A Cis-Acting Element, Directs Pineal-Specific Gene Expression in Zebrafish

Pineal expression-promoting element (PIPE), a cis-acting element, directs pineal-specific gene expression in zebrafish

Yoichi Asaoka*, Hiroaki Mano*, Daisuke Kojima†, and Yoshitaka Fukada‡

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 26, 2002 (received for review July 25, 2002) The pineal gland, sharing morphological and biochemical similar- photoreceptor cells (9–13). These observations illustrate re- ities with the retina, plays a unique and central role in the markable parallels in the pineal and retinal phototransduction photoneuroendocrine system. The unique development of the pathways, but very little is known about the molecular basis that pineal gland is directed by a specific combination of the expressed accounts for the characteristic development of the pineal gland. genes, but little is known about the regulatory mechanism under- The regulatory mechanism responsible for tissue-specific gene lying the pineal-specific gene expression. We isolated a 1.1-kbp expression and its evolutionary background are, therefore, im- fragment upstream of the zebrafish exo-rhodopsin (exorh) gene, portant issues providing clues to the developmental control which is expressed specifically in the pineal gland. Transgenic specifying the pineal or retinal identity. analysis using an enhanced green fluorescent protein reporter In recent years, a number of studies have been reported about gene demonstrated that the proximal 147-bp region of the exorh transcriptional regulation of the retinal genes (reviewed in ref. promoter is sufficient to direct pineal-specific expression. This 14), among which the rhodopsin (rh) promoter has been studied region contains three copies of a putative cone rod homeobox extensively. Biochemical approaches and in vitro transcription (Crx)͞Otx-binding site, which is known to be required for expres- assays have identified several cis-acting DNA elements, such as sion of both retina- and pineal-specific genes. Deletion and muta- Ret-1 (15), BAT-1 (16), and Ret-4 (17). These studies suggest a tional analyses of the exorh promoter revealed that a previously cooperative interplay of multiple cis-acting elements and tran- uncharacterized sequence TGACCCCAATCT termed pineal expres- scription factors for the retinal photoreceptor cell-specific gene sion-promoting element (PIPE) is required for pineal-specific pro- expression. Consistent with this idea, several transcription fac- moter activity in addition to the Crx͞Otx-binding sites. By using the tors have been identified and shown to regulate the rh gene zebrafish rhodopsin (rh) promoter that drives retina-specific ex- expression. Subtraction cDNA cloning resulted in identification pression, we created a reporter construct having ectopic PIPE in the of neural retina leucine zipper (Nrl), a basic leucine zipper rh promoter at a position equivalent to that in the exorh promoter (bZIP) transcription factor that is expressed in all cell layers of by introducing five nucleotide changes. Such a slight modification adult mammalian retina (18, 19). Nrl binds to a well conserved in the rh promoter induced ectopic enhanced green fluorescent cis-acting element Nrl response element (NRE) and transacti- protein expression in the pineal gland without affecting its retinal vates the rh promoter (20, 21). On the other hand, cone rod expression. These results identify PIPE as a critical cis-element homeobox (Crx) is an Otx-related homeodomain protein ex- contributing to the pineal-specific gene expression, in combination pressed exclusively in the retinal photoreceptor cells and pine- with the Crx͞Otx-binding site(s). alocytes (22–24). Crx transactivates the rh promoter by binding to BAT-1 and Ret-4 (22) and is implicated in the regulation of iving organisms use environmental light signals for multiple photoreceptor cell-specific gene expression (25). In addition to Lphysiological functions such as vision, photoentrainment of these transcription factors, Rx and Erx seem to participate in the circadian rhythms, regulation of body color, and detection of regulation of the rh gene expression (26–28). seasonal changes in photoperiod. These diverse functions are In contrast to these advances in the studies on the retina- mediated not only by the retina but also by extra-ocular photo- specific gene expression, transcriptional regulation of the pineal receptive organs, such as the pineal gland. The retina and pineal gene remains poorly understood. The only cis-acting element gland probably arose via divergence from a common ancestral identified so far is a pineal regulatory element (PIRE), which is recognized by Crx (29, 30). PIRE with a consensus sequence of photoreceptive organ, and consistently, the pineal gland acts as ͞ Ј a photosensory organ in the lowest vertebrate (1–3). In the TAATC T is present in 5 -flanking regions of several pineal course of vertebrate evolution, the physiological role of the genes such as rat arylalkylamine-N-acetyltransferase and human pineal gland has been changed from a photosensory organ to a hydroxyindole-O-methyltransferase genes (29). Recently, it has photoendocrinal organ in the lower vertebrates and eventually to been reported that circadian gene expression in the zebrafish pineal complex requires Otx5, which is closely related to Crx a neuroendocrinal organ in mammals (4, 5). Generally, the retina ͞ receives visual images and transmits them to the brain, whereas (31). These studies suggest that a member of the Crx Otx family the primary role of the pineal gland is the rhythmic production and its binding site(s) play a common role in the transcription of of circulating melatonin, which regulates numerous physiological

activities (6). Despite such a dynamic change in the physiological This paper was submitted directly (Track II) to the PNAS ofﬁce. function, the pineal gland displays many similarities to the retina ͞ Abbreviations: rh, rhodopsin; Nrl, neural retina leucine zipper; NRE, Nrl response element; in tissue cellular morphology and biochemical properties (7, 8). Crx, cone rod homeobox; exorh, exo-rhodopsin; PIPE, pineal expression-promoting ele- In fish, amphibians, and lacertilian reptiles, their pineal photo- ment; EGFP, enhanced GFP; dpf, days postfertilization; hpf, hours postfertilization. receptor cells possess well developed and lamellar outer seg- Data deposition: The sequences reported in this paper have been deposited in the GenBank ments, which are homologous to the outer segments of retinal database (accession nos. AB079551 and AB079552). photoreceptor cells. These photoreceptor cells form an ordered *Y.A. and H.M. contributed equally to this work. layer structure in both tissues. Biochemically, the retinal pho- †Present address: Department of Molecular and Cellular Biology, Harvard University, totransduction proteins such as opsin, ␣-subunit of transducin, Cambridge, MA 02138. arrestin, and recoverin also have been localized in the pineal ‡To whom correspondence should be addressed. E-mail: [email protected].

