RESEARCH ARTICLE

Pax6 Is a Direct, Positively Regulated Target of theCircadian Gene ClockRichard Morgan*

Clock is a member of a highly conserved transcription control network that underlies the circadian cycle. During earlyembryogenesis, its expression is developmentally regulated and may be required for the normal development of thehead. In this report, the transcription factor Pax6, a highly conserved regulator of anterior development, is shown tobe a direct target of Clock regulation. Developmental Dynamics 230:643–650, 2004. © 2004 Wiley-Liss, Inc.

Key words: circadian cycle; Clock; head development; Pax6; E-box; promoter

Received 1 December 2003; Revised 3 February 2004; Accepted 25 February 2004

INTRODUCTION

The circadian cycle is the regular,periodic oscillation of cellular andmetabolic activity in anticipation ofdaily environmental changes (re-viewed by Green, 1998; Dunlap,1999; Lakin-Thomas, 2000; Cerma-kian and Sassone-Corsi, 2000). At themolecular level, these oscillationsdepend on the interactions of a rel-atively small number of componentsthat have been highly conserved inevolution.

In mammals, a complex formedby the basic helix-loop-helix (bHLH)transcription factors CLOCK (CLK)and BMAL1 activates the expressionof the Period (Per) and Crypto-chrome (Cry) genes (Antoch et al.,1997; King et al., 1997; Tei et al., 1997;Gekakis et al., 1998; Hogenesch etal., 1998). PER protein binds to caseinkinase I (CKI), masking its nuclear lo-calization signal and retaining it inthe cytoplasm in a phosphorylation-dependent manner (Vielhaber etal., 2000). After a delay, a complex

between PER, CRY, and CKI entersthe nucleus, where CRY inhibits theCLK:BMAL1 complex and the tran-scriptional activation of per and crygenes (Shearman et al., 2000). Thelevel of PER and CRY proteins fallsuntil the CLK:BMAL1 complex is nolonger inhibited, at which point tran-scription of Per and Cry increasesagain. One of the per genes (per2)has also been implicated in a posi-tive role, as the PER2 protein pro-motes activation of the BMAL1 gene(Shearman et al., 2000). Theseevents cause the level of CRY, PER,and BMAL1 proteins to cycle on aroughly 24-hr basis, the latter havingan opposing phase to its two targetgenes (Shearman et al., 1999). Verylittle is known about how this timinginformation is communicated to themany outputs of the clock, althoughin some cases downstream rhythmicgenes have been shown to be un-der the direct transcriptional controlof CLOCK and BMAL1 (Jin et al.,1999).

The primary area for circadiancontrol in adult vertebrates is thesuprachiasmatic nuclei (SCN) inthe brain (reviewed by Weaver,1998). These neurons show verystrong circadian activity and canrelay circadian information toother parts of the nervous systemand, hence, also several target or-gans (Sakamoto et al., 1998; Oishiet al., 1998; Zylka et al., 1998b). Ad-ditionally, many other cells in theadult also express the molecularcomponents of the circadian cy-cle and continue to cycle in theabsence of control by the SCN (Be-sharse and Iuvone, 1983; Cahill andBesharse, 1993; Balsalobre et al.,1998; Whitmore et al., 1998; Zylka etal., 1998b; Yamazaki et al., 2000;Yagita et al., 2001; Green, 2003).This finding applies also to embry-onic cells where the SCN has notyet formed. In particular, zebrafishembryos inherit circadian cycle in-formation through the egg andmaintain the same phase from this

Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London, United KingdomGrant sponsor: the Royal Society; Grant number: 24695.*Correspondence to: Richard Morgan, Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace,London SW17 0RE, UK. E-mail: rmorgan@sghms.ac.uk

DOI 10.1002/dvdy.20097Published online 3 June 2004 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 230:643–650, 2004

© 2004 Wiley-Liss, Inc.

point throughout development(Deluanay et al., 2000).

Despite these advances, relativelylittle is known about the control andfunction of the Clk gene in early de-velopment. The murine and Xeno-pus clock genes (Mclk and Xclk, re-spectively) do not seem to undergocyclical changes in expression earlyon in development (Tei et al., 1997;Shearman et al., 1999; Green et al.,2001). Xclk expression is initially re-stricted to the anterior neural plateand then subsequently expandsalong the whole neural tube. Its tran-scription is activated as an early re-sponse to neural induction and isstrongly up-regulated by the home-odomain-containing transcriptionfactor Otx2 early in gastrulation(Green et al., 2001). This regulation isdirect (i.e., it is independent of fur-ther rounds of transcription andtranslation; Green et al., 2001). Thetranscription of Otx2 is in turn acti-vated by Clk, indicating that a reg-ulatory loop exists between thesetwo genes, the importance of whichis apparent in the low level of Otx2expression and associated defectsin head morphology present in em-bryos where Clk activity has beenblocked (Morgan, 2002). It is alsonoteworthy in this context that a ze-brafish Otx2 homologue, Otx5, re-cently has been shown to regulatethe expression of circadian genes inthe pineal gland (Gamse et al.,2002).

