Autoregulatory loop and retinoic acid repression regulate pou2/pou5f1 gene expression in the zebrafish embryonic brain

RESEARCH ARTICLE

Autoregulatory Loop and Retinoic AcidRepression Regulate pou2/pou5f1 GeneExpression in the Zebrafish Embryonic BrainMst. Shahnaj Parvin,1 Noriko Okuyama,1 Fumitaka Inoue,1 Md. Ekramul Islam,1†

Atsushi Kawakami,2 Hiroyuki Takeda,3 and Kyo Yamasu1*

Zebrafish pou5f1, also known as pou2, encodes a POU-family transcription factor that is transientlyexpressed in the prospective midbrain and anterior hindbrain during gastrulation, governing braindevelopment. In the present study, we found that the main regulatory elements reside in the proximalupstream DNA sequence from �2.2 to �0.12 kb (the �2.2/�0.1 region). The electrophoretic gel mobility shiftassay (EMSA) revealed four functional octamer sequences that can associate with zebrafish Pou2/Pou5f1.The expression of mutated reporter constructs, as well as EMSA, suggested that these four octamersequences operate in a cooperative manner to drive expression in the mid/hindbrain. We also identified aretinoic acid (RA) -responsive element in this proximal region, which was required to repress transcriptionin the posterior part of the embryo. These data provide a scheme wherein pou2/pou5f1 expression inzebrafish embryos is regulated by both an autoregulatory loop and repression by RA emanating from theposterior mesoderm. Developmental Dynamics 237:1373–1388, 2008. © 2008 Wiley-Liss, Inc.

Key words: brain formation; midbrain–hindbrain boundary; pou2; pou5f1; retinoic acid; transgenesis; zebrafish

Accepted 7 March 2008

INTRODUCTION

The POU family of transcription fac-tors is characterized by a structuralmotif called the POU domain, which iscomposed of the POU-specific domainand the POU homeodomain. Membersof this family play important roles inmany aspects of animal development,including brain formation and neuro-genesis (Schonemann et al., 1998).Mouse Oct-3/4, also known as Pou5f1,belongs to class V of the POU family.It is expressed in pluripotent cells,such as inner cell mass (ICM) cells of

the blastocyst, primordial germ cells(PGCs), and embryonic stem (ES)cells, and is thought to play pivotalroles in the maintenance of pluripo-tency (Ovitt and Scholer, 1998; Pesceet al., 1998; Niwa, 2007). Recently,Niwa and his colleagues showed in aseries of experiments, in which Oct-3/4 expression was manipulated by atetracycline-regulated transgene, thatOct-3/4 is a master regulator of pluri-potency that controls lineage commit-ment (Niwa et al., 2000; Pesce andScholer, 2001).

Zebrafish pou2 was originally iden-tified as a gene for a novel POU tran-scription factor with structural fea-tures of both class III and V factors(Takeda et al., 1994). Similar to Oct-3/4, the Pou2 protein associates withan octamer sequence (ATGCAAAT).Based on its structural characteristicsand syntenic relationship, pou2 wasrecently suggested to be a homologueof Oct3/4/Pou5f1 (Belting et al., 2001;Burgess et al., 2002). Maternally de-rived mRNA transcripts for pou2 arepresent in unfertilized eggs, and ma-

1Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama, Japan2Department of Biological Information, Tokyo Institute of Technology, Tokyo, Japan3Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, JapanGrant sponsor: the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI, Grant-in-Aid for ScientificResearch); Grant number: 10220203; Grant number: 15570170; Grant number: 17570170.†Dr. Islam’s present address is Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh.*Correspondence to: Kyo Yamasu, Division of Life Science, Graduate School of Science and Engineering, Saitama University,255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. E-mail: [email protected]

DOI 10.1002/dvdy.21539Published online 11 April 2008 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 237:1373–1388, 2008

© 2008 Wiley-Liss, Inc.

ternal and/or zygotic expression iswidely observed until gastrulation. Bythe end of epiboly, pou2 expression isrestricted to the prospective midbrainand anterior hindbrain, and finallydisappears from the brain duringearly somitogenesis (Takeda et al.,1994; Hauptmann and Gerster, 1995).The overall temporal and spatial ex-pression pattern of Oct-3/4 in mice issimilar to that of pou2 in zebrafish.Oct-3/4 is also ubiquitously expressedin early embryos, both maternally andzygotically, after which it is restrictedto ICM cells, then to epiblast cells, andfinally to PGCs alone (Ovitt and Scho-ler, 1998; Pesce et al., 1998).

Genetic studies showed that pou2 isdisrupted in the spiel-ohne-grenzen(spg) mutant, which does not developthe midbrain–hindbrain boundary(MHB) /isthmus and cerebellum(Schier et al., 1996; Belting et al.,2001; Burgess et al., 2002). The MHBis a potent signaling center that orga-nizes the development of the midbrainand cerebellum. Its positioning in theneural plate depends on the interac-tion of otx2 and gbx1/2, which are ex-pressed in the future fore/midbrainand hindbrain, respectively (Wassefand Joyner, 1997; Kikuta et al., 2003;Rhinn et al., 2003; Hidalgo-Sanchez etal., 2005). At the expression boundaryof these two genes, Fgf8, Pax2, andWnt1 are independently induced nearthe end of gastrulation; this event es-tablishes the MHB and leads to theactivation of downstream genes suchas En and other MHB genes (Naka-mura, 2001; Rhinn and Brand, 2001).This gene cascade, which is conservedin all vertebrates examined thus far,is thought to promote and maintainthe formation of the MHB/isthmic re-gion.

The expression of pou2 in the ze-brafish embryonic brain overlaps thatof pax2a around the MHB. Further-more, pax2a expression is down-regu-lated in spg embryos, whereas pou2expression is not affected in no isth-mus embryos, which display a defectin pax2a. These results indicate thatpou2 is required for pax2a activationin the prospective MHB (Belting et al.,2001; Burgess et al., 2002). Indeed,the MHB enhancer of mouse Pax2 isrecognized by Oct-3/4 (Pfeffer et al.,2002). Meanwhile, spg mutant em-bryos show normal expression of otx2

and gbx1, suggesting that pou2 oper-ates downstream of these genes andcontributes to the establishment ofthe MHB by means of the direct reg-ulation of pax2a.

In addition to defects in the MHB,spg mutant embryos show abnormalmorphology and boundaries of rhom-bomeres in the hindbrain, indicatingthat spg/pou2 is also involved in hind-brain segmentation. Indeed, pou2 istransiently expressed in rhom-bomeres r2 and r4 near the end ofepiboly (Hauptmann and Gerster,1995). In the prospective hindbrain,each rhombomere is specified by acombination of Hox genes (Hox code)and additional regulatory genes, suchas krox20 and velentino (val)/mafB(Theil et al., 2002; Wiellette and Sive,2003). During the establishment ofrhombomeres, pou2 may function up-stream of krox20 and val, leadingto the formation of a gene networkthat controls hindbrain segmentation(Hauptmann et al., 2002b).

In contrast, although a great deal isknown regarding the role of Oct-3/4 inearly mouse embryos and the mainte-nance of ES/EC cells, its role in brainformation remains unclear. In mouseembryos, Oct-3/4 is broadly expressedin the neural plate, and Oct-3/4 over-expression in zebrafish spg embryosrestores MHB development (Scholeret al., 1990; Reim and Brand, 2002).Interestingly, it was recently shownthat class V genes of chick and Xeno-pus are expressed in the anteriorbrain similar to pou2 (Morrison andBrickman, 2006; Lavial et al., 2007).These results raise a possibility thatclass-V POU genes of other verte-brates, including Oct-3/4, are also in-volved in brain development.