15456–15461 ͉ PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.232444199 Downloaded by guest on September 24, 2021 Fig. 1. The proximal promoter sequences of exorh and rh genes of vertebrates. The upstream sequence of the zebrafish exorh (zEx) was aligned with those of rh genes of the zebrafish (zRh), Xenopus (xRh), chicken (cRh), mouse (mRh), rat (rRh), bovine (bRh), and human (hRh). The upstream sequence of the European eel exorh (eEx) was determined in the present study and included in the alignment. Nucleotides conserved among at least six sequences are shown with white characters on black backgrounds. Horizontal lines indicate TATA box, ATG initiation codon, and conserved cis-elements identified previously in the rh promoters. Potential Crx͞Otx-binding sites found in the zebrafish exorh promoter are double-lined. The PIPE sequence in the zebrafish exorh gene is boxed. The nucleotide numbers are relative to the translation initiation site of the zebrafish exorh gene. Accession numbers of the sequences obtained from GenBank are U23808 (xRh), M98497 (cRh), M55171 (mRh), U22180 (rRh), and U49742 (hRh). The bRh sequence was obtained from the original paper (17).

both pineal and retinal genes. It should be stressed, however, that to confirm the sequence with no PCR error. In a similar manner, the transcriptional regulation operated by Crx͞Otx is inadequate a 238-bp region upstream from the ATG initiation codon of the to explain the mechanism segregating the pineal- and retina- European eel exorh gene (GenBank accession no. AB079552) specific gene expression. As yet unidentified transcription fac- was obtained from the eel genomic DNA by using the LA PCR tor(s) and cis-acting element(s) should strictly determine pineal- in vitro Cloning Kit and sequenced. On the other hand, screening specific gene expression, most probably in combination with the of ␭-Fix II zebrafish genomic library resulted in isolation of a Crx͞Otx-dependent regulation. 10.5-kbp fragment containing a 4,558-bp sequence upstream of We previously demonstrated that the zebrafish has two dis- the rh coding region. tinct rhodopsin genes that are highly similar in coding sequence to each other (74% identical) but show unique tissue distribu- Microinjection and Generation of Germ-Line Transgenic Zebrafish. tions (32). One is the canonical rh gene expressed only in the Multiple DNA constructs were generated for microinjection (see retina and the other is exo-rhodopsin (exorh) gene expressed Results and Supporting Methods, which is published as supporting specifically in the pineal gland. A phylogenetic analysis indicated information on the PNAS web site, www.pnas.org). They were that rh and exorh genes were produced by gene duplication that prepared with the Plasmid Midi Kit (Qiagen, Chatsworth, CA). occurred early in the ray-finned fish lineage (32). Such a close Rh(Ϫ1084), Rh(Ϫ1084)͞PIPE, and PIPE-Rh(Ϫ1084) were kinship between the two genes, together with their specificities treated with SacI, and the other constructs were treated with in tissue distribution, prompted us to investigate the evolutionary EcoRI for linearization of each plasmid (cut at a unique site scenario of tissue-specific promoters. Because the proximal located upstream of the promoter). The linearized DNA was promoter sequences of rh genes are highly conserved among purified by phenol-chloroform extraction and subsequently by vertebrates including the zebrafish (33), we expected that the chloroform extraction and precipitated with ethanol. The puri- zebrafish exorh promoter sequence should provide an invaluable fied DNA was dissolved at a final concentration of 25 ng͞␮l information about the mechanism segregating the pineal- and either in distilled water containing 0.05% phenol red (for retina-specific gene expression. The zebrafish is an excellent transient expression assay) or in 0.1 M KCl͞0.05% phenol red animal model suitable for an in vivo promoter analysis with an solution (for production of transgenic fish). Each DNA construct (E)GFP reporter gene, because of its feasibility of transgenesis was microinjected into one-cell-stage embryos of WT zebrafish and transparency of embryos and larvae (34, 35). Taking into by using Transjector 5246 and Micromanipulator 5171 (Eppen- account these advantages, the present study undertook the dorf). F0 founder fish were identified by PCR analysis of the isolation and in vivo analyses of the zebrafish exorh promoter, genomic DNA pool of 2- to 3-day-old F1 embryos with primers and we identified a previously uncharacterized cis-acting ele- specific for the enhanced GFP (EGFP) coding sequence. We ment mediating