山东大学：《发育生物学 Developmental Biology》课程教学资源（参考资料）Avian hairy Gene Expression Identifies a Molecular Clock Linked to Vertebrate Segmentation and Somitogenesis

Cell, Vol 91, 639-648, November 28, 1997, Copyright @1997 by Cell Press Avian hairy Gene Expression Identifies a molecular clock inked to vertebrate Segmentation and Somitogenesis Isabel Palmeirim, Domingos Henrique, t5 are laid down sequentially from a terminal growth zone David Ish-Horowicz, and olivier Pourquietll during the course of development Institut d'Embryologie Cellulaire et Moleculare te embryos, the most obvious metameric du Centre National de la Recherche Scientifique structures are the somites. They constitute the basis of et du College de france the segmental pattern of the body and give rise to the 49 bis avenue de la belle gabrielle axial skeleton, the dermis of the back and all striated 94736 Nogent sur Marne Cedex muscles of the adult body (christ and Ordahl, 1995 FI Individual pairs of somites, located symmetrically on t Imperial Cancer Research Fund either side of the neural tube, emerge from the rostra PO Box 123. 44 Lincolns inn fields nd of the presomitic mesoderm(PSm), while new mes- London wc2A 3PX enchymal cells enter the caudal paraxial mesoderm, as a consequence of gastrulation In the chick embryo, a t Developmental Biology Institute of Marseille(IBDM) somite pair is laid down every 90 min in a rostro-caudal LGPD-UMR CNRS 6545 Campus progression, and a total of 50 somite pairs are formed de Luminy- case 907 during embryogenesis. The presomitic mesoderm ap 13288 Marseille cedex 9 pears as a long strip of mesenchymal tissue, and surgi- France al experiments have shown that approximately 10-12 prospective somites are contained within the 2-day-old chick PSM(Packard, 1976; L P. et al., unpublished data) levelopment. Also, it has been suggested that the PSM lentified and characterized c-hairyl, an includes up to 12"somitomeres, segmented arrange ments of cells that can be visualized using the electron avian homolog of the Drosophila segmentation gene microscope and that may correspond to prospective hairy. c-hairy1 is strongly expressed in the presomitic somites(Meier, 1984) mesoderm, where its mRNA exhibits cyclic waves of Although various models have been proposed to ac expression whose temporal periodicity corresponds count for segmentation in vertebrates little is current to the formation time of one somite(90 min). The ap- known about underlying molecular mechanisms(see parent movement of these waves is due to coordinated Keynes and Stern, 1988: Tam and Trainor, 1994, and pulses of c-hairyl expression, not to cell displacement eferences therein: Discussion below). Numerous verte along the anteroposterior axis, nor to propagation of brate homologs of the Drosophila segmentation genes an activating signal. Rather, the rhythmic c-hairy have been identified but are not expressed during somi- mRNA expression is an autonomous property of the togenesis. However, homologs of the neurogenic genes paraxial mesoderm. These results provide molecular lotch, Delta(Delta like-1: Deltan)and RBPik genes evidence for a developmental clock linked to segmen- which are not involved in segmentation in the fly, have tation and somitogenesis of the paraxial mesoderm, been implicated in vertebrate somitogenesis. Targ and support the possibility that segmentation mecha inactivation of these genes in mice leads to a disruption nisms used by invertebrates and vertebrates have of somitogenesis( Conlon et al. 1995; Oka et al., 1995 been conserved Hrabe de Angelis et al. 1997). Nevertheless, although somitogenesis is disrupted in DeltaT-- mice, paraxial Introduction mesoderm derivatives such as muscles or skeleton re- tain a segmented pattern ( hrabe de angelis et al 1997) Identification and characterization of the Drosophila These results, therefore, support the view that segmen melanogaster segmentation genes has led to a recent tation occurs independently of somitogenesis, and were revival of interest in mechanisms underlying verte- also taken as confirmation of the widely held view that brate segmentation(Lewis, 1978; Nasslein-Volhard and vertebrates 1996: De Robertis, 1997). However, the process of seg- mentation in Drosophila differs significantly from that of of the chick c hairy 1 gene, an avian homolog of the more primitive insects or vertebrates. In long germband Drosophila hairy segmentation gene. In Drosophila sects such as the fly, segments are determined essen- airy is a member of the pair-rule genes, which are the tially simultaneously in a syncytial unicellular embryo. st to reveal the prospective metameric body plan prior to gastrulation. In more primitive short germband the fly(Nusslein -Volhard and Wieschaus, 1980; Ish Horowicz et al. 1985). Here, we show that c- insects like orthopterans, in other arthropods such as mRNA is expressed in a highly dynamic manner crustaceans, and in vertebrates, segment determination occurs in a cellularized embryo, and posterior segments chick PSM, appearing as a caudo-rostral wave is reiterated during the formation of somite. We demonstrate that this wavefront is not due to cell move. s present address: Instituto de Histologia e Embriologia, Faculdade ments within the PSM, nor to the periodic production of an anterior-to-posterior diffusing signal, but is I To whom correspondence should be addressed tonomous property of the cells in this tissue. We show