To fully understand a given regula-tory network, we must elucidate therole of genes that occupy node posi-tions. In the case of mouse Oct-3/4,several reports identified three regu-latory regions (Ovitt and Scholer,1998; Niwa, 2007): the proximal pro-moter, the proximal element (PE), andthe distal element (DE). The proximalpromoter mediates general activationby means of Sp1/Sp3 and repressionby means of retinoic acid (RA). The PEdrives Oct-3/4 expression in the epi-blast and embryonal carcinoma (EC)cells, whereas the DE directs expres-sion in the ICM, ES cells, and PGCs

(Yeom et al., 1996; Niwa, 2007). TheDE also mediates regulation by thecooperative function of Oct-3/4 andSox2, allowing for the formation of anautoregulatory loop (Okumura-Na-kanishi et al., 2005), which is furthersuppressed by Cdx2 that is known tooperate in the differentiation of thetrophoectoderm (Niwa et al., 2005).

Despite the essential role played bypou2 in zebrafish brain formation,however, the regulatory mechanismgoverning its unique and dynamic ex-pression pattern in the brain region isunknown. We used a combination ofreporter analyses and electrophoreticmobility shift assays (EMSA) to iden-tify flanking regulatory DNA se-quences and determine their roles inpou2 regulation. Our results suggestthat pou2 is regulated by means of apositive autoregulatory loop and RA-mediated repression.

RESULTS

Genomic Organization ofZebrafish pou2

A zebrafish genomic library wasscreened by means of plaque hybrid-ization using full-length pou2 cDNAas a probe (Takeda et al., 1994), whichgave rise to four clones (�EP2- �EP5,Fig. 1A and data not shown) that har-bored the pou2 gene. To obtain flank-ing genomic regions far from pou2, wescreened a bacterial artificial chromo-some (BAC) library by means of poly-merase chain reaction (PCR) and ob-tained two BAC clones (233P11,237B24) from which several EcoRIfragments were cloned that encom-passed the flanking region from �24.3to �33.5 (Fig. 1A). The transcriptionalinitiation site was determined by 5�-rapid amplification of cDNA ends(RACE) using total RNA from two- tofour-cell stage embryos. Two tran-scriptional initiation sites were iden-tified at 236 and 197 bp upstream ofthe ATG codon. The relative amountof the RACE products indicated thatthe former site was the primary initi-ation site (Fig. 2). A close view of theupstream sequence revealed four oc-tamer sequences (typically ATG-CAAAT; OS1-4), which are consideredthe binding sites for many POU familytranscription factors (Phillips andLuisi, 2000), as well as a DR2-type

1374 PARVIN ET AL.

RA-responsive element (P2-RARE)that may be the cis-element respon-sive to the RA signal (Ross et al.,2000).

Structural analyses demonstratedthat the gene is composed of five exonsand four introns, as was shown previ-ously for Oct-3/4 (Yeom et al., 1991)(Fig. 1); the two most 5�-terminal in-trons interrupt the POU-specific do-main, the third is located within thelinker, and the last one interrupts thePOU-homeodomain coding region.Comparison with other POU family

genes shows that zebrafish pou2 andmammalian Pou5f1 (human, mouse,and bovine) have very similar exon–intron structures, whereas othersshow no similarity (Fig. 1B,C). Inpou2 and mammalian Pou5f1, the sec-ond to fourth intron divide the gene atexactly the same sites relative to thePOU-specific domain and the POU ho-meodomain, and the site of the firstintron differs by no more than three

codons. Furthermore, the phases of in-tron insertion into the coding regionare identical for the four introns: thefirst, third, and fourth intron inser-tions are located between adjacentcodons, whereas the second intronsexist between the first and secondbase of the corresponding codon(phase 1). Thus, the comparison of thegenomic organization shows that pou2is closely related to mammalian

Fig. 1. Genomic organization of the zebrafish pou2/pou5f1 gene.A: The exon–intron structure of pou2 and the genomic subclones ob-tained from the � phage clones (�EP2, 5) and BAC clone (�24.3/�6.5,12.5/33.5; 237B24) that were examined for regulatory activities by co-injection experiments. Dark gray boxes and light gray boxes representcoding and noncoding sequences of exons, respectively. The bent arrowmarks the transcriptional initiation site. The region from �122 to the ATGcodon, which contains the minimal promoter and is marked with anasterisk, was shown to be unable to drive transcription (Fig. 3A). B:Comparison of the intron insertions between zebrafish pou2 and humanPOU5F1. The top panel shows the alignment of their amino acid se-quences, in which positions of the introns are shown with arrows. Preciseintronic insertions relative to codons are shown for the four sites sepa-rately in the bottom. C: Positions of intronic insertions within the proteinstructure of different POU-family members are shown with arrows. Po-sitions marked with red arrows represent the three sites that are com-pletely conserved between zebrafish pou2 and mammalian Pou5f1.

Fig. 2. The upstream DNA sequence of pou2 that drives the expressionin the brain. The upstream sequence and 5�-untranslated region areshown in black and green, respectively. Four octamer sequences (OS1–4), a DR2-retinoic acid-responsive element (P2-RARE), and the startcodon are shown with blue, orange, and red letters, respectively. Thetwo transcriptional start sites are underlined with thick black bars, andthe 5�-terminal ends of the deletion constructs examined (Fig. 3) aremarked with bent arrows. The two sequences intervening between oc-tamer sequences (IS1 between OS1 and OS2; IS2 between OS2 andOS3) are shown in lowercase letters and marked on the right.

REGULATION OF THE ZEBRAFISH POU5F1 GENE 1375

Pou5f1 (Belting et al., 2001; Burgesset al., 2002).

Upstream 6.5-kb DNA DrivesReporter Expression in theEmbryonic Brain

To identify the cis-region(s) drivingpou2 expression in the mid/hindbrainregion during gastrulation, we co-in-jected various genomic fragmentsfrom �24.3 to �33.5 (Fig. 1A) intofertilized eggs with an egfp reportergene regulated by the minimal pro-moter region of pou2 (GFP-0.1 in Fig.3A; see below). Reporter expressionwas then examined by means of greenfluorescent protein (GFP) fluorescenceduring early somitogenesis. GFP-0.1was hardly expressed on its own. Inzebrafish, the co-injection of regula-tory DNA results in correct expressionof a reporter gene under the control ofan appropriate promoter, probablydue to the rapid concatemerization ofco-injected DNA fragments (Muller etal., 1997; Woolfe et al., 2005; Islam etal., 2006; Inoue et al., 2006, 2008).Although transient expression is inev-itably associated with mosaicism andnonspecific expression, especially inyolk cells, the reporter gene showedsignificant activation in response tothe upstream DNA from �6.5 to �118(�6.5/�0.1; Table 1A). The proximalregion of this DNA from �2,303 to�118 bp (�2.3/�0.1; Figs. 1A, 4D–F)strongly activated GFP-0.1 expres-sion, whereas the distal DNA (�6.5/�2.2; Fig. 4A–C) showed a weaker ac-tivity (Table 1A). For both regions,GFP expression was first detected at50% epiboly in the dorsal region (Fig.4A,D), after which it was observedthroughout the prospective midbrainand hindbrain from the bud to earlysomite stages (Fig. 4B,C,E,F).

To confirm the results obtained byco-injection experiments, we placedthe flanking DNA from �6.5 kb to theATG codon (�6.5/ATG) upstream ofegfp (GFP-6.5, Fig. 3A), and injectedthis construct into 1-cell embryos. Asa result, a significantly large portionof embryos (47%) again showed tran-sient GFP expression in the brain dur-ing early somitogenesis (Fig. 4G). Toavoid mosaicism, which is usually ob-served in transient reporter expres-sion and renders it difficult to specifyexpression boundaries, we established

Fig. 3. Localization of the regulatory activity in the pou2 upstream DNA. A: Deletion constructsexamined for their expression in injected embryos. Positions of octamer sequences and retinoicacid-responsive elements are marked with blue and orange ovals, respectively. B: Subregions fromthe upstream DNA examined for their regulatory activities by co-injection with the GFP-0.1 reporter.A,B: Numbers of injected normal embryos and the rates of expression specifically observed in themid-hindbrain during early somitogenesis are shown on the right.

