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The circadian gene Clock is restricted to the anterior neural plate early in development and is regulated by the neural inducer noggin and the transcription factor Otx2
Mechanisms of Development 101 (2001) 105±110
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The circadian gene Clock is restricted to the anterior neural plate early in development and is regulated by the neural inducer noggin and the transcription factor Otx2 Carla B. Green a, Antony J. Durston b, Richard Morgan c,* a
Department of Biology, NSF Center for Biological Timing, 264 Gilmer Hall, University of Virginia, PO Box 400328 Charlottesville, VA 22904-4328, USA b Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands c Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK Received 6 October 2000; received in revised form 30 November 2000; accepted 30 November 2000
Abstract The circadian cycle is a simple, universal molecular mechanism for imposing cyclical control on cellular processes. Here we have examined the regulation of one of the key circadian genes, Clock, in early Xenopus development. We ®nd that the expression of Clock is dependent on developmental stage, not on time per se, and is mostly restricted to the anterior neural plate. It's expression can be induced by the secreted polypeptide noggin, and subsequently upregulated by Otx2, a transcription factor required for the determination of anterior fate. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Circadian; Clock; Early development; noggin; Otx2; Anterior; Neural plate
1. Introduction The circadian cycle is essentially the regular, periodic oscillation of cellular and metabolic activity in anticipation of daily environmental changes. Although the existence of such cycles have been known for a long time, it is only in the last few years that the underlying molecular processes have been elucidated (reviewed by Green, 1998; Dunlap, 1999; Lakin-Thomas, 2000). They consist of the relatively simple interactions of a few components that have been highly conserved in evolution. In mammals, a complex formed by the proteins CLOCK (CLK) and BMAL1 activates the expression of the Period (Per) and Cryptochrome (Cry) genes (Antoch et al., 1997; King et al., 1997; Tei et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998). After a delay, the CRY protein inhibits the CLK:BMAL1 complex and thus blocks the transcriptional activation of the Per and Cry genes (Kume et al., 1999; Shearman et al., 2000). At the same time though, one of the PERIOD proteins activates the transcription of the Bmal1 gene (Shearman et al., 2000). The level of PER and CRY proteins continue to fall until the CLK:BMAL1 * Corresponding author. Tel.: 144-208-725-5219; fax: 11-44-208-7253326. E-mail address: [email protected] (R. Morgan).
complex is no longer inhibited by CRY, at which point transcription of Per and Cry increases quickly again. This causes the level of CRY, PER and BMAL1 proteins to cycle on a roughly twenty four basis, the latter having an opposing phase to it's two target genes (Shearman et al., 1999). The output of the circadian cycle is the activation or repression of down stream target genes by BMAL1 and possibly also PER. The primary area for circadian control in adult vertebrates is the suprachiasmatic nuclei (SCN) in the brain (reviewed by Weaver, 1998). These neurons show very strong circadian activity, and can relay circadian information to other parts of the nervous system and hence also a number of target organs (Sakamoto et al., 1998; Oishi et al., 1998; Zylka et al., 1998). Additionally, many other cells in the adult also express the molecular components of the circadian cycle, and continue to cycle in the absence of control by the SCN (Balsalobre et al., 1998; Zylka et al., 1998; Yamazaki et al., 2000). This applies also to embryonic cells where the SCN has not yet formed. In particular, zebra®sh embryos inherit circadian cycle information through the egg and maintain the same phase from this point throughout development (Delaunay et al., 2000). The clock can be reset by environmental inputs, of which light seems to be the most important. Light exposure causes an acute induction of Per1 and Per2 and this may be impor-
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00559-1
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tant in the resetting mechanism (Albrecht et al., 1997; Shearman et al., 1997; Zylka et al., 1998). We wished to study the control of the Clock gene in early Xenopus development. The Xenopus homologue of Clock (Xclk) is very similar to the mouse and human genes and is expressed in the retinal photoreceptor cells at a constant level (Anderson and Green, 2000; Zhu et al., 2000). We ®nd that, like the murine clock (Mclk) gene, Xclk does not seem to undergo cyclical changes in expression early on in development (Tei et al., 1997; Shearman et al., 1999). It's expression is initially restricted to the anterior neural plate and then subsequently expands along the whole neural tube. Xclk expression is an early response to neural induction, and is strongly upregulated by the homeodomain containing transcription factor Otx2 early in gastrulation.
gastrulation, Xclk transcripts are localized to the mesodermal cells of the dorsal blastopore lip and the ectodermal cells that lie above it (Fig. 2A). Mesodermal expression is lost during gastrulation, but continues in the ectoderm that reaches the anterior end of the embryo by the late gastrula stage (stage 12.5, Fig. 2B,C). This region is the anterior most part of the neural plate, and expression persists here through neurulation (Fig. 2D) and the tailbud stages (Fig. 2E±H), but extends caudally along the spinal cord after the late neurula stage, forming an apparent gradient of expression decreasing posteriorly along it (Fig. 2G). A transverse section through the ocular region of the forebrain at the late tailbud stage (Fig. 2I) reveals that expression is still restricted to the ectoderm, and is expressed in the eyes as well as the forebrain.