Cell, Vol. 91, 639–648, November 28, 1997, Copyright 1997 by Cell Press Avian hairy Gene Expression Identifies a Molecular Clock Linked to Vertebrate Segmentation and Somitogenesis are laid down sequentially from a terminal growth zone during the course of development. In vertebrate embryos, the most obvious metameric structures are the somites. They constitute the basis of the segmental pattern of the body and give rise to the Isabel Palmeirim,* Domingos Henrique,†§ David Ish-Horowicz,† and Olivier Pourquie´ ‡k *Institut d’Embryologie Cellulaire et Mole´culaire du Centre National de la Recherche Scientifique et du Colle` ge de France 49 bis avenue de la Belle Gabrielle axial skeleton, the dermis of the back, and all striated 94736 Nogent sur Marne Cedex muscles of the adult body (Christ and Ordahl, 1995). France Individual pairs of somites, located symmetrically on † either side of the neural tube, emerge from the rostral Imperial Cancer Research Fund PO Box 123, 44 Lincolns Inn Fields end of the presomitic mesoderm (PSM), while new mes￾London WC2A 3PX enchymal cells enter the caudal paraxial mesoderm, as United Kingdom a consequence of gastrulation. In the chick embryo, a ‡Developmental Biology Institute of Marseille (IBDM) somite pair is laid down every 90 min in a rostro-caudal LGPD-UMR CNRS 6545 Campus progression, and a total of 50 somite pairs are formed de Luminy - case 907 during embryogenesis. The presomitic mesoderm ap￾pears as a long strip of mesenchymal tissue, and surgi- 13288 Marseille cedex 9 cal experiments have shown that approximately 10–12 France prospective somites are contained within the 2-day-old chick PSM (Packard, 1976; I. P. et al., unpublished data). Its length becomes progressively reduced during later Summary development. Also, it has been suggested that the PSM includes up to 12 “somitomeres,” segmented arrange- We have identified and characterized c-hairy1, an ments of cells that can be visualized using the electron avian homolog of the Drosophila segmentation gene, microscope and that may correspond to prospective hairy. c-hairy1 is strongly expressed in the presomitic somites (Meier, 1984). mesoderm, where its mRNA exhibits cyclic waves of Although various models have been proposed to ac- expression whose temporal periodicity corresponds count for segmentation in vertebrates, little is currently to the formation time of one somite (90 min). The ap- known about underlying molecular mechanisms (see parent movement of these waves is due to coordinated Keynes and Stern, 1988; Tam and Trainor, 1994, and pulses of c-hairy1 expression, not to cell displacement references therein; Discussion below). Numerous verte￾along the anteroposterior axis, nor to propagation of brate homologs of the Drosophila segmentation genes an activating signal. Rather, the rhythmic c-hairy have been identified but are not expressed during somi￾mRNA expression is an autonomous property of the togenesis. However, homologs of the neurogenic genes, paraxial mesoderm. These results provide molecular Notch, Delta (Delta like-1:Delta1) and RBPjk genes, evidence for a developmental clock linked to segmen- which are not involved in segmentation in the fly, have tation and somitogenesis of the paraxial mesoderm, been implicated in vertebrate somitogenesis. Targeted and support the possibility that segmentation mecha- inactivation of these genes in mice leads to a disruption nisms used by invertebrates and vertebrates have of somitogenesis (Conlon et al., 1995; Oka et al., 1995; been conserved. Hrabe de Angelis et al., 1997). Nevertheless, although somitogenesis is disrupted in Delta12/2 mice, paraxial Introduction mesoderm derivatives such as muscles or skeleton re￾tain a segmented pattern (Hrabe de Angelis et al., 1997). These results, therefore, support the view that segmen- Identification and characterization of the Drosophila melanogaster segmentation genes has led to a recent tation occurs independently of somitogenesis, and were also taken as confirmation of the widely held view that revival of interest in mechanisms underlying verte- segmentation arose independently in vertebrates and brate segmentation (Lewis, 1978; Nu¨ sslein-Volhard and Wieschaus, 1980; Tautz and Sommer, 1995; Kimmel, invertebrates. In this paper, we report the identification and analysis 1996; De Robertis, 1997). However, the process of seg- of the chick c-hairy1 gene, an avian homolog of the mentation in Drosophila differs significantly from that of Drosophila hairy segmentation gene. In Drosophila, more primitive insects or vertebrates. In long