In this report, Pax6 is also identifiedas a target of Clk regulation. LikeOtx2, Pax6 is a highly conservedtranscription factor with roles in bothforebrain and eye development.Unlike Otx2, though, its regulation byClk is direct and is mediated by ahighly conserved E-box consensussequence in its promoter.

RESULTS

Pax6 Is a Positively RegulatedTarget of Clk/BMAL1

To search for additional downstreamtargets of Clk, we used a differentialdisplay technique to compare geneexpression in embryos that had de-veloped from eggs injected with adominant negative Clk (CL�Q-GFP)RNA to that in untreated controls.

CL�Q-GFP lacks the Q-rich domainrequired for transcriptional activa-tion (Gekakis et al., 1998), which hasbeen replaced with green fluores-cent protein (GFP). CL�Q-GFP canstill bind to the E-box consensus se-quence and BMAL1 protein thoughand, thus, can act in a dominantnegative manner, interfering withthe activation of Xclk target genesby the endogenous XCLK protein(Hayasaka et al., 2002; Morgan,2002).

Our attention was drawn to onetranscript in particular, because itwas significantly reduced in in-jected embryos (Fig. 1). We there-fore cloned and sequenced thecorresponding cDNA from the un-treated, control embryos andfound it to be identical to the tran-scription factor Pax6. To confirmthat Pax6 is indeed up-regulatedby Clk, we injected fertilised eggswith either Clk and BMAL1 RNA, orCLK�Q-GFP RNA and then exam-ined the expression of Pax6 later indevelopment, at the neurula stage(Fig. 2). As predicted, these treat-ments have opposite affects: over-expression of Clk/BMAL1 signifi-cantly increases the expression ofPax6, whilst CLK�Q-GFP reduces it.

Up-Regulation of Pax6 by ClkIs Direct and Independent ofProtein Synthesis

The preceding results indicate thatClk activates Pax6 expression, butthey do not provide any indicationas to whether this repression is direct(i.e., independent of further transla-

Fig. 1. Differential display analysis of embryos injected with CLK�Q-GFP. Fertilised Xeno-pus eggs were injected with varying amounts of CLK�Q-GFP RNA, as indicated. One primeramplified a 230-bp product, the amount of which is reduced in proportion to the amountof CLK�Q-GFP injected. “Gel recovered” shows the original product after gel purification.“Cloned and excised” shows the same product after cloning into vector pGEMT andexcising with the appropriate restriction enzymes. “-RT”, control reaction using the samedifferential display primer but without reverse transcription.

Fig. 2. Reverse transcriptase-polymerasechain reaction (RT-PCR) analysis of RNA ex-tracted from noninjected control (‘NIC'),Clk/BMAL1-, or CLK�Q-GFP-expressing em-bryos. Fertilised eggs were injected with 100pg of each mRNA (as shown above eachlane). Total RNA was extracted at the neu-rula stage and examined for the expressionof Pax6 or ef1� by RT-PCR (the latter is in-cluded as a loading control). “-RT”, PCRamplification without a prior reverse tran-scription step.

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tion) or indirect. To address this ques-tion, we used a fusion between Clkand the human glucocorticoid re-ceptor (CLK-GR, Morgan, 2002). Theglucocorticoid receptor binds theheat shock protein HSP90, prevent-ing it from entering the nucleus. Thissteric hindrance of nuclear entry isrelieved by ligand binding, in thiscase, the glucocorticoid analoguedexamethasone (DEX), which by it-self has no discernible effects on Xe-nopus development (Gammill andSive, 1997). Hence, the CLK-GR con-struct confers DEX dependence onthe activity of CLK (Hooiveld et al.,1999).