the pineal-specific gene expression. We named established three, four, three, one, and three independent trans- it pineal expression-promoting element (PIPE). genic lines of the zebrafish with DNA constructs Ex(Ϫ1055), Ex(Ϫ301), Ex(Ϫ147), Rh(Ϫ1084), and Rh(Ϫ1084)͞PIPE, Materials and Methods respectively. ,Isolation of 5؅-Flanking Regions of the Zebrafish exorh, Zebrafish rh and European Eel exorh Genes. A 1,076-bp region upstream from Results the ATG initiation codon of the zebrafish exorh gene (GenBank Characterization of the 5؅-Flanking Region of the Zebrafish exorh BIOLOGY accession no. AB079551) was obtained by three rounds of Gene. We isolated a 1.1-kbp genomic DNA fragment upstream of PCR-based genome walking with the LA PCR in vitro Cloning the coding region of the zebrafish exorh gene (Fig. 5, which is DEVELOPMENTAL Kit (Takara Shuzo, Kyoto). Subsequent PCR with zebrafish published as supporting information on the PNAS web site). The genome and a pair of primers (5Ј-GCTCA GCTGG CAGTA putative promoter sequence contained five copies of TA- CTACC-3Ј and 5Ј-GCAGC TTCTT GTGCT GCACC-3Ј) am- ATCC͞T sequence, a potential binding site of Crx͞Otx (22, 24) plified a genomic fragment containing a 1,055-bp upstream that contributes to the pineal gene expression (25, 29–31). When region together with a short coding sequence of the zebrafish the sequence in the proximal promoter region (Ϫ175 to Ϫ1 exorh gene. The amplified product was then subcloned into having three potential sites for Crx͞Otx-binding) was aligned pCR2.1-TOPO vector (Invitrogen). Six clones were obtained with those of the rh promoters from various vertebrates (Fig. 1), from three independent amplification reactions and sequenced we found a single conserved site for Crx͞Otx-binding at position

Asaoka et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15457 Downloaded by guest on September 24, 2021 Ϫ142 to Ϫ137 (inverted form). Another site (Ϫ90 to Ϫ85) was same construct. The length-dependent change in signal intensity conserved in the fish rh and exorh genes, and the third site (Ϫ99 suggests that the region between Ϫ1055 and Ϫ148 contributes to to Ϫ94) was found only in the zebrafish exorh gene (Fig. 1). A enhancement of exorh gene expression because of the two TATA-like sequence in the zebrafish exorh promoter was aligned putative Crx͞Otx-binding sites (Fig. 5) and͞or unknown ele- with TATA box conserved among rh genes. On the other hand, ments in this region. Nrl response element (NRE) that is present in many rh genes (Fig. 1) and is important for retinal gene expression was not Internal Deletion and Mutational Analyses Revealed a Positive Reg- found in the zebrafish exorh promoter (Fig. 1 and Fig. 5). ulatory Element in the exorh Promoter. We focused on identification of the pineal-specific cis-acting element(s) presumably A 1,055-bp Fragment Upstream of the exorh-Coding Region Directs present in the short upstream region (Ϫ147 to Ϫ1) of exorh gene. Pineal-Specific Gene Expression. To investigate whether the 1.1-kbp A series of systematic deletion constructs (Exdel-1 to Exdel-8) upstream region of the zebrafish exorh promoter is sufficient to was generated by introducing consecutive 11-bp internal dele- direct pineal-specific gene expression, we generated a reporter tions (del-1 to del-8) into the promoter region of Ex(Ϫ147) construct Ex(Ϫ1055) by ligating EGFP gene to the 1,055-bp construct (Fig. 3A). These deletions cover the 88-bp region fragment upstream from the exorh translation initiation site (Fig. (Ϫ147 to Ϫ60) of the exorh proximal promoter without any 2A) and established three lines of the transgenic zebrafish having overlap nor gap between the neighbors. Embryos microinjected this construct. The transgenic larvae from every line at 7 days with each construct were raised to 5 dpf, and we scored the postfertilization (dpf) showed EGFP fluorescence signals in the percentage of the larvae expressing EGFP fluorescence signals pineal gland with no detectable signal in the other tissues (Fig. in the pineal gland. In this transient expression assay, the 2 B and C). Each of the EGFP-positive pineal cells displayed parental construct Ex(Ϫ147) induced the pineal EGFP expres- highly differentiated morphology with an outer segment-like sion in 40% of 166 larvae examined (Fig. 3A), and six of the eight extrusion (Fig. 2D), a structure characteristic of the (pineal) deletion constructs showed lower percentages (3–23%). Among photoreceptor cell (36). No EGFP signal was detectable within these, Exdel-8 (23%) lacks the TATA-like sequence (Fig. 3A), another type of the pineal neurons that project their axons and Exdel-1 (11%), Exdel-5 (3%) and Exdel-6 (9%) carry a outside the pineal gland (37), and this pineal projection neuron deletion of a potential Crx͞Otx-binding site (Fig. 3A), an ele- never emitted EGFP signals throughout maturation (data not ment that is important for the gene expression in the pineal gland shown). On the other hand, strong EGFP signals were sustained as well as in the retina (25, 29–31). Notably, the promoter in the photoreceptor-like pineal cells of matured transgenic fish activities were significantly reduced in Exdel-2 (7%) and Exdel-3 (6 months old; Fig. 2E), indicating that the 1,055-bp fragment (14%), of which the deleted sequences in tandem (see Fig. 3B) upstream from the exorh translation initiation site is sufficient to showed no homology with any known regulatory element, and it maintain pineal-specific gene expression in the zebrafish. is most probable that the 22-bp region (from Ϫ136 to Ϫ115) Developmental change in EGFP expression pattern was ex- contains a positive regulatory element(s) indispensable for amined in Ex(Ϫ1055) transgenic embryos at earlier stages. The pineal-specific gene expression. EGFP-positive cell in the pineal gland was first detected at To define the element(s), the parental construct Ex(Ϫ147) around 26 hours postfertilization (hpf; Fig. 2 F and G), which is was mutated in the 22-bp region to generate another series of consistent with the previous study showing the differentiation of constructs (Exmut-1 to Exmut-7), in each of which consecutive the pineal photoreceptor cells at a similar timing (38). Intensity 3–4 nucleotides were systematically mutated (Fig. 3B). Every of the pineal EGFP fluorescence signals became stronger at construct was microinjected into the zebrafish embryos for the 43 hpf (Fig. 2 H and I). Although weak fluorescence signals were transient expression assay at 5 dpf, and we observed strikingly observed in the ventral retina at 43 hpf (Fig. 2 H and I), the reduced promoter activities in the four constructs, Exmut-4 to retinal signals disappeared by 7 dpf (Fig. 2 J and K) and were not Exmut-7 (Fig. 3B) that have mutations at the 12-bp cluster (Ϫ126 observed at any later stages. These results indicated that the to Ϫ115) covering del-3 region together with an end of del-2 1,055-bp region of the exorh promoter should be a good target sequence. On the other hand, mutations at positions covering for investigating the regulatory mechanism of pineal-specific most of del-2 region (Ϫ136 to Ϫ127) had less effect on the gene expression. promoter function (Fig. 3B, Exmut-1, -2, and -3). These results of the transient expression assay altogether demonstrate that the The 147-bp Proximal Region of the Zebrafish exorh Promoter Is pineal(-specific) expression of exorh gene highly depends on the Sufficient for Pineal-Specific Gene Expression. To localize cis-acting 12-bp DNA sequence TGACCCCAATCT (Ϫ126 to Ϫ115), DNA element(s) required for the pineal-specific gene expres- which we termed PIPE in this study. sion, we generated two reporter constructs, Ex(Ϫ301) and Ex(Ϫ147), by ligating EGFP gene to 301-bp and 147-bp frag- PIPE Can Direct Gene Expression in Pineal Photoreceptor Cells. PIPE ments of the exorh promoter, respectively (see Fig. 6A, which is may govern directly the pineal-specific gene expression in co- published as supporting information on the PNAS web site). operation with Crx͞Otx-binding sites or, alternatively, enhance Then, the transgenic zebrafish were established for Ex(Ϫ301) the promoter activity without contributing to the tissue-specific and Ex(Ϫ147), respectively. Both of the constructs drove the regulation. Considering these possibilities, we examined the pineal-specific EGFP expression in the transgenic animals (Fig. effect of ectopic placement of PIPE on expression of a gene 6 D–G), and the EGFP-positive cells in these Ex(Ϫ301) and regulated by the rh promoter that structurally resembles the Ex(Ϫ147) transgenic larvae showed morphological features of exorh promoter (Fig. 1) but probably drives retina-specific gene the pineal photoreceptor cell (data not shown). The number of expression in the zebrafish (32). As a control parental construct, the EGFP-positive cells within each pineal gland of these larvae we produced a reporter Rh(Ϫ1084), in which 1,084 bps upstream was also indistinguishable from that of Ex(Ϫ1055) transgenic of the zebrafish rh coding region were fused to the EGFP gene larvae. These results demonstrate that the short promoter region (Fig. 4A), and it was used to generate transgenic fish. Rh(Ϫ1084) of exorh gene (Ϫ147 to Ϫ1) retains the DNA element(s) essential transgenic larvae displayed strong EGFP fluorescence signals for the pineal cell-specific gene expression. Among these larvae, exclusively in the eyes, and no detectable signal was observed in the intensity of the pineal fluorescence signal tended to decrease the pineal gland (Fig. 4 C and D). A detailed examination of as the promoter region of the transgene became shorter (Fig. 6 tissue sections prepared from the transgenic larvae revealed C, E, and G), although the signal intensity varied slightly among localization of EGFP expression in the rod photoreceptor cells individuals from different lines that were produced with the (data not shown), consistent with the previous study that used a

