan, 2008
). Importantly, otd1.6
-GAL4 remains active in otduvi
mutants (), making it useful for expressing various UAS-transgenes in an otduvi
Figure 3 Developing an Otd rescue paradigm. A,B) otd1.6 Gal4 drives reporter expression that mimics endogenous Otd expression in wild type or otduvi photoreceptors. Late 3rd instar imaginal discs whole mounts (A-A”) and cryosections from adult yw; otd (more ...)
To verify that otd1.6
-GAL4 driving a full-length Otd cDNA (otd1.6
>OtdFL) is sufficient to rescue otduvi
mutant eye phenotypes, we compared ommatidia morphology and Rhodopsin gene expression patterns between control, otduvi
, and otduvi
flies carrying the otd1.6
-GAL4 and UAS>OtdFL transgenes (otduvi
>OtdFL, referred to here as Otd rescues). Morphologically, control outer photoreceptor (OPR) rhabdomeric membranes span the entire depth of the retina (~100 μm) (), and their actin-rich surfaces, recognized by fluorescently-labeled phalloidin, form highly regular, trapezoidal arrangement surrounding the smaller rhabdomere of the R7 (or R8) (). In contrast, OPR rhabdomeres in otduvi
flies fail to extend more than 1/3 the depth of the retina (), and these rhabdomeres fail to form properly (Mishra et al., 2010
; Tahayato et al., 2003
; Vandendries et al., 1996
), making individual rhabdomeres difficult to visualize with phalloidin (). Similar to control flies, OtdFL rescues develop fully-elongated and organized rhabdomeres ().
Molecularly, Rh3 and Rh5 expression are expressed in subsets of R7 and R8 cells, respectively, in control flies () but are absent in otduvi
flies (). In addition, Rh6 expression is restricted to a subset of R8 photoreceptors at the base of the retina in control flies (), but is derepressed into OPRs in otduvi
flies ( and , otduvi
) (Tahayato et al., 2003
). In OtdFL rescues, Rh3 and Rh5 are restored to subsets of R7 and R8 cells, respectively (), and Rh6 is no longer expanded into OPRs, restricted to a subset of R8 cells (). Thus, re-expressing the full-length Otd protein is sufficient to rescue otduvi
mutants. We do note, however, that the number of Rh5-expressing cells is reduced in OtdFL rescue flies compared to yw
control or wild-type eyes: only ~15% of all R8 cells express Rh5 in otd1.6
>OtdFL rescue flies (), while ~30% of R8s express Rh5 in yw
or wild-type control flies ( and data not shown). Since the correct ratio of Rh3-expressing R7 cells is achieved in OtdFL rescues, this results in ommatidia that have miscoupling between Rh3-expressing R7s and Rh6-expressing R8s (data not shown). Identical results were observed with two individual OtdFL lines or by expressing both insertions of OtdFL together. Importantly, in otduvi
heterozygotes, we also observe miscoupled Rh3/Rh6 ommatidia and reduced numbers of Rh5-expressing cells, indicating that Otd levels are important for properly establishing the p:y ratio in R8 photoreceptors (). Consistent with this, misexpressing OtdFL in otduvi
heterozygous flies restores correct Rh3/Rh5 coupling (). Thus, we conclude that OtdFL is able to rescue Rh5 expression, but does not reach levels equivalent to wild-type Otd. To further verify that the reduced number of Rh5-expressing cells in OtdFL rescues is not due to an inherent inability of the OtdFL transgene to activate Rh5, we co-expressed OtdFL with Melt, a factor that transforms R8 cells into Rh5-expressing pR8s (Mikeladze-Dvali et al., 2005
). Consistent with a requirement for Otd to activate Rh5, no Rh5 expression is detected in otduvi
mutants that misexpress Melt alone (); however, similar to control eyes misexpressing Melt (), otduvi
mutants co-expressing OtdFL and Melt express Rh5 in almost all R8 cells (). Thus, for the remainder of the studies described here, we report qualitative, not quantitative differences in rhodopsin expression relative to OtdFL rescues. We also verified each construct for their ability to activate Rh5 in the presence of Melt, data which is included in Supplemental Figure S3
Figure 5 A) Rh6 repression in outer photoreceptors requires Otd’s C-terminus. Cryosections of agarose-embedded heads from otduvi flies or those rescued with various Otd deletions (UAS-transgenes listed above; genotypes as in ). Sections were stained (more ...)
Mapping Otd regulatory domains in vivo
Using the rescue paradigm described above, we next tested the same deletion constructs in vivo as we assayed in vitro. In addition, we created two additional constructs, OtdΔABC and OtdHD, to test the contribution of the N-terminus alone to various functions. Below, we describe our findings as they relate to individual aspects of otduvi phenotypes ( & ) and summarize the results in .