2. Results
2.3. Xclk expression can be induced by noggin and further increased by Otx2
2.1. The level of Clock RNA does not cycle in a circadian manner
The ®rst zygotic expression of Xclk is con®ned to the Spemann organizer and overlying ectoderm at the early
To study the role of Xclk in early development we began by examining it's expression from fertilization through to the late tailbud stage (Fig. 1). It is clear that Xclk transcripts are present maternally, at stage 2 (after the second cleavage). This is before the onset of zygotic transcription, which occurs around the time of the mid blastula transition (MBT). The amount of Xclk RNA decreases rapidly after the MBT (stage 8), but then begins to increase later in gastrulation (stage 12). This increase continues steadily throughout neurulation and the tailbud stages. As Xclk is a close homologue of a gene that is required for circadian oscillations in other organisms, we decided to examine whether it might display a temporal dependence in early Xenopus development, rather than depending entirely on developmental stage. In order to do this, we took advantage of the general temperature-insensitivity of circadian clocks (Pittendrigh, 1981), together with the strong temperature-dependence that Xenopus displays in embryonic development. Speci®cally, at 14.58C Xenopus embryos develop far more slowly than at, for example, 21.58C, although the actual developmental progression the embryos follow is identical (Nieuwkoop and Faber, 1994). We examined the amount of Xclk at the same developmental stages in embryos kept at these two different temperatures (Fig. 1). From comparing these two series it is apparent that the amount of Xclk RNA varies only with developmental stage, and is not dependent on the time elapsed since fertilization per se. 2.2. Xclk is expressed initially in the organizer and subsequently in the anterior neural plate To study the spatial distribution of Xclk transcripts during development we performed whole-mount in situ hybridization using an antisense Xclk probe. Shortly after the onset of
Fig. 1. Xclk expression varies with developmental stage, not time. RNA was extracted from embryos at different developmental stages and analyzed for Xclk and Ornithine Decarboxylase (ODC) expression by RT-PCR. The latter gene is expressed at a roughly constant level throughout early development and is used as a loading control. Embryos were cultured either at 14.5 oC or 21.58C, and the time taken to reach each of the developmental stages analyzed is shown. (A) Original RT-PCR data. (B) Semi-quantitative analysis of RT-PCR data. The value represented by the black bars is the Xclk/ODC signal ratio (£100) after background compensation.
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Fig. 2. The expression of Xclk at early developmental stages. (A) Animal to vegetal section through a stage 10 (early gastrula) embryo, the animal pole being uppermost. The arrow head marks the position of the dorsal blastopore lip. (B,C) A stage 12.5 (late gastrula) embryo. (B) Dorsal view. The arrow head marks the blastopore, the future posterior side. Xclk staining is apparent at the opposite end of the embryo (anterior). (C) The same embryo as (B) sectioned along the dotted line. (D) Lateral view of a stage 17 (neurula) embryo, anterior to left. (E) Dorsal view of a stage 22 embryo, and (F) enlargement of the head of the same embryo. (G) Lateral view of a tailbud stage embryo (stage 27), and (H) enlargement of the head of the same embryo. (I) Dorsal±ventral section through the ocular region of the embryo shown in (G), e, eye; fb, forebrain.
gastrula stage (Fig. 2A). This is coincident with another organizer gene, noggin (Smith and Harland, 1992; Smith et al., 1995). Subsequently the expression patterns of these two genes diverge somewhat, with Xclk being strongly expressed in the most anterior part of the neural plate and excluded from the mesoderm. Given this initial coincident expression though, together with numerous previous studies establishing noggin as a secreted polypeptide that can induce ectoderm to differentiate as anterior neural plate (Lamb et al., 1993; Bier, 1997; Weinstein and HemmatiBrivanlou, 1999), we decided to test whether noggin can induce Xclk expression in ectoderm. We injected fertilized Xenopus eggs with noggin RNA and subsequently removed the ectoderm from blastula (stage 9) embryos. We refer to these explants as animal caps. Animal caps from untreated embryos develop as undifferentiated ectoderm, but the presence of noggin RNA within them (or protein added exogenously) induces them to develop as anterior neural tissue (Lamb et al., 1993), expressing genes characteristic of this tissue such as the cell adhesion molecule NCAM (Balak et al., 1987) or the transcription factor opl (Kuo et al., 1998). The results shown in Fig. 3 reveal that noggin has caused neural induction in these animal caps, as demonstrated by the induction of
NCAM and opl. In addition, noggin has also induced Xclk expression, albeit at a low level. The strongest Xclk expression is in the anterior most neural plate and subsequently the forebrain and eyes. This is similar to the ectodermal expression of the homeodomain containing transcription factor Otx2 (Pannese et al., 1995), a highly conserved gene that has an important role in head development. Indeed, at the late gastrula stage the expression of Xclk appears to be identical to the ectodermal expression domain of Otx2 (Fig. 2B,C). Thus, we looked to see whether Otx2 could affect Xclk expression. For this purpose we used of a fusion between Otx2 and the human glucocorticoid receptor (Otx-GR). The glucocorticoid receptor binds heat shock proteins, preventing it from entering the nucleus. This steric hindrance of nuclear entry is relieved by ligand binding, in this case the glucocorticoid analogue dexamethasone (dex), which by itself has no discernible effects on Xenopus development. Hence the Otx-GR construct confers dex dependence on the activity of Otx2, without it seems any perturbation of Otx2 function (Gammill and Sive, 1997). We injected fertilized eggs with Otx-GR RNA and subsequently cut animal caps. Adding dex to activate Otx2 does not induce Xclk expression (Fig. 3), however, in caps that
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simple intracellular system for generating circadian oscillations (Antoch et al., 1997; King et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998; Shearman et al., 1999). Unlike other components of this molecular pathway though, Mclk is not itself cycle under circadian control. This is also true of Xclk in Xenopus retina cells (Zhu et al., 2000), and here we demonstrate that it is also the case in early development (Fig. 1). The fact that Xclk is not under circadian control may make a useful control point in cells. It could for example be regulated by speci®c transcription factors and signalling pathways, which could in turn determine which cells will undergo circadian oscillations but not then interfere with these oscillations once they are in motion. The level of CLOCK protein is probably not limiting once the cycle has started, hence buffering circadian events from changes in the original activating signal.