15458 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.232444199 Asaoka et al. Downloaded by guest on September 24, 2021 Fig. 3. Effects of internal deletions (A) and site-directed mutations (B)inthe exorh promoter sequence. (Right) Bar graphs indicate the percentage of larvae that expressed EGFP ﬂuorescence signals in the pineal gland at 5 dpf, and the number of larvae examined at 5 dpf is shown in parentheses. (A Left) The parental construct Ex(Ϫ147) and its deletion mutants (Exdel-1 to Exdel-8) are schematically depicted. Black and white boxes indicate the positions of three putative Crx͞Otx-binding sites and the TATA-like sequence, respectively. (B Left) The partial nucleotide sequences of the parental construct Ex(Ϫ147) and its seven mutant constructs, Exmut-1 to Exmut-7, with dots representing unaltered nucleotides. Numbers at the top indicate the positions of nucleotides relative to the translation initiation site.

1.2-kbp fragment of the zebrafish rh promoter (33). Then, the parental construct Rh(Ϫ1084) was modified to carry an ectopic PIPE sequence by introducing four nucleotide substitutions and one nucleotide insertion (Fig. 4B) to a region that is aligned with PIPE in the exorh promoter (Fig. 1). Three independent lines of transgenic zebrafish were established with this chimeric construct Rh(Ϫ1084)͞PIPE, and all of the transgenic larvae (at 7 dpf) exhibited EGFP fluorescence signals in the pineal gland in addition to those in the eyes (Fig. 4 E and F), with no detectable signal in the other tissues. The expression pattern of EGFP signals in the pineal gland was indistinguishable from those observed in Ex(Ϫ1055), Ex(Ϫ301), and Ex(Ϫ147) transgenic larvae. The rh promoter acquired the ability to drive ectopic gene expression in the pineal gland by virtue of the mutational change of only several nucleotides, supporting the idea that newly created PIPE is functional as a cis-acting DNA element that induces pineal-specific gene expression. No significant difference was observed in retinal fluorescence signals between Rh(Ϫ1084) and Rh(Ϫ1084)͞PIPE transgenic animals, and this eliminates a negative regulatory role of PIPE for retinal gene Fig. 2. Pineal-speciﬁc EGFP expression in Ex(Ϫ1055) transgenic zebraﬁsh. (A) expression. Schematic representation of Ex(Ϫ1055) construct in a linearized form used for Finally, we asked whether the above mutations introduced into

microinjection. (B) Dorsolateral views (bright field image) of a WT larva Rh(Ϫ1084) might have disrupted an unknown DNA element(s) BIOLOGY (upper) and Ex(Ϫ1055) transgenic larva (lower) at 7 dpf. (C) Fluorescent image of B. The transgene-dependent fluorescence signal was observed specifically DEVELOPMENTAL in the pineal gland of Ex(Ϫ1055) transgenic larva (arrowhead), and autofluo- rescence signals observed in transgenic and WT fish are marked by arrows. (D) (G and I). Arrowheads in G and I indicate EGFP-positive cells in the pineal High-magnification confocal image (dorsal view) of EGFP-positive pineal cells gland, and arrows in I point to EGFP-positive cells in the retina. The embryos of 7-dpf-larva, with anterior to the left. The outer segment-like extrusion is in G and I were photographed under the same exposure conditions. (J) indicated by each arrowhead. (E) Dorsal view of Ex(Ϫ1055) transgenic adult 4Ј,6-diamidino-2-phenylindole staining of a 10-␮m-thick cross-section of the fish illuminated with both tungsten lamp and blue light. EGFP fluorescence head of Ex(Ϫ1055) transgenic larva at 7 dpf. (K) EGFP fluorescent image of J. signals were observed only in the pineal gland throughout its life. (F–I) Frontal [Bars ϭ 1mm(B and C), 10 ␮m(D),2mm(E), and 100 ␮m(F–K).] For a clear views (dorsal up) of living transgenic embryos observed at 28 hpf (F and G)or demonstration, pigmentation of the embryos and larvae was reduced by 43 hpf (H and I) by using Nomarski optics (F and H)orfluorescence microscopy treatment with 0.003% 1-phenyl-2-thiourea (Nacalai Tesque, Kyoto).