TABLE 1A. Genomic Regions Examined for Their Roles inTranscriptional Regulation: Regulatory Activities of the Genomic

Regions Flanking pou2

Regiona

Range Reporter assay

5�-end (kb) 3�-end (kb) Embryos Expressionb (%)

�24.3/�6.3 �24.3 �6.3 145 20�6.5/�0.1 �6.5 �0.12 52 44�6.5/�2.2 �6.5 �2.2 230 33�2.3/�0.1 �2.3 �0.12 153 480.7/3.6 �0.7 �3.6 102 113.4/4.4 �3.4 �4.4 124 44.4/5.4 �4.4 �5.4 96 95.3/6.5 �5.3 �6.5 106 36.4/12.4 �6.4 �12.4 114 512.4/13.3 �12.4 �13.3 61 212.5/33.5 �12.5 �33.5 108 17

aThe genomic regions examined for their regulatory activities by co-injection withGFP-0.1. The two large fragments, �24.3/�6.3 and 12.5/33.5, were obtained byEcoRI digestion from the bacterial artificial chromosome clone, whereas theremaining smaller fragments were prepared from the lambda phage clones byrestriction digestion or polymerase chain reaction.

bRates of transient reporter expression in the mid-hindbrain region at early somitestages.

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transgenic lines harboring GFP-6.5,which showed stable reporter expres-sion (Fig. 5A–H) that recapitulatedendogenous pou2 expression (Fig. 5I–L). We observed the expression of ma-ternally derived GFP in fertilized eggs(Fig. 5A), and maternal and/or zygoticexpression throughout the blastodermuntil 40% epiboly (Fig. 5E). Expres-sion was gradually restricted to thedorsal side by early epiboly (Fig. 5B),and gave rise to a dynamic expressionpattern in the mid- and hindbrainduring gastrulation (Fig. 5C,F–H). Al-though slightly less distinct, this char-acteristic pattern of expression wassimilar to that of endogenous pou2 inthe midbrain and hindbrain (r2 and 4;compare Fig. 5F–H with J–L). Expres-sion was then rapidly down-regulatedto disappear by 24 hpf (Fig. 5D); ex-pression continued only in the tail budand caudal neural tube (Fig. 5H, anddata not shown), consistent with theexpression pattern of endogenouspou2 (Takeda et al., 1994; Haupt-mann and Gerster, 1995).

To determine the portion of �6.5/ATG that is sufficient to recapitulatepou2 expression, we created externaldeletions in GFP-6.5 and examinedtheir effects on reporter expression(Figs. 2, 3A, 4G–J and data notshown). Deletions to �2.3 and �2.2 kbled to slightly higher expression rates,but did not affect the pattern of re-porter expression (Fig. 4G,H, see alsoFig. 7M,N). These results indicatethat the main portion of the regula-tory activity resides in the proximal�2.2/ATG region, which is consistentwith the co-injection experiment de-scribed above. The expression of GFP-2.2 in the dorsal blastoderm and brainwas confirmed by the whole-mount insitu hybridization (WMISH; Fig.4N,O), which also showed that GFP-2.2 is activated in the blastoderm im-mediately after the mid-blastula tran-sition (MBT; Fig. 4M).

Further deletions to �0.2 kb pro-gressively reduced the rate of reporterexpression (Fig. 3A), suggesting thepresence of multiple cis-elements. Wealso conducted co-injection experi-ments in which GFP-0.1 was co-in-jected with various subfragments de-rived from �2.3/ATG (Table 1B, Figs.3B, 4K,L, and data not shown). Exter-nal deletions from the 3�-end of �2.3/�0.1 supported the idea of multiple

Fig. 4. Regulatory activities of the subregions in the upstream DNA of pou2. A–F: Expression ofthe reporter (GFP-0.1) under regulation of the �6.5/�2.2 (A–C) and �2.3/�0.1 (D–F) fragments at50% epiboly (A,D), bud stage (B,E), and three-somite stage (C,F). G–J: Expression of the greenfluorescent protein (GFP) constructs under regulation by different regions of the pou2 upstreamDNA at early somite stages. K,L: Expression of GFP-0.1 co-injected with �2.3/�0.1 (K) and�2.3/�0.4 (L) at early somite stages. M–O: mRNA expression of GFP-2.2, as revealed by whole-mount in situ hybridization, at the 1-k cell (M), 50% epiboly (N), and bud (O) stages. GFP expressionin the dorsal blastoderm and head regions are marked with white curves. A–L,N: Lateral views withanterior to the top and dorsal to the right. M: Animal view. O: Dorsal view with anterior to the top.Scale bar � 200 �m.


cis-elements. Based on these results,we concluded that several positiveregulatory elements exist in the �2.2/�0.1 region. Interestingly, the foursubregions that together representthe entire �2.2/�0.1 region showed noregulatory activities when co-injectedseparately (�2.3/�1.8, �1.8/�0.9,�0.9/�0.4, �0.4/�0.1, Fig. 3B).

In this proximal region, we identi-fied four consensus binding sites(OS1–4; Figs. 2, 3A) for the POU fam-ily proteins (octamer sequences, typi-cally ATGCAAAT) that were previ-ously demonstrated to bind to thePou2 protein (Takeda et al., 1994). Todetermine whether these were thepredicted cis-elements, we examinedthe expression of mutated GFP-2.2(Fig. 6), in which the intervening se-quences (IS1 and IS2, Table 1B) weredeleted or base substitutions were in-troduced into the octamer sequences(Table 2A). The deletion of IS1 and/orIS2 (GFP�IS1, GFP�IS2, GFP�IS12)had little effect on expression in themid/hindbrain, indicating that theseregions are dispensable (Figs. 6,7A–C, and data not shown). In con-trast, the disruption of any octamersequence (�OS1–4, Figs. 6, 7D–G)drastically reduced reporter gene ex-pression in the brain, and the disrup-tion of additional sequences enhancedthis effect only slightly (�2OS–�4OS),showing that these four sites operatein a highly cooperative manner (Figs.6, 7H–J). Unexpectedly, the disrup-tion of octamer sequences led to exten-sive ectopic reporter expression in theposterior region, including the tail

Fig. 5. Stable expression of GFP-6.5 in transgenic embryos. A–C: Expression of GFP-6.5 asvisualized with green fluorescent protein (GFP) fluorescence in a fertilized egg (A), or in developingembryos at the shield (B) and three-somite (C) stages. D: GFP expression could not be detected at28 hours postfertilization (hpf) in the head. GFP expression in the dorsal blastoderm and headregions is marked with white curves. E–H: Expression of GFP-6.5 mRNA, detected by whole-mountin situ hybridization, in embryos at 40% epiboly (E), 90% epiboly (F), bud (G), and three-somite (H)stages. I–L: Endogenous expression of pou2 at 50% epiboly (I), 90% epiboly (J), bud (K), andthree-somite stages (L). A,E,I: Lateral views with animal poles to the top. B–D,H: Lateral views withanterior to the top and dorsal to the right (B,C,H) or with anterior to the left and dorsal to the top(D). F,G,J–L: Dorsal views with anterior to the top. cnt, caudal neural tube; hb, hindbrain; lls, laterallongitudinal stripe; mb, midbrain; mls, medial longitudinal stripe. Scale bar � 200 �m.

Fig. 6.