germband hairy is a member of the pair-rule genes, which are the insects such as the fly, segments are determined essen- first to reveal the prospective metameric body plan of tially simultaneously in a syncytial unicellular embryo, the fly (Nu¨ sslein-Volhard and Wieschaus, 1980; Ish- prior to gastrulation. In more primitive short germband Horowicz et al., 1985). Here, we show that c-hairy1 insects like orthopterans, in other arthropods such as mRNA is expressed in a highly dynamic manner in the crustaceans, and in vertebrates, segment determination chick PSM, appearing as a caudo-rostral wave, which occurs in a cellularized embryo, and posterior segments is reiterated during the formation of every somite. We demonstrate that this wavefront is not due to cell move- § ments within the PSM, nor to the periodic production Present address: Instituto de Histologia e Embriologia, Faculdade de Medicina, Av. Prof. Egas Moniz, 1699 Lisboa Codex, Portugal. of an anterior-to-posterior diffusing signal, but is an au￾kTo whom correspondence should be addressed. tonomous property of the cells in this tissue. We show

RY 1 XAIR￥1 Hλ PP D30Q9A二二二P c=HATR了1 PG8-------------- 基A￥12吉5 TR工BHA工RY1 释 I RY 1 Se-e-- HAI RY I m52:!你 E VIORVPMEOO PL S LVIKK SE c·HAIR1 H粪IRY1 恚灘 Figure 1. Sequence Analysis of c-hairy7 and the insect hairy protein 二 highest homology with the Xenopus x-hairy he zebrafish Hero, and the mammalian He eads) and the orange do 1996)(between white arrowheads) are we as the tetrapeptide WRPW at the carboxyl terminus, essential to recruit the corepressor Groucho and exert its negative effect on the transcriptional apparatus(Paroush et al., 1994 that blocking protein synthesis in embryo explants leads screen a random-primed CDNA library prepared from to an arrest of somitogenesis but that the oscillations chick embryonic mRNA, and several positive clones of c-hairy1 expression persist. This provides evidence were isolated. Sequence analysis of a fraction of these against the cyclic c-hairy 1 expression being under nega CDNAs revealed that they arise from a new gene, named tive autoregulatory control. Together, these results dem C-hairy. Comparison with other vertebrate Hairy-like onstrate that cells of the PSM undergo a defined and genes reveals that c-hairy] is most similar to the Xeno- constant number of c-hairy1 expression cycles between pus laevis hairy, the mammalian HES, and the zebrafish emergence from the primitive streak and incorporation Herb genes( Figure 1) into a somite. The rhythmic oscillations of the c-hairyl The putative c-hairy1 protein is 291 amino acids long ssenger RNA in prospective somitic cells provide the including a bHLH domain and the tetrapeptide WRPw first molecular evidence in favor of a developmental at the carboxyl terminus, which are characteristic fea clock involved in vertebrate segmentation ures of the hairy-related class of bHLH transcription factors in flies and vertebrates(Figures 1 and 2). Analysis Results of the c-hairy1 sequence suggests that it belongs to a ubgroup of the WRPw-containing bHLH proteins. Identification of an Avian hairy Homolog(c-hairyn) which includes mammalian HESI and HES2, Xenopus Expressed in the Paraxial Mesoderm X-hairy1, zebrafish Her6, and the fly and tribolium ha To identify chick homologs of the fly pair-rule gene he (Figure 1). The Enhancer-of-split and the zebrafish Her1 we used a PCR-based approach with degenerate oligo- genes are only distantly related to these hairy-like g nucleotides that correspond to sequences conserved( Figure 2). In Drosophila, these proteins act as transcrip- between the two hairy-like genes in Drosophila (hairy tional repressors in a variety of developmental contexts and deadpan). An initial PCR fragment was used to(Ohsako et al. 1994; Paroush et al. 1994: Van Doren et

Cell 640 Figure 1. Sequence Analysis of c-hairy1 Comparison of the c-hairy1 protein sequence with that of other vertebrate homologs belonging to the Hairy/Enhancer-of-split (HES) family and the insect hairy proteins using the ClustalX programme (Higgins et al., 1996). c-hairy1 shows highest homology with the Xenopus x-hairy1, the zebrafish Her6, and the mammalian HES genes. The bHLH domain (between black arrowheads) and the orange domain (Fisher et al., 1996) (between white arrowheads) are well conserved between all the HES proteins, as well as the tetrapeptide WRPW at the carboxyl terminus, essential to recruit the corepressor Groucho and exert its negative effect on the transcriptional apparatus (Paroush et al., 1994; Fisher et al., 1996). that blocking protein synthesis in embryo explants leads screen a random-primed cDNA library prepared from to an arrest of somitogenesis but that the oscillations chick embryonic mRNA, and several positive clones of c-hairy1 expression persist. This provides evidence were isolated. Sequence analysis of a fraction of these against the cyclic c-hairy1 expression being under nega- cDNAs revealed that they arise from a new gene, named tive autoregulatory