Asaoka et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15459 Downloaded by guest on September 24, 2021 advantage of zebrafish transgenesis technology. This may represent an excellent example of zebrafish transgenesis to delineate a cis-acting DNA element in vivo, and the identification of PIPE corroborates the prominent aspect of the strategy (39). We used the rapidity of the transient expression assay for scanning the exorh promoter region (Fig. 3) while we established multiple transgenic lines to prove eventually the crucial role of PIPE in the pineal-specific gene expression (Fig. 4). Our systematic deletion analysis revealed that, just like regulation of the rh promoter, the pineal-specific activity of the exorh promoter required the putative Crx͞Otx-binding sites to be present in the proximal region (Fig. 3A). This finding is consistent with previous studies on several genes expressed in the pineal gland of the rat, mouse, chicken, and zebrafish (25, 29–31). In addition to the Crx͞Otx-binding sites, our analysis identified PIPE as a major contributor to the pineal-specific gene expression, suggesting strongly that the pineal-specific expression of the zebrafish exorh gene is mediated by a combination of those cis-acting elements and presumably by multiple transcription factors. Such a mode of transcriptional regulation operated by multiple cis-elements and transcription factors is generally seen in a variety of tissue-specific promoters including the rh promoter, which contains a number of evolutionarily conserved cis-acting elements such as BAT-1, Ret-4, and NRE (16, 17, 20, 21). Indeed, rh gene is transactivated cooperatively by Crx and Nrl proteins through these sites (22). We speculate that the Crx͞Otx-binding site would provide a basis for photoreceptor cell (cell type)-specific expression in both the retina and pineal gland, whereas NRE and PIPE would serve for determination of tissue-specificity, i.e., expression in the retina and pineal gland, respectively. It is possible that PIPE also plays a role in transcriptional regulation of other genes expressed in the zebrafish pineal gland. For example, we found a 12-bp DNA sequence (TGAC- CCCTCTCT) similar to PIPE in the proximal promoter region of the zebrafish floating head, an important gene that is expressed Fig. 4. Ectopic gene expression in the pineal gland driven by the zebrafish from early stages in the pineal gland and is required for rh chimeric promoter carrying the PIPE sequence. (A) Schematic representa- differentiation of most pineal neurons (37). On the other hand, tion of the constructs, Rh(Ϫ1084), Rh(Ϫ1084)͞PIPE, and PIPE-Rh(Ϫ1084). (B) Comparison of PIPE and nearby sequence in Ex(Ϫ1055) with those in the PIPE or PIPE-related sequence is not found in a 2.1-kbp corresponding region of Rh(Ϫ1084) or Rh(Ϫ1084)͞PIPE. Bold characters in fragment upstream of the chicken pineal-specific gene, pinopsin Rh(Ϫ1084)͞PIPE represent five nucleotides (four substitutions and a single (12, 40), and PIPE may be present at a more distal region or insertion) modified from Rh(Ϫ1084) to create an ectopic PIPE sequence. (C–F) downstream of the translation initiation site. Further sequence Dorsal views (anterior up) of Rh(Ϫ1084) (C and D) and Rh(Ϫ1084)͞PIPE (E and analysis of pineal-specific genes in other vertebrates as well as in F) transgenic larvae at 7 dpf. Nomarski (C and E) and fluorescent (D and F) the zebrafish should help to evaluate the general role of PIPE in images were taken at the same focal plane without moving the larvae. EGFP pineal-specific gene expression. Ϫ ͞ fluorescence signals in the pineal gland of Rh( 1084) PIPE transgenic larva PIPE is most likely recognized by the transcription factor(s) are marked by an arrowhead (F). (F Inset) High-magnification image of the expressed specifically in the pineal gland, but none of the EGFP-positive pineal structure. The larvae in C–F were not treated with 1- phenyl-2-thiourea, so that strong EGFP fluorescence signals in the pigmented pineal-specific transcription factors has been identified yet. One eyes (D and F) are only visible through the pupil. [Bars ϭ 100 ␮m(C–F), 20 ␮m noticeable feature of PIPE is a CCAAT motif (Fig. 5), a binding (F Inset).] site for many transcription factors such as CBF͞NF-Y, CTF͞ NF1, and C͞EBP (41). Thus, it is possible that PIPE serves as a functional CCAAT box for the pineal-specific