We injected fertilised eggs withCLK-GR and BMAL1 RNA and al-lowed them to develop to the mid-neurula stage. The embryos werethen treated with DEX in the pres-ence or absence of cycloheximide(CHX), which blocks protein synthe-sis. We examined the expression ofOtx2 and Pax6 by reverse transcrip-tase-polymerase chain reaction (RT-PCR) of RNA subsequently extractedfrom these embryos (Fig. 3). Otx2 ispositively regulated by Clk throughan indirect mechanism (i.e., onethat requires intervening protein syn-thesis steps, Morgan, 2002). Thus, ac-tivating the CLK-GR construct shouldup-regulate Otx2 expression, exceptin the presence of CHX, and this isindeed the case (Fig. 3). The activa-

tion of CLK-GR by DEX results in astrong up-regulation of Pax6. There isalso a very strong up-regulation ofPax6 when DEX and CHX are addedtogether, suggesting that a directmechanism is involved, at least inpart.

Conserved E-Box ConsensusBinding Site Present in thePax6 Promoter Can MediateTranscriptional Activation byClk

Previous studies have shown thatCLK binds to a specific sequence ofnucleotides present in the promoterof the Per gene, termed the E-box(Darlington et al., 1998). The E-boxconsensus is also present in the pro-moter of the Xenopus Pax6 gene(Fig. 4), and in the promoter of Pax6homologues from other species. Ineach case, it is present in the sameorientation and is located at ap-proximately the same distance fromthe transcription start site. Further-more, in each of these promoters,the putative E-box is flanked by aseries of conserved nucleotides(Fig. 4).

To determine whether this con-served sequence sites could medi-ate transcriptional activation by Clk,we cloned them into a position im-mediately 3� to a luciferase (luc) re-

porter gene, driven by a SV40 pro-moter (PX6EBOX�, Fig. 5). Thispromoter drives expression of luc inXenopus embryos (Fig. 4; Etkin andBalcells, 1985). Coinjecting Clk andBMAL1 RNA with the PX6EBOX� con-struct results in a significant increasein luc activity. As a control, we usedsite-directed mutagenesis to alterthe Pax6 E-box consensus bindingsequence in PX6EBOX�. This secondconstruct (PX6EBOX�) is not af-fected by Clk/BMAL1 coinjection.Additionally, we coinjectedPX6EBOX � with CLK�Q-GFP RNA;this significantly reduces PX6EBOX�activity (Fig. 5).

To test the specificity of the puta-tive binding site for Clk, mRNAs forthree other bHLH transcription fac-tors, NeuroD1, Hes, and Id1 (Cai etal., 2000; Kageyama et al., 1997,2000; Ross et al., 2003), were alsocoinjected with the PX6EBOX� re-porter construct (Fig. 5). None ofthese caused a significant increasein reporter activity.

DISCUSSION

The noncircadian control of circa-dian genes such as Clk early in de-velopment has raised the possibilitythat they may have a role in embry-onic patterning. Indeed, this hasbeen shown previously to be thecase for both the timeless gene(Gotter et al., 2000; Rothenfluh et al.,2000) and the Clk gene itself (Mor-gan, 2002). Here, it is shown that Clkcan regulate the transcription of thekey developmental gene Pax6, atranscription factor with both apaired box and a homeodomainthat is expressed in several differentembryonic tissues, including the eye,brain, and pancreas (reviewed bySimpson and Price, 2002). Studies ofmouse Pax6 mutants have estab-lished that it has a role in the devel-opment of each of these organs.Most notably, Pax6 mutations leadto a severely reduced eye in mice(Hill et al., 1991), a phenomena thatis mirrored in humans where muta-tions in Pax6 lead to eye defects,including aniridia (Ton et al., 1991).Pax6 is also required for eye devel-opment in Drosophila (Quiring et al.,1994), and ectopic expression ofPax6 in the ectoderm of Xenopus

Fig. 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA ex-tracted from control (“untreated”) or CLK-GR–expressing embryos. The embryos weretreated with dexamethasone (DEX) and cycloheximide (CHX), either alone or in combi-nation, as shown. Ef1� is included as a loading control. “-RT”, PCR amplification without aprior reverse transcription step. The Pax6:Ef1� signal ratio is shown for each sample.

Pax6 IS REGULATED BY Clock 645

embryos results in the formation ofadditional eye structures (Onuma etal., 2002).

The highly conserved develop-mental role of Pax6 has promptedseveral studies on its transcriptional

regulation in both Drosophila andmice, and the most significant find-ings of these are summarised in Fig-

Fig. 4. Alignment of putative E-box consensus and surrounding nucleotides from the Pax6 promoter of the Xenopus, mouse, human,Drosophila, and C. elegans genes. The grey shading indicates the E-box. Reprinted from Trends in Genetics, Volume 20 in “Conservationof Sequence and Function in the Pax6 regulatory elements” © 2004 with permission from Elsevier.