Fig. 6. Localization of the cis-elements withinthe upstream DNA that regulates expressionin the embryonic brain. Alterations, such assequence deletion and base substitution,were introduced into the pou2 upstream�2.2/ATG DNA within GFP-2.2, which wereexamined for their expression in injected em-bryos. The GFP-2.2 constructs lacking IS1,IS2, and both IS sequences are referred to asGFP�IS1, GFP�IS2, and GFP�IS12, respec-tively. Those lacking one of the four octamersequences are called GFP�OS1– 4, and thoselacking two, three, and four octamer se-quences are GFP�2OS, GFP�3OS, andGFP�4OS, respectively. Numbers of injectednormal embryos and rates of GFP expression(%) in the brain and posterior embryonic re-gion were shown in the right panel. N.D., notdetermined.

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bud, which was eliminated by the re-moval of IS2 (Fig. 7K,L). These resultssuggest that the posterior expressionof pou2 is driven by cis-regions in IS2,and that this expression is repressedor restricted to smaller regions by theoctamer sequences (and proteins asso-ciated with these sites).

Binding of Pou2 WithOctamer Sequences in theProximal Regulatory Region

Oligonucleotides were designed againstthe octamer sequences and labeled withdigoxigenin (DIG, Table 2A). An exam-ination of binding activity to Pou2 pro-

duced doublet bands and a lower mobil-ity band in all cases, including thereference octamer probe (R-Oct; Fig.8A), although the relative intensity dif-fered among bands. Complex formationbetween each oligo and Pou2 was inhib-ited by the cognate oligonucleotides andother octamer sequences, confirmingthe specificity of these binding reactions(Fig. 8B,C; data not shown for OS2 and4). Furthermore, the competing activi-ties of respective oligonucleotides wereseverely affected by base substitution inthe octamer sequences (Table 2A; Fig.8D), which was shown above to abro-gate the regulatory activities of the�2.2/ATG region in GFP-2.2.

Because the reporter assay sug-gested considerable functional cooper-ativity among the four octamer sites,we examined the binding activities ofincreasing amounts of Pou2 to theproximal region, from which the twointervening sequences had been de-leted to produce a size suitable forEMSA (4�OS). We observed the for-mation of a single large complex with-out intermediate sizes (Fig. 8E). Fur-thermore, as the octamer sequences in4�OS were disrupted sequentially(3�OS, 2�OS, and 1�OS), the com-plexes were reduced gradually in size,and finally disappeared (0�OS, Fig.8F). These together suggested a possi-bility that the binding of Pou2 with4�OS is cooperative. It should bementioned that the binding affinityseems significantly reduced, as can beseen from the increase in amount ofthe free probes. Finally, we directlycompared the competing activity of4�OS and 1�OS against the bindingof Pou2 with DIG-labeled 4�OS,showing that 4�OS was significantlymore effective than the additive ef-fects of 1�OS (data not shown), whichis also consistent with the idea thatPou2 cooperatively associates with�2.2/�0.1.

pou2 Is Regulated by Meansof an Autoregulatory Loopin Embryos

The results described above show thatPou2 may activate pou2 by means ofthe proximal region. Indeed, we co-injected pou2 mRNA and GFP-2.2 intoembryos, finding that pou2 mRNA sig-nificantly up-regulated and expandedGFP-2.2 expression at both the shield

TABLE 1B. Genomic Regions Examined for Their Roles inTranscriptional Regulation: Range of the Genomic Regions Within the

�2.3/ATG Region

Regiona

Range

5�-end (bp) 3�-end (bp)

�2.3/ATG �2303 �236�2.3/�0.1 �2303 �118�2.3/�0.4 �2303 �421�2.3/�0.8 �2303 �844�2.3/�1.8 �2303 �1827�1.8/�0.8 �1847 �844�0.9/�0.4 �863 �421�0.4/�0.1 �440 �118IS1 �2063 �1596IS2 �1500 �316

aSubfragments obtained from �2.3/ATG by polymerase chain reaction and examinedfor their regulatory activities, and the two sequences intervening between OS1 andOS2 or between OS2 and OS3 (IS1 and 2) that were removed from GFP-2.2 toevaluate their roles in GFP-2.2 regulation.

TABLE 2. Oligonucleotides Used for the EMSA Assay and BaseSubstitutiona

Oligos Sequenceb

(A) Octamersequences

OS1 5�-GCTATGGCCTATGCAAATAGCCTTTATT-3�OS1m 5�-GCTATGGCCTAgGacAATAGCCTTTATT-3�OS2 5�-ATGTATGCTTATGCATATTCAAAAAAAA-3�OS2m 5�-ATGTATGCTTcgGaATATTCAAAAAAAA-3�OS3 5�-AAATACATGAATTTGCATTGTAAATACT-3�OS3m 5�-AAATACATGAATTgtCcTTGTAAATACT-3�OS4 5�-CTTTTATAAGATGCAAATCTATACAGAT-3�OS4m 5�-CTTTTATAAGAgGacAATCTATACAGAT-3�R-Oct 5�-GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3�(B) RARERf-RARE 5�-TCGAGGGTAGGGTTCACCGAAAGTTCACTC-3�P2-RARE 5�-ACCAAGTTCATTCACAAATTCACAGTCAGC-3�P2-RAREm 5�-ACCAAGTTCATTCACAgAaTtcCAGTCAGC-3�

aEMSA, electrophoretic gel mobility shift assay; RARE, retinoic acid-responsiveelement.

bSequences of the oligos used for EMSA or base substitution in the GFP-2.2 construct.The consensus sequences for POU and RAR/RXR factors are underlined, and thesubstituted bases are shown in lower cases. In EMSA, oligos of the complementarysequences were also synthesized to prepare double-stranded probes.


and bud stages, as determined by GFPfluorescence and mRNA expression(Fig. 7M–R). These results suggest

that pou2 is regulated by means of anautoregulatory loop in embryos.

To confirm that pou2 is under auto-

regulation, GFP-2.2 expression wasexamined in embryos in which pou2was knocked down by a morpholinooligo (MO-pou2). As expected, GFP-2.2 expression in the brain was signif-icantly down-regulated, whereas, un-expectedly, the posterior expression ofGFP-2.2 was up-regulated both interms of the intensity (Fig. 7S,T) andthe expression rate (Table 3; 46% to75%). This ectopic expression trig-gered by MO-pou2 was abrogated bydeletion of IS2 from the GFP construct(GFP�IS2, GFP�IS12; Fig. 7U; Table3; and data not shown). Finally, theposterior expression of GFP observedfor GFP�4OS was little affected (Ta-ble 3 and data not shown). In all caseswhen MO-pou2 was injected, morethan two thirds of embryos showedhindbrain defects characteristic of spgmutants (Belting et al., 2001; Burgesset al., 2002), confirming the efficacy ofpou2 knockdown. Taken together, theresults suggest that pou2 expressionis maintained by autoregulation in thebrain. Furthermore, pou2 is probablyinvolved in the restriction of its ex-pression to a small portion in the cau-dal neural tube as well, by means ofrepressing the function of IS2.