expression. This responsible for pineal-specific repression of the rh gene expres- idea is, however, at odds with our sequence analysis of the exorh sion. To demonstrate the positive regulatory role of PIPE more promoter of the European eel, Anguilla anguilla, in which a directly, we generated an additional chimeric construct PIPE- Ϫ Ϫ CCAAT motif is not found at the equivalent site (Fig. 1). The Rh( 1084), which has one copy of PIPE upstream of Rh( 1084) consensus sequence between the zebrafish PIPE and the PIPE- A Ϸ construct (Fig. 4 ). In transient expression assay, 60% of like element in the eel exorh is TGACCNNAATCN. The con- larvae injected with PIPE-Rh(Ϫ1084) expressed EGFP fluores- served nine nucleotides rather than the CCAAT motif might be cence signals both in the pineal gland and in the retina at 5 dpf. Control injection of the parental construct Rh(Ϫ1084) induced essential for binding of a common transcription factor(s), al- no detectable expression of EGFP signals in the pineal gland at though it remains to be determined whether the PIPE-like 5 dpf. Together, these results demonstrate that PIPE can direct element in the eel exorh promoter serves for pineal-specific gene expression in the pineal gland. We concluded that PIPE is expression. the major contributor to the pineal-specific gene expression We previously suggested that teleost exorh and rh genes were mediated by the zebrafish exorh promoter. produced from an ancestral gene by gene duplication that occurred in the ray-finned fish lineage before the teleost radi- Discussion ation (32). It is conceivable that the ancestral gene was expressed In the present study, we accomplished detailed analyses of the in both the retina and pineal gland, and that the two descendent exorh promoter without assistance of in vitro assay, by taking genes, rh and exorh genes, became segregated from one another

15460 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.232444199 Asaoka et al. Downloaded by guest on September 24, 2021 with respect to the localization and function. This possibility is the eel exorh promoter has a sequence highly similar to NRE at consistent with the duplication-degeneration-complementation a site corresponding to that of rh NRE (Fig. 1), although no (DDC) model (42), which predicts that complementary non- NRE-like sequence is found in the zebrafish exorh promoter. functionalization of the cis-acting elements can produce the These observations seem to support the idea that a functional spatio-temporal partitioning of the ancestral functions between NRE was originally present in the ancestral exorh promoter and the descendents. The present study showed complementary then lost in the Euteleost lineage after the divergence between absence of the cis-elements, PIPE and NRE, in the proximal the Anguilliformes and the Euteleostei. Further study of the promoters of the zebrafish rh and exorh genes, respectively, as molecular basis for similarity and difference between the pineal predicted by the DDC model. This model leads to the specula- and retinal gene expression could provide an important insight tion that the promoter region of the common ancestor of rh and into the evolution of the vertebrate photoreceptive organs. exorh genes formerly contained both PIPE and NRE. Subse- quent loss of a functional PIPE in the rh promoter could restrict We thank Dr. K. Kawakami (National Institute of Genetics) for helpful the rh gene expression to the retina, whereas loss of NRE might advice about maintenance of fish. We thank Dr. H. Okamoto (Brain lead to the pineal-specific expression of exorh gene. Consistently, Science Institute, RIKEN) for providing the zebrafish strain Michigan a sequence comparison revealed significant homology in nucle- and the zebrafish genomic library. We are grateful to Dr. David R. Hyde (University of Notre Dame) for his kind gifts of antibodies to the otide sequence between PIPE and the equivalent site in the zebrafish opsins. We also thank Dr. T. Okano for helpful comments and zebrafish rh promoter (Fig. 1), and experimentally we showed discussion and K. Imazato for assistance with fish maintenance. This that only several nucleotide changes within the site of the rh work was supported in part by Grants-in-Aid from the Japanese Ministry promoter induced the pineal expression (Fig. 4). Interestingly, of Education, Science, Sports, and Culture.