Fig. 3. Xclk is induced by noggin in animal caps. Caps were taken from either untreated embryos or from embryos injected RNA encoding noggin or OtxGR, as shown. Dexamethasone (dex) was used to activate OTX protein, and cyclohexamide (chx) to block protein synthesis. Subsequently, caps were analyzed for the expression of a number of genes by RT-PCR. 2RT, control PCR reaction on extracted RNA without reverse transcription.
also contain noggin RNA a very strong increase in Xclk was observed. Use of the Otx-GR construct has the distinct advantage of allowing us to determine whether Xclk is a direct target of Otx2 transcriptional activation. By pre-incubating caps with cyclohexamide (chx), a drug that blocks protein synthesis, followed by Otx2 activation by dex, only genes that are dependent on the addition of active OTX protein alone should be transcribed (Gammill and Sive, 1997). We thus treated noggin and Otx-GR containing caps with both dex and chx, under these conditions Xclk is still transcribed, indicating that it is a direct target of Otx (Fig. 3). As a control, we looked to see if two genes expressed speci®cally in the cement gland were also induced, XCG and XAG. Both have previously been shown to be upregulated by Otx2, but whilst XCG is activated directly XAG is not (Gammill and Sive, 1997). As expected, both XCG and XAG are induced by Otx2 in our study, but only the induction of XAG is blocked by cyclohexamide. Chx and dex alone have no effect on the expression on any of the genes examined (Fig. 3). 3. Discussion 3.1. The Clock gene is required for the generation of circadian oscillations but is not under circadian control The Xclk gene is very closely related to the mouse Clock gene (Zhu et al., 2000), functional studies of which have demonstrated that it is a central component of the relatively
3.2. Xclk expression can be induced by noggin and up regulated by Otx If Xclk expression represents a possible start up event for circadian oscillations, where in the embryo does it start? We have demonstrated that Xclk expression is found initially in the organizer region and overlying ectoderm, coincident with the neural inducer noggin. Further, noggin can induce Xclk expression in ectodermal explants along with markers of neural plate (Fig. 3), including the cell adhesion molecule NCAM. Interestingly, NCAM is required for photic resetting of the circadian clock in mice, and a mutation in NCAM that blocks it's conjugation to polysialic acid results in the gradual running down of the circadian cycle in the SCN (Shen et al., 1997; Glass et al., 2000). The expression of Xclk can be upregulated directly by Otx2, a homeodomain containing transcription factor (Fig. 3). Otx2 is expressed in the most anterior region of both Xenopus and mouse embryos in cells that are destined to form the forebrain and other anterior structures (Lamb et al., 1993; Pannese et al., 1995; Blitz and Cho, 1995; Boncinelli et al., 1993). In Xenopus, Otx2 has been shown to directly induce genes required for cement gland formation, an organ that is considered to be the most anterior tissue of the frog embryo (Gammill and Sive, 1997). Mice lacking Otx2 gene function do not form a recognizable forebrain (Ang et al., 1996) and Drosophila embryos with a null mutation for the homologous gene orthodenticle lack the anterior most portion of the head (Finkelstein and Perrimon, 1990; Finkelstein et al., 1990). As would be expected if noggin and Otx were regulators of Xclk, it's expression becomes con®ned later on in gastrulation to the most anterior neurectoderm (Fig. 2). Is circadian activity restricted to these cells? In zebra®sh this is indeed the case, here the cyclical expression of the period gene is predominantly in the eyes and forebrain with a phase that seems to be maternally inherited (Delaunay et al., 2000).