RA Represses pou2 byMeans of the RA-ResponsiveElement in the ProximalRegion

In previous studies, the pou2-express-ing region expanded during earlysomitogenesis after treatment with10�7 M RA (Hauptmann and Gerster,1995). At this stage, RA is primarilysynthesized by the raldh2 gene prod-uct, retinaldehyde dehydrogenase 2,in the posterior non-axial mesoderm,from which RA emanates anteriorly(Begemann et al., 2001; Grandel et al.,2002). The authors suggested thatthis RA concentration transforms theanterior rhombomeres (r1–r3) to r4. Incontrast, the expression of mousePou5f1/Oct-3/4 is down-regulated inES and EC cells after RA treatment(Ovitt and Scholer, 1998). Therefore,we reexamined the effect of RA onpou2 expression in embryos. We foundthat, although 10�7 M RA expandedpou2 expression anteriorly, as previ-ously observed, 10�6 M RA abrogatedanterior pou2 expression in the mid/hindbrain (Fig. 9A–C). In parallel ex-

Fig. 7. Roles of the octamer sequences in the transcriptional regulation by the proximal upstreamDNA. A–L: GFP-2.2 or its mutated constructs were introduced into embryos and examined for theirgreen fluorescent protein (GFP) expression at early somite stages. Deletion of the interveningsequences little affected the expression (B,C), while disruption of any of the four octamer se-quences abrogated the brain expression, but gave rise to ectopic expression in the caudal region(thick arrows, D–J). Deletion of IS2 from GFP-2.2 lacking the octamer sequences eliminated theectopic expression in the caudal region (K,L). M–R: The expression of GFP-2.2, which wasrestricted to the dorsal blastoderm at 50% epiboly (M), and to the brain at the bud (O) andthree-somite stages (N), was expanded significantly by pou2 overexpression by mRNA injection(P–R), as was revealed both by GFP fluorescence (M,N,P,Q) and whole-mount in situ hybridization(O,R). S–U: The expression of GFP-2.2 (S,T) and GFP�IS2 (U) in embryos injected with MO-con (S)or MO-pou2 (T,U).

1380 PARVIN ET AL.

periments, RA significantly repressedotx2 in the fore/midbrain (data notshown; Kudoh et al., 2002), as well asthe expression of gbx2 and pax2a atthe midbrain–hindbrain boundary(MHB; Fig. 9D,E and data not shown;see also Kikuta et al., 2003), confirm-ing the patterning role of RA in theMHB region.

In addition, we identified a se-quence in the proximal region of pou2(�115 to �102, CATTCACAAATTCA)that is similar to the DR2-type RARE(Figs. 2, 10B; Ross et al., 2000). Fur-

thermore, we found that exogenousRA repressed the transient expres-sion of GFP-2.2 in late gastrulae(Fig. 9F–I) and early-somite stageembryos (data not shown), indicat-ing that RA affects pou2 expressionat the transcriptional level throughthe �2.2/ATG region (Table 4). Ofinterest, a base substitution intro-duced into the RARE in GFP-2.2(Fig. 10A,B, Table 2B) expanded re-porter expression in the blastodermmargin and the tail bud in late gas-trulae (Fig. 9L,M), suggesting that

P2-RARE represses the �2.2/ATGregion in the posterior region. Thesame base substitution disruptedRARE’s sensitivity to RA, confirmingits role in the RA-mediated regula-tion of pou2 (Fig. 9N,O; Table 4).These results also suggest that therepression of pou2 in the posteriorregion by RA through the RARE con-tributes to the refinement of pou2expression in embryos. Indeed, whenembryos were treated with diethyl-aminobenzaldehyde (DEAB), an in-hibitor of Raldh2-mediated RA syn-thesis, the expression of GFP-2.2was expanded in the posterior region(Fig. 9J,K).

In EMSA, the RAR/RXR complex(zebrafish RARaa and RXRg; Hale etal., 2006; Tallafuss et al., 2006) boundto P2-RARE, as well as to the RARE inthe human RAR gene (Rf-RARE; Sunet al., 2000). Binding was effectivelycompeted by an excess amount of bothP2-RARE and Rf-RARE (Fig. 10C).Furthermore, P2-RARE competitiondecreased in response to a base sub-stitution in the consensus sequence,which also abrogated RA-responsive-ness in the �2.2/ATG region (Fig.10B,C; Table 2B).

DISCUSSION

Relationship BetweenZebrafish pou2 and OtherClass V POU Family Genes

Despite the structural similarity andapparent synteny relationship, whichwas suggested previously, the func-tional equivalence between zebrafishpou2/pou5f1 and mouse Oct-3/4 stillremains obscure. In Oct-3/4 mutantmouse embryos, severe anomalieswere observed at the very early stages(blastocyst) when ICM cells are nolonger pluripotent and restricted todifferentiation along the extraembry-onic trophoblast lineage (Nichols etal., 1998). The most striking anomalyin zebrafish spg mutant embryos oc-curs during the development of themidbrain, MHB, and hindbrain (Belt-ing et al., 2001; Burgess et al., 2002;Hauptmann et al., 2002a), which isconsistent with the brain-specific ex-pression of pou2 during epiboly. Theanalysis of embryos lacking both ma-ternal and zygotic pou2 activities (MZ-spg) revealed that a combination of

Fig. 8. Binding activities of the octamer sequences in the pou2 regulatory region with the Pou2protein. A: Digoxigenin (DIG) -labeled probes for the four octamer sequences (OS1–4) and R-Octsequence generated shifted bands in the presence of Pou2. The fast-migrating doublet bands markedwith large arrowheads were generated by all the probes, whereas the slower-migrating bands, shownwith small arrowheads, were seen only when the pou2 octamer sequences were used. B,C: Binding ofPou2 with DIG-labeled OS1 (B) and OS3 (C) was competed out with 100-fold molar excess of theunlabeled oligos (OS1–4 and R-Oct). D: Binding of Pou2 with the four octamer probes was competedwith 100-fold excess of cognate oligos (OS1–4), but not at all or only partially by mutated oligos(OS1m–4m). E: Binding of Pou2 with 4�OS DNA containing the four octamer sequences (OS1–4)generated a single shifted band with no intermediate bands. F: Binding of Pou2 with 4�OS, 3�OS,2�OS, 1�OS, and 0�OS, which included decreasing numbers of the OS sequences, was examined,showing a gradual decrease in size of the complex and an increase in the amount of free probes.


endodermal pou2 expression andcasanova/sox32 expression was re-quired for the specification ofendoderm (Lunde et al., 2004; Reim etal., 2004). However, the phenotypesobserved in MZ-spg mutants were notas dramatically aberrant as those ob-served in embryos with the targeteddisruption of Oct-3/4. In addition, Oct-3/4 seems to be a negative regulator ofthe endoderm. In mouse ES cells,knockdown of Oct-3/4 elicited differ-entiation to the primitive endoderm(Niwa et al., 2000; Hay et al., 2004).Indeed, Oct-3/4 inhibited the FoxD3-driven activation of the endodermalpromoters for FoxA1 and FoxA2 (Guoet al., 2002). Finally, MZ-spg mutantsshowed no defects in germ cell devel-opment. Of interest, Xenopus andchick possess class V POU genes withsimilarities to Oct-3/4 (Xlpou25/91and cPouV, respectively), which act asrepressors of commitment duringgerm layer specification; these factorsalso substitute for Oct-3/4 in self-re-newal of ES cells (Morrison and Brick-man, 2006; Lavial et al., 2007). How-ever, zebrafish pou2 did not functionlike Oct-3/4 as a self-renewal factor, fur-ther rendering the relationship amongthe vertebrate class V genes enigmatic.

However, we showed that thegenomic organization of pou2 is sim-ilar to that of mammalian Pou5f1/Oct-3/4. This structural similarity ingenomic organization supports theidea that pou2 and Oct-3/4 areclosely related among the POU-fam-ily proteins, despite the discrepan-

cies regarding their functionalequivalence. It should be noted thatOct-3/4 is widely expressed in theneural plate by E8.0, before rapiddown-regulation (Scholer et al.,1990; Reim and Brand, 2002). In ad-dition, Oct-3/4 overexpression intransgenic mouse embryos altersmid- and hindbrain patterning(Ramos-Mejia et al., 2005). Similarto the pou2 gene, Xlpou25/91 andcPouV, which are also widely ex-pressed in early embryos and thenrapidly down-regulated at laterstages, show transient expression inthe anterior neural plate (Morrisonand Brickman, 2006; Lavial et al.,2007). Furthermore, we showed thathigh concentration of RA repressespou2 expression, as is known for Oct-3/4 in ES and EC cells. Thus, wepostulate that pou2 and the pou5f1genes from other vertebrates, includ-ing Oct-3/4, may share functionalsimilarities in brain formation.