control. Together, these results dem- c-hairy1. Comparison with other vertebrate Hairy-like onstrate that cells of the PSM undergo a defined and genes reveals that c-hairy1 is most similar to the Xeno￾constant number of c-hairy1 expression cycles between pus laevis hairy1, the mammalian HES, and the zebrafish emergence from the primitive streak and incorporation Her6 genes (Figure 1). into a somite. The rhythmic oscillations of the c-hairy1 The putative c-hairy1 protein is 291 amino acids long, messenger RNA in prospective somitic cells provide the including a bHLH domain and the tetrapeptide WRPW first molecular evidence in favor of a developmental at the carboxyl terminus, which are characteristic fea￾clock involved in vertebrate segmentation. tures of the hairy-related class of bHLH transcription factors in flies and vertebrates (Figures1 and 2). Analysis Results of the c-hairy1 sequence suggests that it belongs to a subgroup of the WRPW-containing bHLH proteins, Identification of an Avian hairy Homolog (c-hairy1) which includes mammalian HES1 and HES2, Xenopus Expressed in the Paraxial Mesoderm X-hairy1, zebrafish Her6, and the fly and tribolium hairy To identify chick homologs of the fly pair-rule gene hairy, (Figure 1). The Enhancer-of-split and the zebrafish Her1 we used a PCR-based approach with degenerate oligo- genes are only distantly related to these hairy-like genes nucleotides that correspond to sequences conserved (Figure 2). In Drosophila, these proteins act as transcrip￾between the two hairy-like genes in Drosophila (hairy tional repressors in a variety of developmental contexts and deadpan). An initial PCR fragment was used to (Ohsako et al., 1994; Paroush et al., 1994; Van Doren et

Molecular Clock Linked to Vertebrate Segmentation This dynamic expression sequence is reiterated dur- ng the formation of every somite and can be repre sented as a cycle of three successive stages( Figure 3 bottom). In stage L, c-hairy] transcripts are detected in broad domain comprising the posterior 70% of the PSM(corresponding to at least eight prospective so mites) and in a narrow band in the ective caudal art of the forming somite(somite O). In stage ll, the osterior band of C-hairyl expression has narrowed to about 3 somite-equivalents in length and has moved E45 anteriorly, so it now lies in the rostral half of the PSM In stage Ill, the c-hairy1 expression domain becomes narrower than a somite-equivalent and moves further anteriorly, forming a stripe coincident with the caudal part of prospective somite O Transitions between these stages are observed, indi cating that c-hairy1 is expressed as a continuous and dynamic sequence rather than abrupt switches from one tage to the other. For example, the broad caudal stripe bserved in stage i begins to appear during stage Ill (Figure 3C), indicating that stage ll is indeed a precursor Figure 2. Phylogenetic Tree Analysis of b-HLH Proteins of the Hairy to the next stage I and that the anterior c-hairy1 st and Enhancer-of-Split Families Indicate that C-Hairyl Belongs to in stage Ill is a precursor to the stripe in somite 0 of he Hairy Family of Proteins stage I. In addition, the intensity of c-hairy1 expression g the Distances and Growtree progam- es of the GCG package Version 9.0(Madison, Wisconsin). Another increases between stage I and stage Ill. Out of 71 em ed tree calculated on a Power Macintosh using the Clus- bryos analyzed, 24 were found in stage I, 22 in stage I genset al., 1996 generated a substantially simi- ind 25 in stage Ill. Based on a cycle time of 90 min, we (not shown). All compared sequences are accessible in estimate that each stage lasts about 30 min The reiterated patterns of c-hairy1 expression at the different stages examined suggest that the wavefront of C-hairyI in the unsegmented mesoderm occurs in Trib, Tribolium Castaneum: Hes 1. Hes2 Hes3, and Hes5 are the rat a cyclic fashion correlated with somite formation. To investigate this further, we cultured bilaterally divided avian embryos in vitro, under conditions where the PSM yields at least three new somites according to in vivo al. 1994 Jimenez et al. 1996 Fisher et al. 1996). Given kinetics (one somite per 90 min). The caudal parts of the structural conservation, it is likely that c-hairy1 func 2-day-old embryos including the PSM were removed he c-hairyi gene was analyzed during chick embryonic alves. One embryonic half was fixed immediately, and development and was detected in several tissues(data he other half cultured on a filter for 30-270 min prior not shown). In this paper, we focus our attention on to fixation. Both halves were then hybridized with the mesoderm expression, in particular on the presomitic C-hairy7 probe, and the expression pattern on the two mesoderm, where c-hairy] revealed a very dynamic sides was compared( Figure 4) mRNA expression pattern After culturing for 30 to 60 min, the patterns of c-hairyt expression in the cultured and uncultured presomitic Cyclic c-hairy] mRNA Expression in the