C.B. Green et al. / Mechanisms of Development 101 (2001) 105±110
Subsequently Xclk expression expands caudally towards the posterior end of the embryo, but strikingly it's expression is still restricted to the nervous system even up to very late developmental stages (Fig. 2). The only expression outside the neural plate is seen in the mesodermal cells of the organizer mesoderm, but this may be a response to the relatively high level of noggin expressed there. 3.3. Why is Xclk expression restricted to the anterior neural plate in early development? Vertebrate embryos have only one function, and that is to form the adult body. In general, but in amphibians in particular, this process is unaffected by external in¯uences short of those that are actually injurious. Hence light, sound and other stimuli that affect the behavior of the adult have no perceptible effect. The metabolic processes that drive these developmental processes do not need to respond to the regular, repeated events that the circadian cycle anticipates. Indeed, circadian in¯uences could interfere with the complex controls that govern development, and it may thus be prudent not to have global circadian regulation at this stage. Why is the circadian cycle maintained in the anterior neural plate at this time? This question has not yet been addressed, but it is perhaps hardly surprising to ®nd expression in these cells that will form the eyes and brain, tissues that will be ®rst to perceive the unceasingly changing world. 4. Materials and methods 4.1. Embryos, explants and over expression studies In vitro fertilization, embryo culture, staging, microinjection and culture of explants were carried out as previously described (Pannese et al., 1995). Dexamethasone and cyclohexamide treatments were performed as previously described (Gammill and Sive, 1997). Capped RNA transcripts of otx2-GR and noggin were prepared using the Ambion in vitro transcription kit (Ambion, USA). 4.2. Constructs Xclk in situ probe: bases 551±1261 of Xclk (accession number AF227985) were subcloned in to vector pGEMTEASY (Promega). For antisense in situ probes this was linearized with NcoI and transcribed with SP6 polymersase. 4.3. RT-PCR PCR assays with reverse transcription (RT-PCR) were carried out in the exponential phase of ampli®cation as previously described (Busse and Seguin, 1993), with minor modi®cations. PCR primers are; for Xopl (accession number AF028805) U 5 0 CCG CAA CCG GGG CTT CGG 3 0 and D 5 0 GAA GCC CAG CCT CAT CTG 3 0 , for Xclk (accession number AF227985) U 5 0 CGC GCT ACT ATA
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TCA CCT ACC 3 0 and D 5 0 GAT CCG AGT TCT CTG CTC C 3 0 and for XAG U 5 0 CAA AGA AGC AGG CAG GAA CAA CT 3 0 and D 5 0 ATC GTC AGC GTC ATA GGC ATA CAT 3 0 . The other primers used are described on the internet at http://vize222.zo.utexas.edu Semi-quantitative analysis of RT-PCR data was performed using a Fluoroimager 595 (Molecular Dynamics), staining gels with Vista Green (Amersham) at 1:10 000 in order to visualize the bands. Background was subtracted using the area on the gel immediately above each of the relevant bands as a reference. The signal from each data point was compared to the corresponding ODC signal. 4.4. Whole-mount in situ hybridization An antisense, DIG-labeled transcript of Xclk was prepared in vitro according to the manufacturers instructions (Roche). The in situ protocol used has been described previously (Harland, 1991). Roche BM Purple was used as a precipitating reagent for alkaline phosphatase. Acknowledgements We thank Stephan Wacker for supplying embryos and Hans Jansen for quantifying the RT-PCR data shown in Fig. 1. A.J.D. thanks the E.U. HPRN programme for support. R.M. thanks St. George's Hospital Medical School for support. References Albrecht, U., Sun, Z.S., Eichele, G., Lee, C.C., 1997. A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91, 1055±1064. Anderson, F.E., Green, C.B., 2000. Symphony of rhythms in the Xenopus laevis retina. Microsc. Res. Tech. 50, 360±372. Ang, S.L., Jin, O., Rhinn, M., Daigle, N., Stevenson, L., Rossant, J., 1996. A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122, 243±252. Antoch, M.P., Song, E.J., Chang, A.M., Vitaterna, M.H., Zhao, Y., Wilsbacher, L.D., Sangoram, A.M., King, D.P., Pinto, L.H., Takahashi, J.S., 1997. Functional identi®cation of the mouse circadian Clock gene by transgenic BAC rescue. Cell 89, 655±667. Balak, K., Jacobson, M., Sunshine, J., Rutishauser, U., 1987. Neural cell adhesion molecule expression in Xenopus embryos. Dev. Biol. 119, 540±550. Balsalobre, A., Damiola, F., Schibler, U., 1998. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929±937. Bier, E., 1997. Anti-neural-inhibition: a conserved mechanism for neural induction. Cell 89, 681±684. Blitz, I.L., Cho, K.W.Y., 1995. Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. Development 121, 993±1004. Boncinelli, E., Gulisano, M., Broccoli, V., 1993. Emx and Otx homeobox genes in the developing mouse brain. J. Neurobiol. 24, 1356±1366. Busse, U., Seguin, C., 1993. Molecular analysis of the Wnt-1 proto-oncogene in Ambystoma mexicanum (axolotol) embryos. Differentiation 53, 7±15.