To understand the evolution of theclass V POU genes, in addition tofunctional comparison, phylogeneticrelationship should be compared fur-ther in detail among different genesand vertebrate species, using the ac-cumulating database of genomic se-quences. In addition, it should benoted that ES cells, which show manyof the characteristics of mouse EScells, have been obtained from te-leosts, including the zebrafish andmedaka (Fan and Collodi, 2006; Hongand Schartl, 2006). Such cells could begood models to test if fish class V POU

genes are also involved in the mainte-nance of pluripotency.

Transcriptional Regulationby Upstream DNA From�6.5 kb to the Start Codon

By observing the transient and stableexpression of GFP constructs, weidentified a region of DNA upstreamof the pou2 gene that nearly recapitu-lates the maternal expression pattern,as well as the characteristic expres-sion transiently observed during epi-boly in the neural plate. First, femaletransgenic fish harboring the GFP-6.5construct produced embryos that ex-pressed GFP/egfp ubiquitously in eggsand early embryos. Second, similar toendogenous pou2, we observed stableand transient reporter expression inthe midbrain, the anterior hindbrain,and in medial longitudinal stripe cellsduring late epiboly. Third, GFP-6.5expression decreased rapidly afterearly somitogenesis, but it was main-tained in the caudal neural tube/tailbud during somitogenesis. However,note that the reporter constructshowed slightly wider expression withindistinct boundaries in the braincompared with endogenous pou2. Fur-thermore, the medial longitudinalstripe was not split like the equivalentexpression of pou2 (Hauptmann andGerster, 1995). Finally, the constructwas not expressed in the lateral longi-tudinal stripe, showing that more dis-tant regions are required to com-pletely recapitulate the endogenous

TABLE 3. Knock Down of pou2 Affects the Expression of GFP-2.2a

Construct Morpholino Embryos

Expression (%)b

PhenotypecBrain Posterior

GFP-2.2 Control 130 77 46 0 (n � 125)MO-pou2 67 12 75 91 (n � 33)

GFP�IS2 Control 56 66 14 0 (n � 55)MO-pou2 104 6 1 67 (n � 61)

GFP�IS12 Control 41 76 10 0 (n � 38)MO-pou2 99 0 0 94 (n � 93)

GFP�4OS Control 50 26 72 0 (n � 50)MO-pou2 77 12 55 84 (n � 61)

aEmbryos were injected with MO-pou2 or MO-con together with the GFP constructs, and examined for GFP expression at theearly-somite stages. GFP, green fluorescent protein.

bRates of restricted GFP expression in the brain or posterior embryonic region are shown.cPercentage of 24-hours postfertilization (hpf) embryos that survived after observation of GFP fluorescence at early somite stagesand showed hindbrain defects characteristic of spg mutants are indicated. Numbers in parentheses show alive embryos at 24 hpf.

1382 PARVIN ET AL.

expression. Of interest, the mRNA ex-pression of GFP-2.2 suggests that thezygotic expression of pou2, which isdifficult to differentiate from the highlevel of maternal expression, is initi-ated at the 1-k cell stage or the MBT(Kane and Kimmel, 1993) in the blas-toderm.

Autoregulation of pou2Through Multiple OctamerSequences in Upstream DNA

Functional dissection of the �6.5/ATGregion through extended reporter as-says revealed a 2.1-kb region (�2.2/�0.1), which was responsible for themajority of regulatory activity in thebrain. Any additional deletion intro-duced into this proximal region led to

Fig. 9.

Fig. 10.

Fig. 9. Repression of pou2 and GFP-2.2 ex-pression by retinoic acid (RA). The mRNA ex-pression of pou2 and pax2a in uninjected em-bryos (A–E) and green fluorescent protein (GFP)constructs in injected embryos (F–O) was ex-amined by whole-mount in situ hybridization.A–C: Expression of endogenous pou2 mRNA atthe bud stage in the mid–hindbrain (A) wasexpanded by 10�7 M RA (B), whereas re-pressed effectively by 10�6 M RA (C). D,E: Ex-pression of pax2a mRNA at the bud stage in theMHB region (D) was repressed by 10�6 M RA(E). F–I: Expression of GFP-2.2 in injected lategastrulae, which was seen in the mid–hindbrainregion (F,G), was effectively repressed by 10�6

M RA (H,I). J,K: GFP-2.2 expression in injectedembryos expanded laterally in the posterior re-gion by diethylaminobenzaldehyde (DEAB)treatment. L,M: Expression of GFP-2.2�RAREin injected embryos was expanded laterallynear the blastoderm margin compared with thatof GFP-2.2. N,O: Expression of GFP-2.2�RARE in embryos treated with 10�6 M of RA.The observed expression pattern was indistin-guishable from that in untreated embryos (L,M).A–F,H,J,L,N: Dorsal views with anterior to thetop. RARE, retinoic acid-responsive element.

Fig. 10. Binding of the DR2-type retinoic acid-responsive element (RARE) with the RAR/RXRcomplex. A: Schemes of the GFP-2.2 con-structs that are intact or possesses a disruptedRARE (GFP-2.2�RARE). B: Sequences of thethree oligos, Rf-RARE, P2-RARE, and P2-RAREm, are aligned. Sequences correspondingto the RARE consensus, which is shown at thebottom, are underlined. The bases substitutedin P2-RAREm compared with the original oligo(P2-RARE) are shown with lower-case letters.C: Electrophoretic gel mobility shift assay(EMSA) showing specific binding of RAR/RXRwith P2-RARE. Both P2-RARE and Rf-RAREformed complexes with RAR/RXR, which werecompeted out by a 100-fold molar excess ofP2-RARE and/or Rf-RARE, but not by P2-RAREm.


the progressive reduction of expres-sion, indicating the presence of multi-ple regulatory cis-elements. Four oc-tamer sequences were scatteredthroughout this region, prompting usto evaluate their roles in transcrip-tional regulation. The EMSA showedthat these sequences, OS1–4, specifi-cally bind to Pou2 in vitro. The intro-duction of base substitutions demon-strated that any one of the fouroctamer sequences is required for thefunction of the �2.2/�0.1 region,whereas two large sequences, IS1 andIS2, are dispensable. Indeed, we foundthat the overexpression of pou2 bymeans of mRNA injection results inthe global up-regulation of GFP-2.2 inearly embryos, showing that pou2 ex-pression is a limiting factor and suffi-cient for driving GFP-2.2 throughoutthe embryo. Furthermore, the expres-sion of GFP-2.2 was disrupted in thebrain by the knockdown of pou2,which is consistent with the down-reg-ulation of pou2 in spg embryos (Bur-gess et al., 2002). Therefore, it ishighly likely that the pou2 gene prod-uct positively regulates the pou2 genethrough an autoregulatory loop. Evena single disruption led to the abro-gation of the �2.2/�0.1 activity,suggesting that these four octamer se-quences operate in a highly coopera-tive manner. The mechanism of thisfunctional cooperativity remains to bedelineated in the future, but it shouldbe mentioned that this cooperativityseems to be compatible with the bind-ing behavior of Pou2 with the frag-ment containing four octamer se-quences (4�OS), which suggestscooperativity in the binding reaction.

We cannot exclude a possibilitythat, in addition to Pou2, other POU

factors are additionally involved inthe regulation of pou2 in the brainthrough the octamer sequences. Inthis regard, Brn3 proteins, class-IVPOU factors that regulate brain for-mation and recognize the octamer se-quence, may be candidate regulators,although they are expressed more spe-cifically in the brain at later stages(brn3a, brn3b, brn3c; see The Ze-brafish Information Network; http://zfin.org/).