Fig. 5. The Xenopus Pax6 promoter contains an E-box binding site that can mediate the transcriptional activation of a luciferase (luc)reporter construct. A: The reporter constructs were based on the pGL3 vector, which contains the luc gene under the control of theubiquitously active SV40 promoter. The putative E-box consensus from Pax6 was cloned immediately 5� to the luc gene, as shown. Thenucleotide sequence of the putative E-box consensus (PX6EBOX�) and a nonbinding variant used as a control (PX6EBOX�) are shown.B: Coinjection of Clk and BMAL1 mRNA blocks PX6EBOX� reporter activity. The PX6EBOX� reporter construct was injected into fertilisedeggs together with Clk and BMAL1 mRNA (the amounts shown are in picogrammes [pg]), or with 100 pg of CLK�Q-GFP. “no promoter”,control luciferase construct lacking the SV40 promoter. Five hundred picogrammes of mRNA for the human Id1, NeuroD1, and Hes geneswere also injected with the PX6EBOX� construct, as indicated.

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ure 6. To describe its regulation ascomplex would be something of anunderstatement. In mice, Pax6 tran-scription can start from three sitesassociated with the P0, P1, and P�promoters (Kammandel et al., 1999;Anderson et al., 2002). These promo-tors are under the control of at leastsix different enhancers, locatedboth upstream and downstream ofeach promoter. One of these en-hancers directs Pax6 expression inthe pancreas (Kammandel et al.,1999; Xu et al., 1999), whilst another,located between P0 and P1, drivesexpression in the brain (Kammandelet al., 1999). The others regulate dif-ferent aspects of Pax6 expression inthe developing eye (Williams et al.,1998; Xu et al., 1999; Kammandel etal., 1999; Griffin et al., 2002). There isa striking degree of enhancer conser-vation between different vertebrates,whereby, for example, Fugu (pufferfish) regulatory elements can substi-tute for mouse enhancer function(Kammandel et al., 1999). The func-tional conservation of Pax6 regulationalso extends to Drosophila, as demon-strated by the ability of a fly Pax6 en-

hancer to function in a mouse em-bryo and vice versa (Xu et al., 1999).

The conservation of Pax6 regula-tion is reflected in the relatively highdegree of sequence conservationbetween enhancers in different ver-tebrate species and to a lesser ex-tent between the vertebrates andDrosophila (Xu et al., 1999). No se-quence identity is apparent thoughbetween the noncoding regions ofthe Caenorhabditis elegans Pax6gene and those of other species,and combined with a lack of obvi-ous homologous structures, it mightbe concluded that the C. elegansPax6 gene is regulated by a distinctmechanism (Chisholm and Horvitz,1995; Zhang and Emmons, 1995). Acloser look at the C. elegans andmouse Pax6 genes reveals that theE-box consensus defined in this re-port is located between the P0 andP1 regions in the mouse gene, and isalso present in the C. elegans pro-moter. This sequence is bipartite andis located in the same orientationand a similar position relative to thepromoter region in each of the fivespecies considered here. These

properties make it fundamentallydifferent from the E-box elementsfound in other genes known to beregulated by Clk, such as Per (Dar-lington et al., 1998).

Whilst the Pax6 promoter “E-box”identified here can mediate the ac-tivation of Pax6 transcription by Clk,it is also possible that other transcrip-tion factors could interact with thissite, although the three tested here(Id1, Hes, and NeuroD1) do not (Fig.5). The relatively specific interactionof Clk with this element raises thequestion of why a component of cir-cadian regulation also regulates theexpression of genes involved in thedetermination of cell identity in theearly neural plate (i.e., Pax6 andOtx2; Morgan, 2002). An exact an-swer to this question is beyondpresent understanding, but one pos-sibility would be that Clk is used in-dependently for two different func-tions at the same embryonic stage.This finding would not be withoutprecedent as, for example, the tran-scription factor Hoxb4 is involved inboth promoting cell proliferationand cell death in different popula-