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The circadian gene Clock is restricted to the anterior neural plate early in development and is regulated by the neural inducer noggin and the transcription factor Otx2 Carla B. Green a, Antony J. Durston b, Richard Morgan c,* a
Department of Biology, NSF Center for Biological Timing, 264 Gilmer Hall, University of Virginia, PO Box 400328 Charlottesville, VA 22904-4328, USA b Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands c Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK Received 6 October 2000; received in revised form 30 November 2000; accepted 30 November 2000
Abstract The circadian cycle is a simple, universal molecular mechanism for imposing cyclical control on cellular processes. Here we have examined the regulation of one of the key circadian genes, Clock, in early Xenopus development. We ®nd that the expression of Clock is dependent on developmental stage, not on time per se, and is mostly restricted to the anterior neural plate. It's expression can be induced by the secreted polypeptide noggin, and subsequently upregulated by Otx2, a transcription factor required for the determination of anterior fate. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Circadian; Clock; Early development; noggin; Otx2; Anterior; Neural plate
1. Introduction The circadian cycle is essentially the regular, periodic oscillation of cellular and metabolic activity in anticipation of daily environmental changes. Although the existence of such cycles have been known for a long time, it is only in the last few years that the underlying molecular processes have been elucidated (reviewed by Green, 1998; Dunlap, 1999; Lakin-Thomas, 2000). They consist of the relatively simple interactions of a few components that have been highly conserved in evolution. In mammals, a complex formed by the proteins CLOCK (CLK) and BMAL1 activates the expression of the Period (Per) and Cryptochrome (Cry) genes (Antoch et al., 1997; King et al., 1997; Tei et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998). After a delay, the CRY protein inhibits the CLK:BMAL1 complex and thus blocks the transcriptional activation of the Per and Cry genes (Kume et al., 1999; Shearman et al., 2000). At the same time though, one of the PERIOD proteins activates the transcription of the Bmal1 gene (Shearman et al., 2000). The level of PER and CRY proteins continue to fall until the CLK:BMAL1 * Corresponding author. Tel.: 144-208-725-5219; fax: 11-44-208-7253326. E-mail address: [email protected] (R. Morgan).
complex is no longer inhibited by CRY, at which point transcription of Per and Cry increases quickly again. This causes the level of CRY, PER and BMAL1 proteins to cycle on a roughly twenty four basis, the latter having an opposing phase to it's two target genes (Shearman et al., 1999). The output of the circadian cycle is the activation or repression of down stream target genes by BMAL1 and possibly also PER. The primary area for circadian control in adult vertebrates is the suprachiasmatic nuclei (SCN) in the brain (reviewed by Weaver, 1998). These neurons show very strong circadian activity, and can relay circadian information to other parts of the nervous system and hence also a number of target organs (Sakamoto et al., 1998; Oishi et al., 1998; Zylka et al., 1998). Additionally, many other cells in the adult also express the molecular components of the circadian cycle, and continue to cycle in the absence of control by the SCN (Balsalobre et al., 1998; Zylka et al., 1998; Yamazaki et al., 2000). This applies also to embryonic cells where the SCN has not yet formed. In particular, zebra®sh embryos inherit circadian cycle information through the egg and maintain the same phase from this point throughout development (Delaunay et al., 2000). The clock can be reset by environmental inputs, of which light seems to be the most important. Light exposure causes an acute induction of Per1 and Per2 and this may be impor-
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00559-1
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tant in the resetting mechanism (Albrecht et al., 1997; Shearman et al., 1997; Zylka et al., 1998). We wished to study the control of the Clock gene in early Xenopus development. The Xenopus homologue of Clock (Xclk) is very similar to the mouse and human genes and is expressed in the retinal photoreceptor cells at a constant level (Anderson and Green, 2000; Zhu et al., 2000). We ®nd that, like the murine clock (Mclk) gene, Xclk does not seem to undergo cyclical changes in expression early on in development (Tei et al., 1997; Shearman et al., 1999). It's expression is initially restricted to the anterior neural plate and then subsequently expands along the whole neural tube. Xclk expression is an early response to neural induction, and is strongly upregulated by the homeodomain containing transcription factor Otx2 early in gastrulation.
gastrulation, Xclk transcripts are localized to the mesodermal cells of the dorsal blastopore lip and the ectodermal cells that lie above it (Fig. 2A). Mesodermal expression is lost during gastrulation, but continues in the ectoderm that reaches the anterior end of the embryo by the late gastrula stage (stage 12.5, Fig. 2B,C). This region is the anterior most part of the neural plate, and expression persists here through neurulation (Fig. 2D) and the tailbud stages (Fig. 2E±H), but extends caudally along the spinal cord after the late neurula stage, forming an apparent gradient of expression decreasing posteriorly along it (Fig. 2G). A transverse section through the ocular region of the forebrain at the late tailbud stage (Fig. 2I) reveals that expression is still restricted to the ectoderm, and is expressed in the eyes as well as the forebrain.