Figure 4 Mapping domains within Otd involved in Rh3 and Rh5 activation. Whole-mounted retinas or agarose-embedded cryosections from freshly eclosed flies were co-immunostained with A) R7 opsins Rh3 (purple, and black-and-white channel below) and Rh4 (green) or (more ...) Rh3 activation
All constructs except OtdΔNC, OtdΔAB, and OtdHD rescue Rh3 expression. However, empty R7 cells are more frequently observed with OtdΔC, OtdΔA, and OtdΔABC (circles, ) than OtdFL, OtdΔN, and OtdΔB, suggesting these are less effective in Rh3 activation. We also note that all Rh3-expressing cells in OtdΔABC rescues co-express Rh4 (arrows, ), and are restricted to the dorsal region of the eye, suggesting that OtdΔABC is only able to rescue Rh3 in a recently-described specialized subset of dorsal ommatidia that weakly co-express Rh3 with Rh4 (Mazzoni et al., 2008
). Interestingly, although OtdΔABC is able to restore at least some Rh3 expression, no Rh3 is detected in rescues with OtdΔAB. This is not likely due to an inability of OtdΔAB to recognize and regulate gene targets since this construct activates transcription more strongly than OtdFL in S2 cells, and is the strongest activator of Rh5 expression in R8 cells (see below). Other possibilities for this finding are described in the Discussion. Nevertheless, based on the findings that OtdΔN, OtdΔC, and OtdΔABC maintain at least one activation domain identified in vitro
and each can activate Rh3, while a construct lacking both the N- and C-terminus (OtdΔNC) shows no Rh3 rescue, these data support the possibility that both the N- and C-terminus contribute to Rh3 activation in vivo
, and that the N-terminus is sufficient to partially activate Rh3.
Rh5 is re-expressed in otduvi
mutants rescued with Otd factors that lack the A, B, or AB domains, but is absent when rescued with constructs lacking either the N- or C-terminus. However, distinct phenotypes are observed in rescues with OtdΔN and OtdΔC. First, in OtdΔN rescues, all R8 cells express Rh6 and very few empty R8 cells are detected, whereas in OtdΔC rescues, Rh6 is restricted to a subset of R8 cells, similar to wild-type animals, and cells expressing neither Rh5 or Rh6 are frequently observed (circles, ). These data suggest that in OtdΔC rescues, the pale subset of ommatidia is largely maintained, whereas in OtdΔN rescues, the yellow subset is expanded. Second, co-expressing OtdΔN with Melt shows very weak, but detectable Rh5, whereas no Rh5 is observed with constructs lacking the C-terminus (Supp Fig 3
), indicating that OtdΔN maintains some ability to activate Rh5, while OtdΔC does not. Interestingly, in flies rescued with OtdΔA, ΔB, and ΔAB flies, we note a unique and consistent phenotype: Rh5 and Rh6 are co-expressed in a subpopulation of R8 cells. In OtdΔA flies, all cells expressing Rh5 also express Rh6, whereas in OtdΔB and OtdΔAB flies, a subset of R8 cells express only Rh5, a subset expresses both Rh5 and Rh6, and a subset expresses only Rh6. Together, these data suggest that the C-terminus is essential for Rh5 expression, that the N-terminus is important to prevent Rh6 expression in pR8s and can weakly activate Rh5, and that the A and B regions are involved in preventing co-expression of R8 opsins.
Rh6 repression in OPRs
As previously mentioned, Otd is necessary for preventing Rh6 expression from being inappropriately expressed in outer photoreceptors (OPRs). Surprisingly, we find that this opsin is not localized to the actin-rich rhabdomeric membrane like most Rhodopsin proteins in control or otduvi mutants, but instead is present in OPR cytoplasm (). Proper repression of Rh6 in OPRs is observed when rescued with Otd proteins lacking the N, A, B, or AB regions ( and data not shown), but Rh6 expression in OPR cytoplasm is maintained in flies rescued with constructs lacking the C-terminus, including OtdDelta;C, OtdDelta;NC, OtdDelta;ABC, and OtdHD (). Thus, the C-terminus is necessary to repress Rh6 in OPRs.
Each deletion construct previously tested in vitro maintains at least some ability to restore proper photoreceptor morphogenesis (). However, only OtdΔN rescues similarly to OtdFL, whereas rescues with OtdΔC, OtdΔNC, OtdΔA, OtdΔB, OtdΔAB, and OtdΔABC only partially rescue trapezoid formation (best seen in sections at the R7 layer) and rhabdomere elongation (best seen in sections at the R8 layer). We note, though, that constructs lacking the C-terminus show fewer rhabdomeres reaching the R8 layer compared to constructs that retain this domain, suggesting that the C-terminus is particularly important for rhabdomere elongation. Since the smallest construct, OtdΔABC, is still able to partially rescue morphogenesis, and since the only common region in all of the deletions is the homeodomain itself, we also tested the ability of the homeodomain of Otd alone (OtdHD) to rescue morphogenesis. As shown in HD, distinct rhabdomeres are present in the R7 layer in OtdHD rescues that are more defined than in otduvi mutants, and more rhabdomeres have extended into the R8 layer than otduvi mutants, suggesting that the homeodomain can, albeit weakly, rescue some aspects of photoreceptor development. However, since no other constructs except OtdΔN rescue rhabdomeres to the same extent as OtdFL, it is likely that Otd utilizes multiple domains for regulating photoreceptor morphogenesis.