Fig. 6. Regulatory sites in the Pax6 gene. The genomic map of the Pax6 gene is shown. Enhancer regions identified to date are shownas dark grey ovals, and the regions in which each directs expression of a reporter construct is indicated above. Exons are shown asvertical bars (light grey), and the three promoter regions at which transcripts P0, P1, and P� start are shown in the enlargement of thisregion, together with the exons present in each transcript and the sites at which these transcripts are expressed in the mouse embryo. Thevertical, starred black bar indicates the location of the highly conserved region reported here. AC, amacrine cells; CB, ciliary body; CJ,conjunctiva; CNS, central nervous system; HB, hindbrain; LG, lacrimal gland; NR, neural retina; NTDP, nonterminally differentiatedneurones; OR, olfactory region; PLR, pigmented layer of retina; TL, telencephalon. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

Pax6 IS REGULATED BY Clock 647

tions of cells in the same embryo(Morgan et al., 2004). An alternativepossibility is that the shared regula-tory properties of Clk allow circadiancycling and neural patterning to becoordinated both spatially and tem-porally. Further studies may help todistinguish between these fascinat-ing possibilities.

EXPERIMENTAL PROCEDURES

Differential Display Analyses

Fertilised Xenopus eggs were in-jected with 500 pg of CLK�Q-GFPRNA. DEX was added at either stage7 or 11, and the embryos were thenallowed to develop until stage 17. Atthis point, total RNA was extractedand 1 �g was used to make cDNAby reverse transcription using a poly-deoxythymidine primer (T15). Twopercent of this reaction was thenrandomly amplified by PCR using asingle primer (5� CAG ATT GGT GCTGGA TAT GC 3�), with two rounds ofamplification at 94°C for 30 sec,45°C for 30 sec, and 72°C for 60 sec,and then 30 rounds of amplificationat 94°C for 30 sec, 60°C for 30 sec,and 72°C for 60 sec. The PCR prod-ucts were resolved by electrophore-sis on 2% agarose for 4 hr at 200 V(4°C) and visualised by ethidium bro-mide staining. Differentially displayedbands of interest were cut out the geland the PCR products were ex-tracted by Qiaquick PCR PurificationKit (Qiagen) and eluted in 50 �l ofwater. The purified PCR productswere PCR reamplified and gel purifiedif necessary, cloned into pGEM-T easyvector (Promega), and sequenced.

RNA Extraction and RT-PCR

Total RNA was extracted from wholeembryos using the QuickPrep TotalRNA extraction kit (Amersham Phar-macia Biotech, Inc.). A total of 3 �gof RNA was used in subsequent re-verse transcription reactions. Thiswas mixed with a poly T15 oligo to 5�g/ml and heated to 75°C for 5 min.After cooling on ice, the followingadditional reagents were added:dNTPs to 0.4mM, RNase OUT (Pro-mega) to 1.6 �g/�l, Moloney murineleukemia virus reverse transcriptase(M-MLRvT) RnaseH- point mutant(Promega) to 8 �g/�l and the ap-

propriate buffer (supplied by themanufacturer) to �1 concentration.The mixture was incubated for 1 hr at37°C, heated to 70°C for 2 min andcooled on ice.

PCR reactions were all performedin a total volume of 40 �l. For each,we used 1 �l of the M-MLRvT reac-tion (as described above), 0.2 nmolof each primer and 20 �l of Redimixpremixed PCR components (Sigma).All reactions were cycled at 94°C for30 sec, 60°C for 30 sec, and 72°C for60 sec. Thirty cycles were used for allprimer sets except those for ef1�, forwhich 23 cycles were used. Theprimers used for XPax6 amplificationwere as follows: forward XPax6F, 5�GCC ACA TTC CCA TTA GCA GT 3�;reverse, XPax6R, 5� GCC ACA TTCCCA TTA GCA GT 3�. The sequencesof the other primer pairs can befound on the internet at http://www.sghms.ac.uk/depts/anatomy/pages/richhmpg.htm/

QPCR was performed using theSYBR green labelling kit from Sigma,using ROX as the internal standarddye. Thermal cycling and fluores-cence detection was by a MX4000(Stratagene, Inc., La Jolla, CA).Semiquantitative data were ob-tained by using measurements threecycles after reactions had risenabove the baseline, and whereclearly in exponential increase. Ef1�was used as a loading control, andall values are presented as a ratio oftarget to ef1� signal.

Embryo Culture andMicroinjection

These procedures were performedas previously described (Sive et al.,2000).