2. Results
2.3. Xclk expression can be induced by noggin and further increased by Otx2
2.1. The level of Clock RNA does not cycle in a circadian manner
The ®rst zygotic expression of Xclk is con®ned to the Spemann organizer and overlying ectoderm at the early
To study the role of Xclk in early development we began by examining it's expression from fertilization through to the late tailbud stage (Fig. 1). It is clear that Xclk transcripts are present maternally, at stage 2 (after the second cleavage). This is before the onset of zygotic transcription, which occurs around the time of the mid blastula transition (MBT). The amount of Xclk RNA decreases rapidly after the MBT (stage 8), but then begins to increase later in gastrulation (stage 12). This increase continues steadily throughout neurulation and the tailbud stages. As Xclk is a close homologue of a gene that is required for circadian oscillations in other organisms, we decided to examine whether it might display a temporal dependence in early Xenopus development, rather than depending entirely on developmental stage. In order to do this, we took advantage of the general temperature-insensitivity of circadian clocks (Pittendrigh, 1981), together with the strong temperature-dependence that Xenopus displays in embryonic development. Speci®cally, at 14.58C Xenopus embryos develop far more slowly than at, for example, 21.58C, although the actual developmental progression the embryos follow is identical (Nieuwkoop and Faber, 1994). We examined the amount of Xclk at the same developmental stages in embryos kept at these two different temperatures (Fig. 1). From comparing these two series it is apparent that the amount of Xclk RNA varies only with developmental stage, and is not dependent on the time elapsed since fertilization per se. 2.2. Xclk is expressed initially in the organizer and subsequently in the anterior neural plate To study the spatial distribution of Xclk transcripts during development we performed whole-mount in situ hybridization using an antisense Xclk probe. Shortly after the onset of
Fig. 1. Xclk expression varies with developmental stage, not time. RNA was extracted from embryos at different developmental stages and analyzed for Xclk and Ornithine Decarboxylase (ODC) expression by RT-PCR. The latter gene is expressed at a roughly constant level throughout early development and is used as a loading control. Embryos were cultured either at 14.5 oC or 21.58C, and the time taken to reach each of the developmental stages analyzed is shown. (A) Original RT-PCR data. (B) Semi-quantitative analysis of RT-PCR data. The value represented by the black bars is the Xclk/ODC signal ratio (£100) after background compensation.
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Fig. 2. The expression of Xclk at early developmental stages. (A) Animal to vegetal section through a stage 10 (early gastrula) embryo, the animal pole being uppermost. The arrow head marks the position of the dorsal blastopore lip. (B,C) A stage 12.5 (late gastrula) embryo. (B) Dorsal view. The arrow head marks the blastopore, the future posterior side. Xclk staining is apparent at the opposite end of the embryo (anterior). (C) The same embryo as (B) sectioned along the dotted line. (D) Lateral view of a stage 17 (neurula) embryo, anterior to left. (E) Dorsal view of a stage 22 embryo, and (F) enlargement of the head of the same embryo. (G) Lateral view of a tailbud stage embryo (stage 27), and (H) enlargement of the head of the same embryo. (I) Dorsal±ventral section through the ocular region of the embryo shown in (G), e, eye; fb, forebrain.
gastrula stage (Fig. 2A). This is coincident with another organizer gene, noggin (Smith and Harland, 1992; Smith et al., 1995). Subsequently the expression patterns of these two genes diverge somewhat, with Xclk being strongly expressed in the most anterior part of the neural plate and excluded from the mesoderm. Given this initial coincident expression though, together with numerous previous studies establishing noggin as a secreted polypeptide that can induce ectoderm to differentiate as anterior neural plate (Lamb et al., 1993; Bier, 1997; Weinstein and HemmatiBrivanlou, 1999), we decided to test whether noggin can induce Xclk expression in ectoderm. We injected fertilized Xenopus eggs with noggin RNA and subsequently removed the ectoderm from blastula (stage 9) embryos. We refer to these explants as animal caps. Animal caps from untreated embryos develop as undifferentiated ectoderm, but the presence of noggin RNA within them (or protein added exogenously) induces them to develop as anterior neural tissue (Lamb et al., 1993), expressing genes characteristic of this tissue such as the cell adhesion molecule NCAM (Balak et al., 1987) or the transcription factor opl (Kuo et al., 1998). The results shown in Fig. 3 reveal that noggin has caused neural induction in these animal caps, as demonstrated by the induction of
NCAM and opl. In addition, noggin has also induced Xclk expression, albeit at a low level. The strongest Xclk expression is in the anterior most neural plate and subsequently the forebrain and eyes. This is similar to the ectodermal expression of the homeodomain containing transcription factor Otx2 (Pannese et al., 1995), a highly conserved gene that has an important role in head development. Indeed, at the late gastrula stage the expression of Xclk appears to be identical to the ectodermal expression domain of Otx2 (Fig. 2B,C). Thus, we looked to see whether Otx2 could affect Xclk expression. For this purpose we used of a fusion between Otx2 and the human glucocorticoid receptor (Otx-GR). The glucocorticoid receptor binds heat shock proteins, preventing it from entering the nucleus. This steric hindrance of nuclear entry is relieved by ligand binding, in this case the glucocorticoid analogue dexamethasone (dex), which by itself has no discernible effects on Xenopus development. Hence the Otx-GR construct confers dex dependence on the activity of Otx2, without it seems any perturbation of Otx2 function (Gammill and Sive, 1997). We injected fertilized eggs with Otx-GR RNA and subsequently cut animal caps. Adding dex to activate Otx2 does not induce Xclk expression (Fig. 3), however, in caps that
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simple intracellular system for generating circadian oscillations (Antoch et al., 1997; King et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998; Shearman et al., 1999). Unlike other components of this molecular pathway though, Mclk is not itself cycle under circadian control. This is also true of Xclk in Xenopus retina cells (Zhu et al., 2000), and here we demonstrate that it is also the case in early development (Fig. 1). The fact that Xclk is not under circadian control may make a useful control point in cells. It could for example be regulated by speci®c transcription factors and signalling pathways, which could in turn determine which cells will undergo circadian oscillations but not then interfere with these oscillations once they are in motion. The level of CLOCK protein is probably not limiting once the cycle has started, hence buffering circadian events from changes in the original activating signal.