Paraxial mesoderm always differ( Figure 4A, n= 18), demonstrat Mesoderm is correlated with somite formation ing the extremely dynamic nature of the expression of Analysis of the c-hairy1 mRNA expression pattern in the for 90 min, the time required to form one somite, the araxial mesoderm was carried out by whole mount in c-hairyl expression patterns in the PSMs of cultured situ hybridization of embryos containing between 1 and and uncultured halves are identical, reflecting the cyo 25somites.c-hairylexpression is detected in the caudal property of this expression pattern( Figure 4B; n= 25) part of somites at all stages examined, where it persists he same rhythmicity of c-hairy] expression profile is in the caudal sclerotome for at least 15 hr(Figure 3 and so observed when half embryos are cultured for 270 data not shown). By contrast, c-hairyT expression in the in, corresponding to the time required to form three presomitic mesoderm is highly dynamic, as reflected somites in vivo(n = 3; data not shown). Therefore, the by the variety of expression patterns that are seen in wavefront of c-hairy1 expression in the PSM occurs embryos with an identical number of somites. The do- in a cyclic fashion, with a periodicity that correlates main of c-hairy7 expression has the appearance of a precisely with somite formation wavefront beginning in the broad, caudal PSM, pro- gressing anteriorly and intensifying into the narrow ante- The Wave of c-hairy1 mRNA Expression in rior PSM(Figure 3, top). Finally, in each cycle, expression the Presomitic Mesoderm Is Independe decays sharply throughout the PSM except for a thin of Cell Movement stripe corresponding to the posterior part of the forming Several mechanisms could account for the kinetics of somite(somite O c-hairy1 expression in the PSM. One simple possibility

Molecular Clock Linked to Vertebrate Segmentation 641 This dynamic expression sequence is reiterated dur￾ing the formation of every somite and can be repre￾sented as a cycle of three successive stages (Figure 3, bottom). In stage I, c-hairy1 transcripts are detected in a broad domain comprising the posterior 70% of the PSM (corresponding to at least eight prospective so￾mites) and in a narrow band in the prospective caudal part of the forming somite (somite 0). In stage II, the posterior band of c-hairy1 expression has narrowed to about 3 somite-equivalents in length and has moved anteriorly, so it now lies in the rostral half of the PSM. In stage III, the c-hairy1 expression domain becomes narrower than a somite-equivalent and moves further anteriorly, forming a stripe coincident with the caudal part of prospective somite 0. Transitions between these stages are observed, indi￾cating that c-hairy1 is expressed as a continuous and dynamic sequence rather than abrupt switches from one stage to the other. For example, the broad caudal stripe observed in stage I begins to appear during stage III (Figure 3C), indicating that stage III is indeed a precursor Figure 2. Phylogenetic Tree Analysis of b-HLH Proteins of the Hairy to the next stage I and that the anterior c-hairy1 stripe and Enhancer-of-Split Families Indicate that c-Hairy1 Belongs to in stage III is a precursor to the stripe in somite 0 of the Hairy Family of Proteins stage I. In addition, the intensity of c-hairy1 expression The tree was generated using the Distances and Growtree progam- increases between stage I and stage III. Out of 71 em- mes of the GCG package Version 9.0 (Madison, Wisconsin). Another bryos analyzed, 24 were found in stage I, 22 in stage II, bootstrapped tree calculated on a Power Macintosh using the Clus- and 25 in stage III. Based on a cycle time of 90 min, we talX programme (Higgins et al., 1996) generated a substantially simi￾estimate that each stage lasts about 30 min. lar tree (not shown). All compared sequences are accessible in Genbank. Espl, Enhancer-of-split; HES, Hairy and Enhancer-of-split The reiterated patterns of c-hairy1 expression at the genes; Her, Hairy and Enhancer-of-split related genes; X, Xenopus; different stages examined suggest that the wavefront M, mouse; Zf, zebrafish; Hu, human; Dm, Drosophila Melanogaster; of c-hairy1 in the unsegmented mesoderm occurs in Trib, Tribolium Castaneum; Hes1, Hes2, Hes3, and Hes5 are the rat a cyclic fashion correlated with somite formation. To genes. investigate this further, we cultured bilaterally divided avian embryos in vitro, under conditions where the PSM yields at least three new somites according to in vivo al., 1994; Jime´ nez et al., 1996; Fisher et al., 1996). Given kinetics (one somite per 90 min). The caudal parts of the structural conservation, it is likely that c-hairy1 func- 2-day-old embryos including the PSM were removed tions similarly during chick development. Expression of and separated surgically along the midline into two the c-hairy1 gene was analyzed during chick embryonic halves. One embryonic half was fixed immediately, and development and was detected in several tissues (data the