Of interest, the co-injection of ei-ther double-stranded OS1 or R-Octwith GFP-0.1 gave rise to only faintreporter expression with little spa-tial restriction (data not shown),suggesting that the octamer se-quences function in concert with ad-ditional factors that recognize the in-tervening sequences. It is well-known that Oct-3/4 cooperates withSox transcription factors in the reg-ulation of several genes (Pesce andScholer, 2001), and Pou2 cooperateswith a Sox protein (Casanova/Sox32)in endoderm differentiation (Lundeet al., 2004; Reim et al., 2004). Wefound several Sox consensus se-quences in the �2.2/�0.1 region(data not shown), and sox2 and otherB1 group sox genes, including sox3,sox19a, and sox19b, are broadly ex-pressed in the neural plate duringepiboly and at later stages (Okuda etal., 2006). The involvement of thesesox genes in pou2 regulation is nowunder investigation. Although un-identified so far in zebrafish, Nanog,which is an NK2-type homeodomainprotein, also functions as a cofactorof Oct-3/4 for the maintenance ofpluripotency (Wang et al., 2006;Niwa, 2007), and their involvement

in pou2 regulation deserves atten-tion.

Negative Regulation Refinespou2 Expression in theNeural Plate

The autoregulatory loop controllingpou2 suggests that maternal Pou2protein initiates zygotic pou2 expres-sion. Indeed, the proximal region wasactivated as early as the MBT stage.Once activated, pou2 expression islikely to be maintained through thesame regulatory loop. However, pou2expression is actually down-regulatedduring epiboly in the forebrain andthe neural plate posterior to r4, and iteventually disappears in the majorityof the neural plate after the end ofepiboly; the only remaining expres-sion is observed in the caudal neuraltube. This dynamic expression pat-tern suggests that repressive factorsspatially restrict pou2 expression. RAis a potent substance that plays im-portant roles in many aspects of ver-tebrate development and is involvedin neural plate patterning in early em-bryos (Ross et al., 2000; Moens andPrince, 2002). A previous studyshowed that 10�7 M RA expands pou2expression in the hindbrain, mostlikely as a result of transforming theanterior hindbrain to r4 (Hauptmannand Gerster, 1995). Here, we foundthat 10�6 M RA effectively repressespou2 in the mid/hindbrain. BecauseRA is synthesized by the raldh2 geneproduct during epiboly in the blasto-derm margin (Begemann et al., 2001;Grandel et al., 2002), RA is a promis-ing candidate repressor in the poste-rior region. Indeed, our results showthat 10�6 M RA effectively represses

TABLE 4. Role of the RARE in the Regulation of pou2a

Construct RA (�M) Embryos

Expression (%)b

Brain Posterior Nonspecific

GFP-2.2 0 35 91 45 91 32 0 0 13

GFP-2.2�RARE 0 19 78 74 211 44 83 61 11

aEmbryos injected with GFP-2.2 or GFP-2.2�RARE were treated with RA from the 50% epiboly and examined for the expression ofthe transcripts in late gastrulae. GFP, green fluorescent protein; RA, retinoic acid; RARE, retinoic acid-responsive element.

bRates of restricted reporter expression in the mid–hindbrain and tail bud region, as well as those of nonspecific ubiquitousexpression, are shown.

1384 PARVIN ET AL.

GFP-2.2 expression, similar to endog-enous pou2 expression. Furthermore,we identified a DR2-type RARE (P2-RARE) in the proximal region thatspecifically associates with the RAR/RXR complex. The disruption of thissequence abrogated the sensitivity ofGFP-2.2 to RA, showing that P2-RARE is required for RA responsive-ness. Importantly, the expression ofGFP-2.2 expanded ventrally, espe-cially in the blastoderm margin, whenthe RARE was disrupted (GFP-2.2�RARE). In this regard, it shouldbe mentioned that, from late epibolyto early somitogenesis, at least tworar genes (rara and rarb) are report-edly expressed in the neural plate,and all the rxr genes examined showubiquitous expression (Hale et al.,2006; Tallafuss et al., 2006), render-ing the neural plate responsive to RA.Thus, RA (by means of RARE) likelyrepresses the expression of intactGFP-2.2, and possibly endogenouspou2, in the posterior neural plateduring epiboly.

The disruption of the four octamersequences, as well as the knockdownof pou2, also resulted in the posteriorexpansion of GFP-2.2 expression, indi-cating that, in addition to RA, pou2itself may repress itself in the poste-rior region, restricting the expressionto narrow portions in the posterior re-gion. Thus, it appears that pou2 func-tions differently in its own regulationin the anterior and posterior embry-onic regions. In mice, repression ofOct-3/4 also involves DNA methyl-ation of the promoter sequence in EScells, trophoblast cell lines, and mouseembryos (Ben-Shushan et al., 1993;Hattori et al., 2004). Although thismechanism requires several days toinduce down-regulation in case of Oct-3/4, such epigenetic repression mayalso be a factor in pou2 down-regula-tion during somitogenesis.

Activation of Expression inthe Posterior Region

The posterior expansion of GFP-2.2expression in the absence of either theoctamer sequences, functional Pou2,or P2-RARE indicates the presence ofregulatory element(s) that drive geneexpression in the posterior region. Inline with this, pou2 is expressed dur-ing somitogenesis in the caudal neural

tube, although in a restricted region.Indeed, we detected stable expressionof GFP-6.5 in the caudal region, simi-lar to endogenous pou2. Furthermore,Xenopus class V POU genes (Xl-Pou25/91) are also expressed in thecaudal end of the neural tube (Morri-son and Brickman, 2006). Because thedeletion of IS2 eliminated the poste-rior expression of GFP-2.2 that wasobserved when octamer sequences aredisrupted or pou2 was knocked down,the IS2 region likely harbors the reg-ulatory region responsible for poste-rior expression, although the physio-logical significance of this posteriorexpression is unknown. In this regard,it should be noted that Cdx2 forms arepressor complex with Oct-3/4 andnegatively regulates Oct-3/4 andCdx2, forming a negative regulatoryloop (Niwa et al., 2005). Mouse Cdx4was shown to have similar function,and cdx4 is expressed in the posteriorregion of zebrafish embryos, includingthe neural plate (Shimizu et al., 2006).Therefore, the repressive effect of theoctamer sequences and pou2 on theposterior expression of GFP-2.2 couldbe explained similarly, which is to bedefined in the future. Whatever themechanism, it is probable that thepresence of repressive regulation en-sures caudally restricted expression ofpou2 in the neural tube.

Comparison With theRegulatory Mechanism ofOct-3/4

Previous research into the regulationof Oct-3/4 transcription in EC cells,ES cells, and mouse embryos led tothe identification of three separateregulatory elements (Ovitt and Scho-ler, 1998; Niwa, 2007), as mentionedin the Introduction: the promoter re-gion, the PE, and the DE. It should bementioned that down-regulation ofOct-3/4 at the later stages of mousedevelopment is recapitulated in ESand EC cells that are induced to dif-ferentiate by RA (Ovitt and Scholer,1998, and references therein), andthis effect was mediated by the RAREin the promoter-enhancer region ofOct-3/4 (Okazawa et al., 1991; Pikar-sky et al., 1994).

We focused on the regulation ofpou2 in the brain, which has not yetbeen examined for Oct-3/4. This lack

of information made it difficult tocompare the regulatory mechanismof these two genes directly. Althoughwe failed to detect meaningful se-quence similarities in the flankingregions of pou2 and Oct-3/4 (from�10 kb to �10 kb) by means of theVISTA or PipMaker analyses (datanot shown), we did note several com-mon features. First, both genes areactivated by means of interactionwith their own products, forming anautoregulatory loop. Second, RA re-presses the expression of both genesthrough upstream RARE(s), al-though the positions and sequencesof these RAREs show some varia-tion. Third, both genes are under theregulation of TATA-less promoters.Finally, the main regulatory activityresides within immediately up-stream DNA sequences of similarsizes (5– 6 kb). Further analysesand comparison of these two genesand their regulatory mechanismswill provide insight into the regula-tion of class V POU genes, whichplay pivotal roles during early verte-brate embryogenesis. This informa-tion can aid in the development ofregeneration therapy and contributeto our understanding of vertebrateevolution, which involves the alter-ation of early developmental regula-tory mechanisms in common ances-tors.