Luciferase Reporter Constructs

The putative E-box binding consen-sus and its surrounding sequences inthe Pax6 promoter region werecloned into the XbaI site of the pGL3luciferase reporter construct (Pro-mega), immediately 3� to the lucreading frame (Fig. 5). To do this, thefollowing oligos were synthesized:PX6EBOX�U 5� CTAGT CAAATGGCAC GTGGG GAGAA GTG 3�;PX6EBOX�D 5� CTAGC CTCCCCACGT GCCAT TTGAA TCA 3�;

PX6EBOX�U 5� CTAGG GAACCGGCAC GGGGC CAGAA GTG 3�;PX6EBOX�D 5� CTAGC CTGGCCCCGT GCCGG TTCGG TCC 3�.Each of these four oligos were phos-phorylated in separate reactions us-ing polynucleotide kinase (PNK), us-ing the protocol recommended bythe manufacturer, and then the two(�) and (�) oligos were annealedby mixing half of each PNK reactiontogether, heating to 90°C for 5 min,and then cooling on ice. The an-nealed PX6EBOX� and PX6EBOX�oligos were then ligated into pGL3,which had been restricted with XbaI,dephosphorylated using calf intesti-nal phosphatase (Promega), andpurified using the Concert PCR puri-fication system (Life Technologies).PX6EBOX� and PX6EBOX� cloneswere selected that contained onlyone copy of the insert, and thesewere checked by sequencing. Thechosen clones were then purified byusing the Plasmid Midi Kit (Qiagen).

PX6EBOX� and PX6EBOX� wereinjected into fertilised Xenopus eggs(100 pg in 5 nl), using the further re-finements described by Mayor et al.(1993). Luciferase activity was mea-sured as previously described (Mor-gan et al., 1999a).

Cycloheximide and DEXTreatments of Clock/GR-Injected Embryos

These were performed as described(Gammill and Sive, 1997). Embryoswere incubated with cycloheximidefor 30 min before the addition ofDEX. RNA was extracted from em-bryos 2 hr after DEX treatment, bywhich point the untreated controlembryos had reached stage 17.

ClkGR and CL�Q-GFPExpression Constructs

For ClkGR, the full-length Xclk read-ing frame (accession no. AF227985)was amplified from the Xclk cDNAcloned in pBlueScript (Zhu et al.,2000). This PCR product had XhoIand BamHI linkers, allowing it to beligated into vector CS2� containingthe human GR ligand binding do-main (Morgan et al., 1999b). The re-sulting construct, ClkGRCS2�, was a

648 MORGAN

Xclk/GR fusion (the two sequencesbeing in frame with each other).

For CL�Q-GFP (Hayasaka et al.,2002; kindly provided by C.B. Green),the first 1,629 nucleotides of the Xclkreading frame were amplified byPCR and ligated into vector pEGFP-1(Clontech). This construct lacks thepoly-Q transcriptional activation re-gion of Xclk, which has been re-placed with the GFP encoding por-tion of the pEGFP-1 vector. Theresulting CL�Q-GFP reading frametogether with the SV40 poly A regionfrom pEGFP-1was then amplified byPCR, and the resulting product wascloned into pGEMT-easy (Promega)to give construct CL�Q-GFPpGT.

NeuroD1, Hes, and Id1 Clones

These sequences were amplifiedfrom human embryonic cDNA by us-ing the following primers:NeuroD1(NM_002500) – forward, 5�GCCAT GACCA AATCG TACAGCGAG 3�, reverse 5� TGTTT TTAATTTTTT AATCT AATCA TGAAA TATGGCAT 3�; Hes(NM_005524) - forward: 5�GCCAT GCCAG CTGAT ATAATGGAG 3�, reverse 5� TGTTT TTAATTTTTT AATCT CAGTT CCGCCACGGC CTC 3�; Id1(BC030148) - for-ward: 5� GCCAT GGCGG CGGAGCCGAA CAAG 3�, reverse: 5� TGTTTTTAAT TTTTT AATTC AGTAG GAACCGTAGC GATC3�. The PCR productswere cloned into pGEMTeasy to giveclones pGEMTeNeuroD1, pGEMTe-Hes, and pGEMTeId1, respectively.

Transcription of Capped mRNAfor Microinjection

ClkGR RNA was transcribed fromClkGRCS2� using SP6 polymeraseafter linearising with NotI, and CL�Q-GFP RNA was transcribed by using T7RNA polymerase from CL�Q-GFP-pGT after linearising with BamHI.NeuroD1, Hes, and Id1 mRNA weretranscribed by using SP6 RNA poly-merase from their respectivepGEMTeasy clones after linearisingwith NcoI. The transcription reactionwas performed as previously de-scribed (Sive et al., 2000). RNA waspurified by using the RNeasy kit (Qia-gen).

ACKNOWLEDGMENTThe author thanks Carla Green forthe CL�Q-GFP clone used in thisstudy.

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