1. Pu, G. A. & Dowling, J. E. (1981) J. Neurophysiol. 46, 1018–1038. 24. Furukawa, T., Morrow, E. M. & Cepko, C. L. (1997) Cell 91, 531–541. 2. Cole, W. C. & Youson, J. H. (1982) Am. J. Anat. 165, 131–163. 25. Furukawa, T., Morrow, E. M., Li, T., Davis, F. C. & Cepko, C. L. (1999) Nat. 3. Tamotsu, S. & Morita, Y. (1986) J. Comp. Physiol. A 159, 1–5. Genet. 23, 466–470. 4. Collin, J. P., Voisin, P., Falcoń, J., Faure, J. P., Brisson, P. & Defaye, J. R. 26. Mathers, P. H., Grinberg, A., Mahon, K. A. & Jamrich, M. (1997) Nature 387, (1989) Arch. Histol. Cytol. 52, 441–449. 603–607. 5. Shedpure, M. & Pati, A. K. (1995) Indian J. Exp. Biol. 33, 625–640. 27. Martinez, J. A. & Barnstable, C. J. (1998) Biochem. Biophys. Res. Commun. 250, 6. Falcoń, J. (1999) Prog. Neurobiol. 58, 121–162. 175–180. Biol. Cell 89, 7. Meissl, H. (1997) 549–554. 28. Kimura, A., Singh, D., Wawrousek, E. F., Kikuchi, M., Nakamura, M. & 8. Kusmic, C. & Gualtieri, P. (2000) Micron 31, 183–200. Shinohara, T. (2000) J. Biol. Chem. 275, 1152–1160. 9. Mirshahi, M., Faure, J. P., Brisson, P., Falcoń, J., Guerlotte, J. & Collin, J. P. 29. Li, X., Chen, S., Wang, Q., Zack, D. J., Snyder, S. H. & Borjigin, J. (1998) Proc. (1984) Biol. Cell 52, 195–198. Natl. Acad. Sci. USA 95, 1876–1881. 10. Vigh-Teichmann, I. & Vigh, B. (1990) J. Pineal Res. 8, 323–333. 11. Korf, H. W., White, B. H., Schaad, N. C. & Klein, D. C. (1992) Brain Res. 595, 30. Bernard, M., Dinet, V. & Voisin, P. (2001) J. Neurochem. 79, 248–257. 57–66. 31. Gamse, J. T., Shen, Y. C., Thisse, C., Thisse, B., Raymond, P. A., Halpern, M. E. 12. Okano, T., Yoshizawa, T. & Fukada, Y. (1994) Nature 372, 94–97. & Liang, J. O. (2002) Nat. Genet. 30, 117–121. 13. Yoshikawa, T., Yashiro, Y., Oishi, T., Kokame, K. & Fukada, Y. (1994) Zool. 32. Mano, H., Kojima, D. & Fukada, Y. (1999) Mol. Brain Res. 73, 110–118. Sci. 11, 675–680. 33. Kennedy, B. N., Vihtelic, T. S., Checkley, L., Vaughan, K. T. & Hyde, D. R. 14. Livesey, F. J. & Cepko, C. L. (2001) Nat. Rev. Neurosci. 2, 109–118. (2001) J. Biol. Chem. 276, 14037–14043. 15. Morabito, M. A., Yu, X. & Barnstable, C. J. (1991) J. Biol. Chem. 266, 34. Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y. & Eguchi, G. (1997) Dev. 9667–9672. Biol. 192, 289–299. 16. DesJardin, L. E. & Hauswirth, W. W. (1996) Invest. Ophthalmol. Visual Sci. 37, 35. Long, Q., Meng, A., Wang, H., Jessen, J. R., Farrell, M. J. & Lin, S. (1997) 154–165. Development (Cambridge, U.K.) 124, 4105–4111. 17. Chen, S. & Zack, D. J. (1996) J. Biol. Chem. 271, 28549–28557. 36. Kusmic, C., Barsanti, L., Passarelli, V. & Gualtieri, P. (1993) Micron 24, 18. Swaroop, A., Xu, J., Pawar, H., Jackson, A., Skolnick, C. & Agarwal, N. (1992) 279–286. Proc. Natl. Acad. Sci. USA 89, 266–270. 37. Masai, I., Heisenberg, C. P., Barth, K. A., Macdonald, R., Adamek, S. & 19. Liu, Q., Ji, X., Breitman, M. L., Hitchcock, P. F. & Swaroop, A. (1996) Wilson, S. W. (1997) Neuron 18, 43–57. Oncogene 12, 207–211. 38. Heisenberg, C. P., Brand, M., Jiang, Y. J., Warga, R. M., Beuchle, D., van 20. Kumar, R., Chen, S., Scheurer, D., Wang, Q. L., Duh, E., Sung, C. H., Eeden, F. J. M., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, Rehemtulla, A., Swaroop, A., Adler, R. & Zack, D. J. (1996) J. Biol. Chem. 271, M., et al. (1996) Development (Cambridge, U.K.) 123, 191–203. 29612–29618. 21. Rehemtulla, A., Warwar, R., Kumar, R., Ji, X., Zack, D. J. & Swaroop, A. 39. Meng, A., Tang, H., Ong, B. A., Farrell, M. J. & Lin, S. (1997) Proc. Natl. Acad. (1996) Proc. Natl. Acad. Sci. USA 93, 191–195. Sci. USA 94, 6267–6272. 22. Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gilbert, 40. Takanaka, Y., Okano, T., Yamamoto, K. & Fukada, Y. (2002) J. Neurosci. 22, D. J., Jenkins, N. A. & Zack, D. J. (1997) Neuron 19, 1017–1030. 4357–4363. 23. Freund, C. L., Gregory-Evans, C. Y., Furukawa, T., Papaioannou, M., Looser, 41. Maity, S. N. & de Crombrugghe, B. (1998) Trends Biochem. Sci. 23, 174–178. J., Ploder, L., Bellingham, J., Ng, D., Herbrick, J. S., Duncan, A., et al. (1997) 42. Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. & Postlethwait, J. Cell 91, 543–553. (1999) Genetics 151, 1531–1545. BIOLOGY DEVELOPMENTAL

Asaoka et al. PNAS ͉ November 26, 2002 ͉ vol. 99 ͉ no. 24 ͉ 15461 Downloaded by guest on September 24, 2021