other half cultured on a filter for 30–270 min prior not shown). In this paper, we focus our attention on to fixation. Both halves were then hybridized with the mesoderm expression, in particular on the presomitic c-hairy1 probe, and the expression pattern on the two mesoderm, where c-hairy1 revealed a very dynamic sides was compared (Figure 4). mRNA expression pattern. After culturing for 30 to60 min, the patterns of c-hairy1 expression in the cultured and uncultured presomitic mesoderm always differ (Figure 4A, n 5 18), demonstrat- Cyclic c-hairy1 mRNA Expression in the Paraxial ing the extremely dynamic nature of the expression of Mesoderm Is Correlated with Somite Formation this mRNA. However, when half embryos are cultured Analysis of the c-hairy1 mRNA expression pattern in the for 90 min, the time required to form one somite, the paraxial mesoderm was carried out by whole mount in c-hairy1 expression patterns in the PSMs of cultured situ hybridization of embryos containing between 1 and and uncultured halves are identical, reflecting the cyclic 25somites. c-hairy1 expression is detected in thecaudal property of this expression pattern (Figure 4B; n 5 25). part of somites at all stages examined, where it persists The same rhythmicity of c-hairy1 expression profile is in the caudal sclerotome for at least 15 hr (Figure 3 and also observed when half embryos are cultured for 270 data not shown). By contrast, c-hairy1 expression in the min, corresponding to the time required to form three presomitic mesoderm is highly dynamic, as reflected somites in vivo (n 5 3; data not shown). Therefore, the by the variety of expression patterns that are seen in wavefront of c-hairy1 expression in the PSM occurs embryos with an identical number of somites. The do- in a cyclic fashion, with a periodicity that correlates main of c-hairy1 expression has the appearance of a precisely with somite formation. wavefront beginning in the broad, caudal PSM, pro￾gressing anteriorly and intensifying into the narrow ante- The Wave of c-hairy1 mRNA Expression in rior PSM (Figure3, top). Finally,in each cycle,expression the Presomitic Mesoderm Is Independent decays sharply throughout the PSM except for a thin of Cell Movement stripe corresponding to the posterior part of the forming Several mechanisms could account for the kinetics of somite (somite 0). c-hairy1 expression in the PSM. One simple possibility

15S 16S 17s S1 St S SI- n in the Presomitic Mesoderm Defines a Highly Dynamic Caudal-to-Rostral Expression Sequence Reiterated (Top)In situ hybridization with c-hairy1 probe showing the different categories of c-hairyl expression patterns in embryos aged of 15(A, B, nd C), 16(D, E, and F), and 17(G, H, and n) somites. Rostral to the top Bar=200 ottom) Schematic representation of the correlation between c-hairy1 expression in the PSM with the progression of somite formation. While a new somite is forming from the rostral-most PSM(somite 0: So), a narrow stripe of c-hairyf is observed in its caudal aspect, and a larg caudal expression domain extends rostrally from the tail bud region (stage I: A, D, and G). As somite formation proceeds, as evidenced by the visualization of the appearing caudal fissure, the c-hairyl expression expands anteriorly, the caudal-most domain disappears, and c-hairyl ars as a broad stripe in the rostral PSM (stage ll; B, E, and H) When somite O is almost formed, the stripe has considerably narrowed, and c-hairyl is detected in the caudal part of the pros (stage Ill; C, F, and n) while a new caudal expression domain arises from the tail bud region (in C can be seen the beginning of stage I of the next cycle). This highly dynamic sequence of c-hairy1 expression in the PSM was observed at all stages of somitogenesis examined (from 1 to 25 somites), suggesting a cyclic expression of the c-hairy1 mRNA correlated with somite formation. Arrowheads point to the most recently mpletely formed somite(somite l: S) is that the wavefront reflects extensive caudo-rostral patterns differ( Figure 5). This experiment clearly indi- movement of c-hairyl expressing cells during somite cates that the progression of the -hairy] wavefront formation. This appears unlikely because previous work occurs independently of cell movement. It also confirms has indicated that cell movement within the psm is re and extends the results of cell grafting experiments in stricted (Tam and Beddington, 1986; Stern et al., 1988) he mouse and of tracer injection into single PSM cells If the c-hairy 1-expressing cells in stage ll were to derive which demonstrated that their progeny never encom- from cells in stage I, they would have to move across pass more than two consecutive segments(Tam and about 50% of the ps M, a distance greater than 450 um, Beddington, 1986; Stern et aL., 1988) in less than 30 min To exclude the possibility that cell migration contrib- Rhythmic Expression of c-hairy1 Is an Autonomous es to the dynamics of c-hairy1 expression, we have Property of the Presomitic