Fig. 3. Xclk is induced by noggin in animal caps. Caps were taken from either untreated embryos or from embryos injected RNA encoding noggin or OtxGR, as shown. Dexamethasone (dex) was used to activate OTX protein, and cyclohexamide (chx) to block protein synthesis. Subsequently, caps were analyzed for the expression of a number of genes by RT-PCR. 2RT, control PCR reaction on extracted RNA without reverse transcription.
also contain noggin RNA a very strong increase in Xclk was observed. Use of the Otx-GR construct has the distinct advantage of allowing us to determine whether Xclk is a direct target of Otx2 transcriptional activation. By pre-incubating caps with cyclohexamide (chx), a drug that blocks protein synthesis, followed by Otx2 activation by dex, only genes that are dependent on the addition of active OTX protein alone should be transcribed (Gammill and Sive, 1997). We thus treated noggin and Otx-GR containing caps with both dex and chx, under these conditions Xclk is still transcribed, indicating that it is a direct target of Otx (Fig. 3). As a control, we looked to see if two genes expressed speci®cally in the cement gland were also induced, XCG and XAG. Both have previously been shown to be upregulated by Otx2, but whilst XCG is activated directly XAG is not (Gammill and Sive, 1997). As expected, both XCG and XAG are induced by Otx2 in our study, but only the induction of XAG is blocked by cyclohexamide. Chx and dex alone have no effect on the expression on any of the genes examined (Fig. 3). 3. Discussion 3.1. The Clock gene is required for the generation of circadian oscillations but is not under circadian control The Xclk gene is very closely related to the mouse Clock gene (Zhu et al., 2000), functional studies of which have demonstrated that it is a central component of the relatively
3.2. Xclk expression can be induced by noggin and up regulated by Otx If Xclk expression represents a possible start up event for circadian oscillations, where in the embryo does it start? We have demonstrated that Xclk expression is found initially in the organizer region and overlying ectoderm, coincident with the neural inducer noggin. Further, noggin can induce Xclk expression in ectodermal explants along with markers of neural plate (Fig. 3), including the cell adhesion molecule NCAM. Interestingly, NCAM is required for photic resetting of the circadian clock in mice, and a mutation in NCAM that blocks it's conjugation to polysialic acid results in the gradual running down of the circadian cycle in the SCN (Shen et al., 1997; Glass et al., 2000). The expression of Xclk can be upregulated directly by Otx2, a homeodomain containing transcription factor (Fig. 3). Otx2 is expressed in the most anterior region of both Xenopus and mouse embryos in cells that are destined to form the forebrain and other anterior structures (Lamb et al., 1993; Pannese et al., 1995; Blitz and Cho, 1995; Boncinelli et al., 1993). In Xenopus, Otx2 has been shown to directly induce genes required for cement gland formation, an organ that is considered to be the most anterior tissue of the frog embryo (Gammill and Sive, 1997). Mice lacking Otx2 gene function do not form a recognizable forebrain (Ang et al., 1996) and Drosophila embryos with a null mutation for the homologous gene orthodenticle lack the anterior most portion of the head (Finkelstein and Perrimon, 1990; Finkelstein et al., 1990). As would be expected if noggin and Otx were regulators of Xclk, it's expression becomes con®ned later on in gastrulation to the most anterior neurectoderm (Fig. 2). Is circadian activity restricted to these cells? In zebra®sh this is indeed the case, here the cyclical expression of the period gene is predominantly in the eyes and forebrain with a phase that seems to be maternally inherited (Delaunay et al., 2000).