EXPERIMENTALPROCEDURES

Animals

Adult zebrafish were maintained at27°C in a 14-hr light/10-hr dark cycle,and embryos were raised at 28.5°Cuntil appropriate stages. Morphologi-cal features and hours postfertiliza-tion (hpf) were used to stage embryos(Kimmel et al., 1995). For RA treat-ment, the embryos were incubatedwith 1 �M RA (all-trans retinoic acid;Sigma) from 6 to 7 hpf, washed inwater three times, and allowed to de-velop to the bud stage. The embryoswere also treated with 20 �M DEAB(Nakalai Tesque) from 30% epiboly.

Cloning Genomic DNA forpou2

The zebrafish genomic phage library(�FIX II, kindly donated by Dr. Hitoshi


Okamoto) was screened by means ofplaque hybridization using pou2 cDNAas a probe (Takeda et al., 1994). Toclone the far upstream or downstreamDNA of pou2, the zebrafish genomicBAC library (BUSM-1, created byDr. C. Amemiya) was screened bymeans of PCR using two primers forexon 1: 5�-ATGTTCATGCCATACCG-GTCAGTG-3� and 5�-TAACGTGGC-CATTAGCGTGGATGT-3�. DNA fromone pou2 BAC clone (237B24) was di-gested with EcoRI, and the fragmentswere subcloned and then examined fortranscriptional regulation.

Determination of theTranscriptional InitiationSite

Total RNA from 2- to four-cell stageembryos was subjected to 5� RACE us-ing a 5� RACE kit (Gibco BRL) accord-ing to the manufacturer’s protocol.The 5� RACE products were subclonedand sequenced. Positions within thegenome are shown relative to themain transcriptional start site (seethe Results section).

Plasmid Construction

Genomic DNA from �6.5 kb to imme-diately upstream of the start codon(�6.5/ATG) was amplified by means ofPCR using lambda phage clone DNA(clone �EP5) as a template and thefollowing primers: forward, 5�-ATC-GGATATGAGCATCCGT-3�; reverse,5�-CTTTCCGCTAAAAAGGTTGTTGA-G-3�. The amplified fragment was sub-cloned into a pGEM-T vector (Pro-mega), and the insert was cloned intothe multicloning site (MCS) of pEGFP-1in the forward orientation (pGFP-6.5).Likewise, genomic DNA from �2.2 bpto the start codon (�2.2/ATG) was am-plified by means of PCR (forward, 5�-TCGGGCTCTTCTGGCACAAA-3�) andthen subcloned into the MCS ofpEGFP-1 (pGFP-2.2; Fig. 3).

Inverse PCR was used to delete sub-regions of �2.2/ATG in pGFP-2.2 asfollows. The pGFP-2.2 DNA was am-plified by means of PCR using oppo-sitely oriented 5�-phosphorylatedprimers that flanked the targeted de-letion sequences (IS1 and IS2; Fig. 6;Table 1B). The products were self-li-gated so that the flanked regions weredeleted in all resultant constructs.

The second repetitive sequence in theRARE within pGFP-2.2 was replacedwith an irrelevant sequence (EcoRIsequence) by means of inverse PCRfollowed by self-ligation (Fig. 10A,B;Table 2B). The octamer sequences inpGFP-2.2 were disrupted by base sub-stitution with mutagenic primers (Ta-ble 2A), using the Transformer Site-Directed Mutagenesis Kit (Clontech,Fig. 6). It was shown by EMSA thatthe same base substitutions disruptthe binding activities of the octamersequences (Fig. 8D). Throughout thestudy, PCR for plasmid constructionwas conducted using LA Taq (Takara)for high fidelity, and sequencing wasperformed to confirm the structure ofall recombinant plasmids.

Reporter Assay by Transientand Stable Expression

For the microinjection of reporter con-structs (GFP-6.5 and GFP-2.2), DNAfrom the 5� ends of the regulatory re-gion to the site immediately down-stream of the polyadenylation site inpEGFP1 was amplified by means ofPCR using LA Taq, excluding thebackbone plasmid DNA. The externaldeletion of the upstream DNA in theGFP construct was performed by am-plifying the DNA of the GFP-6.5 DNAbetween given sites in the upstreamDNA and the site downstream to thepoly(A) addition site (Fig. 3A). The re-porter DNA was separated by agarosegel electrophoresis and extracted us-ing the QIAEX II Gel Extraction Kit(Qiagen). The DNA was then solubi-lized in sterilized water and pressure-injected into one-cell stage embryos(10 pg/embryo).

In co-injection experiments, thegenomic fragments of interest (Figs.1A, 3B; Table 1A,B) were preparedfrom the plasmids by means of exci-sion or PCR using the appropriateprimers, and co-injected into embryosas mixtures with the egfp gene underthe regulation of the pou2 minimalpromoter (GFP-0.1), as was previouslydescribed (Inoue et al., 2006, 2008).

Transgenic fish lines were gener-ated as previously described (Inoue etal., 2006). Injected or transgenic em-bryos were allowed to develop to thedesired stages and then observed un-der a fluorescence stereomicroscope

(MZ FLIII, Leica) equipped with aGFP2 filter.

Whole-Mount In SituHybridization (WMISH)

DIG-labeled RNA probes were synthe-sized using T3 or T7 RNA polymerase(Stratagene) and the DIG RNA Label-ing Mix (Roche Diagnostics) accordingto the manufacturers’ protocols.WMISH was performed as previ-ously described (Schulte-Merker etal., 1992).

Electrophoretic MobilityShift Assay (EMSA)

The cDNA sequences of pou2, raraa,and rxrg were subcloned into pTnT(Promega), and gene products weresynthesized from these plasmids invitro using the TnT Coupled Reticulo-cyte Lysate System (Promega). Dou-ble-stranded oligonucleotides were la-beled with DIG by means of TerminalTransferase (Roche Diagnostics) andused as probes. The �2.2/�0.1 DNAlacking IS1 and IS2 (4�OS) was am-plified from GFP�IS12 and used as aprobe in EMSA to examine the coop-erativity among octamer sequences.The 4�OS DNA lacking octamer se-quences (3�OS, 2�OS, 1�OS, 0�OS)were similarly amplified fromGFP�IS12 with mutated octamer se-quences (Fig. 6). The binding reac-tions, electrophoresis of the DNA-pro-tein complexes, and complex detectionwere conducted using the DIG GelShift Kit, 2nd Generation (Roche Di-agnostics). As references, a 39-bp oligocontaining the octamer sequence(Roche Diagnostics, here referred to asR-Oct) and a 30-bp oligo (Rf-RARE)including the DR5-RARE from humanRAR (Sun et al., 2000) were used(Table 2).

Morpholino Oligonucleotides

The morpholino oligonucleotide (Gene-Tools Inc.) targeted to the start site ofthe pou2 coding region (MO-pou2) wasinjected into one-cell stage embryos(200 pg/embryo). MO-pou2 had the se-quence 5�-CGCTCTCTCCGTCATCTT-TCCGCTA-3�; and the control morpho-lino oligonucleotide (MO-con) had thesequence 5�-CCTCTTACCTCAGTTA-CAATTTATA-3�.

1386 PARVIN ET AL.

ACKNOWLEDGMENTSWe thank Dr. Hitoshi Okamoto forproviding us with the lambda phagelibrary and Dr. M. Nikaido for helpfuldiscussion and technical advices. Wealso thank Ms. Akiko Ishioka for hertechnical assistance.

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Autoregulatory loop and retinoic acid repression regulate pou2/pou5f1 gene expression in the zebrafish embryonic brain