Mesoderm arked small clusters of cells at the same anteroposte- What might drive the caudal-to-rostral wavefront of rior level in both the left and right PSM with Dil. The C-hairy1 expression in the PSM? One possibility is that caudal part of these embryos was then separated into it results from a periodic signal originating at the poste- its two halves as described previously, and one half was rior end of the PSM, which spreads and activates immediately fixed while the other was cultured for 30 C-hairy1 in successively more anterior cells. This relay lin prior to fixation. The Dil was then photoconverted hypothesis predicts that a discontinuity within the PSM to an insoluble DAB precipitate, and both halves were would interrupt spreading of the signal and halt the ante hybridized with the c-hairy7 probe In all observed cases rior progression of c-hairy1 expression. To test this idea (n=8), Dil labeled cells are found at exactly the same c-hairy expression was assayed in half embryos in level in the two halves whereas the c-hairy1 expression which the caudal part of the PSM including the tailbud

Cell 642 Figure 3. c-hairy1 mRNA Expression in the Presomitic Mesoderm Defines a Highly Dynamic Caudal-to-Rostral Expression Sequence Reiterated during Formation of Each Somite (Top) In situ hybridization with c-hairy1 probe showing the different categories of c-hairy1 expression patterns in embryos aged of 15 (A, B, and C), 16 (D, E, and F), and 17 (G, H, and I) somites. Rostral to the top. Bar 5 200 mm. (Bottom) Schematic representation of the correlation between c-hairy1 expression in the PSM with the progression of somite formation. While a new somite is forming from the rostral-most PSM (somite 0:S0), a narrow stripe of c-hairy1 is observed in its caudal aspect, and a large caudal expression domain extends rostrally from the tail bud region (stage I; A, D, and G). As somite formation proceeds, as evidenced by the visualization of the appearing caudal fissure, the c-hairy1 expression expands anteriorly, the caudal-most domain disappears, and c-hairy1 appears as a broad stripe in the rostral PSM (stage II; B, E, and H). When somite 0 is almost formed, the stripe has considerably narrowed, and c-hairy1 is detected in the caudal part of the prospective somite (stage III; C, F, and I) while a new caudal expression domain arises from the tail bud region (in C can be seen the beginning of stage I of the next cycle). This highly dynamic sequence of c-hairy1 expression in the PSM was observed at all stages of somitogenesis examined (from 1 to 25 somites), suggesting a cyclic expression of the c-hairy1 mRNA correlated with somite formation. Arrowheads point to the most recently completely formed somite (somite I:SI). is that the wavefront reflects extensive caudo-rostral patterns differ (Figure 5). This experiment clearly indi￾movement of c-hairy1 expressing cells during somite cates that the progression of the c-hairy1 wavefront formation. This appears unlikely because previous work occurs independently of cell movement. It also confirms has indicated that cell movement within the PSM is re- and extends the results of cell grafting experiments in stricted (Tam and Beddington, 1986; Stern et al., 1988). the mouse and of tracer injection into single PSM cells, If the c-hairy1-expressing cells in stage II were to derive which demonstrated that their progeny never encom￾from cells in stage I, they would have to move across pass more than two consecutive segments (Tam and about 50% of the PSM, a distance greater than 450 mm, Beddington, 1986; Stern et al., 1988). in less than 30 min. To exclude the possibility that cell migration contrib- Rhythmic Expression of c-hairy1 Is an Autonomous utes to the dynamics of c-hairy1 expression, we have Property of the Presomitic Mesoderm marked small clusters of cells at the same anteroposte- What might drive the caudal-to-rostral wavefront of rior level in both the left and right PSM with DiI. The c-hairy1 expression in the PSM? One possibility is that caudal part of these embryos was then separated into it results from a periodic signal originating at the poste￾its two halves as described previously, and one half was rior end of the PSM, which spreads and activates immediately fixed while the other was cultured for 30 c-hairy1 in successively more anterior cells. This relay min prior to fixation. The DiI was then photoconverted hypothesis predicts that a discontinuity within the PSM to an insoluble DAB precipitate, and both halves were would interrupt spreading of thesignal and halt the ante￾hybridized with the c-hairy1 probe. In all observed cases rior progression of c-hairy1 expression. To test this idea, (n 5 8), DiI labeled cells are found at exactly the same c-hairy1 expression was assayed in half embryos in level in the two halves whereas the c-hairy1 expression which the caudal part of the PSM including the tailbud