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Subsequently Xclk expression expands caudally towards the posterior end of the embryo, but strikingly it's expression is still restricted to the nervous system even up to very late developmental stages (Fig. 2). The only expression outside the neural plate is seen in the mesodermal cells of the organizer mesoderm, but this may be a response to the relatively high level of noggin expressed there. 3.3. Why is Xclk expression restricted to the anterior neural plate in early development? Vertebrate embryos have only one function, and that is to form the adult body. In general, but in amphibians in particular, this process is unaffected by external in¯uences short of those that are actually injurious. Hence light, sound and other stimuli that affect the behavior of the adult have no perceptible effect. The metabolic processes that drive these developmental processes do not need to respond to the regular, repeated events that the circadian cycle anticipates. Indeed, circadian in¯uences could interfere with the complex controls that govern development, and it may thus be prudent not to have global circadian regulation at this stage. Why is the circadian cycle maintained in the anterior neural plate at this time? This question has not yet been addressed, but it is perhaps hardly surprising to ®nd expression in these cells that will form the eyes and brain, tissues that will be ®rst to perceive the unceasingly changing world. 4. Materials and methods 4.1. Embryos, explants and over expression studies In vitro fertilization, embryo culture, staging, microinjection and culture of explants were carried out as previously described (Pannese et al., 1995). Dexamethasone and cyclohexamide treatments were performed as previously described (Gammill and Sive, 1997). Capped RNA transcripts of otx2-GR and noggin were prepared using the Ambion in vitro transcription kit (Ambion, USA). 4.2. Constructs Xclk in situ probe: bases 551±1261 of Xclk (accession number AF227985) were subcloned in to vector pGEMTEASY (Promega). For antisense in situ probes this was linearized with NcoI and transcribed with SP6 polymersase. 4.3. RT-PCR PCR assays with reverse transcription (RT-PCR) were carried out in the exponential phase of ampli®cation as previously described (Busse and Seguin, 1993), with minor modi®cations. PCR primers are; for Xopl (accession number AF028805) U 5 0 CCG CAA CCG GGG CTT CGG 3 0 and D 5 0 GAA GCC CAG CCT CAT CTG 3 0 , for Xclk (accession number AF227985) U 5 0 CGC GCT ACT ATA
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TCA CCT ACC 3 0 and D 5 0 GAT CCG AGT TCT CTG CTC C 3 0 and for XAG U 5 0 CAA AGA AGC AGG CAG GAA CAA CT 3 0 and D 5 0 ATC GTC AGC GTC ATA GGC ATA CAT 3 0 . The other primers used are described on the internet at http://vize222.zo.utexas.edu Semi-quantitative analysis of RT-PCR data was performed using a Fluoroimager 595 (Molecular Dynamics), staining gels with Vista Green (Amersham) at 1:10 000 in order to visualize the bands. Background was subtracted using the area on the gel immediately above each of the relevant bands as a reference. The signal from each data point was compared to the corresponding ODC signal. 4.4. Whole-mount in situ hybridization An antisense, DIG-labeled transcript of Xclk was prepared in vitro according to the manufacturers instructions (Roche). The in situ protocol used has been described previously (Harland, 1991). Roche BM Purple was used as a precipitating reagent for alkaline phosphatase. Acknowledgements We thank Stephan Wacker for supplying embryos and Hans Jansen for quantifying the RT-PCR data shown in Fig. 1. A.J.D. thanks the E.U. HPRN programme for support. R.M. thanks St. George's Hospital Medical School for support. References Albrecht, U., Sun, Z.S., Eichele, G., Lee, C.C., 1997. A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91, 1055±1064. Anderson, F.E., Green, C.B., 2000. Symphony of rhythms in the Xenopus laevis retina. Microsc. Res. Tech. 50, 360±372. Ang, S.L., Jin, O., Rhinn, M., Daigle, N., Stevenson, L., Rossant, J., 1996. A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122, 243±252. Antoch, M.P., Song, E.J., Chang, A.M., Vitaterna, M.H., Zhao, Y., Wilsbacher, L.D., Sangoram, A.M., King, D.P., Pinto, L.H., Takahashi, J.S., 1997. Functional identi®cation of the mouse circadian Clock gene by transgenic BAC rescue. Cell 89, 655±667. Balak, K., Jacobson, M., Sunshine, J., Rutishauser, U., 1987. Neural cell adhesion molecule expression in Xenopus embryos. Dev. Biol. 119, 540±550. Balsalobre, A., Damiola, F., Schibler, U., 1998. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929±937. Bier, E., 1997. Anti-neural-inhibition: a conserved mechanism for neural induction. Cell 89, 681±684. Blitz, I.L., Cho, K.W.Y., 1995. Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. Development 121, 993±1004. Boncinelli, E., Gulisano, M., Broccoli, V., 1993. Emx and Otx homeobox genes in the developing mouse brain. J. Neurobiol. 24, 1356±1366. Busse, U., Seguin, C., 1993. Molecular analysis of the Wnt-1 proto-oncogene in Ambystoma mexicanum (axolotol) embryos. Differentiation 53, 7±15.
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