Molecular Clock Linked to Vertebrate Segmentation 301 Figure 4. Cyclic Expression of c-hairy1 RNA in the Presomitic Meso- Figure 5. Cell Movements Do Not Account for c-hairyl Expression derm Correlates with somite formation Kinetics he caudal regions of 15-to 20-somite embryos (inclu divided into two halves after Dil labeling of a small group of cells into two halves. One half (left side)was immediately fixed, and the at the same anteroposterior level in the left and right PSM. One half /bridized with c-hairyl probe (A) Experimental half-embryo cultured for 30 min A different expres- hybridized with c-hairy1 probe after photoconversion of the Dil. In ion pattern is observed between the two halves, indicating the tremely dynamic nature of C-hairyl expressi while in(B), it progresses from stage ll to stage Ill. Cells labeled (B)Experimental half-embryo cultured for 90 min( the time required with Dil appear brown owing to the formation of DAB precipita for the formation of one somite). The same expression pattern is nd are indicated by white arrows. In both ex found in both halves, indicating that c-hairy1 expression pattern cated at exactly the same anteroposterior level after a 30 min cycles over a period exactly corresponding to somite formation Open arrowhead, sor ds, segmented somites. Ros- indicating that expression dynamics are not due to cell movements tral to the top Bar 350 um in the PSM. Asterisk marks the last formed somite. Bar= 150 um was surgically ablated(n= 8). The same expression their circuitry involves unstable components that are pattern is observed in ablated and unoperated halves ubject to negative autoregulation (reviewed in Sas- even after extended culture( Figures 6A-6C). Therefore, sone-Corsi, 1994: Dunlap, 1996). The dynamic pattern cycling of c-hairy1 expression in the rostral PSM is inde- of c-hairy1 expression and the likelihood that c-hairy1 pendent of the presence of a caudal PSM, and the pro- is a transcriptional repressor led us to ask whether gression of the c-hairyl-expressing wavefront during c-hairyl is itself a central component of the clock mech- somite formation is not related to the spreading of a anism or if its cyclical transcription reflects an output signal originating in the posterior part of the embryo and from the clock. to address these questions, we exam- travelling anteriorly along the cells in the PSM. ined the effects of blocking protein synthesis on c-hairyl These experiments suggest that the dynamic c-hairyl expression lence reflects an autonomous property Half-embryo explants were incubated in cyclohex of the PSM. We therefore studied the c-hairyl expres- mide for up to 90 min while the contralateral half was sion pattern in explant cultures of presomitic mesoderm fixed immediately. When explants are cultured for less isolated from all the surrounding tissues that might be than 75 min, the fixed and incubated halves show differ providing extrinsic signals. The presomitic mesoderm ent patterns, indicating that inhibiting protein synthesis of one- half of 15-to 25-somite embryos was separated loes not block c-hairy1 oscillations(n =4/4: Figure from ectoderm, endoderm, neural tube, notochord lat 7A). We confirmed this result by studying half-embryos eral plate, and tail bud while the other half remained cultured for equal times in the presence or absence of intact. The two halves were cultured separately for peri- cycloheximide. For the first 60 min of culture, treated ods between 30 and 180 min(n=31). c-hairy1 expres and untreated halves show the same patterns of c-hairy sion patterns are similar in both types of explant( Figures expression(n=11/11: Figures 7C and 7D), suggesting 6D-6F), suggesting that the kinetics of c-hairy1 expres- that the periodicity of c-hairy1 pulsing is initially inde- sion are independent of surrounding tissues, and derive pendent of de novo protein synthesis autonomously from the PSM. Moreover, these cultures Nevertheless, protein synthesis may be required for ing that cycling in the caudal PsM does not depend on ultured in cycloheximide for 90 min(one somite equiva a signal from the node( Figure 6) nt usually show a different pattern of expression from halves fixed immediately(n=6/9; Figure 7B). Also Periodic Oscillations of c-hairyl Are Independent C-hairy1 expression in half-embryos cultured for 90 min of Protein Synthesis or more in the presence of cycloheximide often differs The above results show that c-hairy1 mRNA is ex- from that in the matched half-embryos incubated with pressed cyclically in cells of the PSM and are consistent out the drug(n=8/16; Figures 7E and 7F). Thus, a 90 with clock models for somitogenesis(see Discussion). min periodicity is not maintained in such longer term Studies of other clock control mechanisms indicate that cultures

Molecular Clock Linked to Vertebrate Segmentation 643 Figure 4. CyclicExpression of c-hairy1RNA in thePresomitic Meso- Figure 5. Cell Movements Do Not Account for c-hairy1 Expression derm Correlates with Somite Formation Kinetics The caudal regions of 15- to 20-somite embryos (including the pre- The caudal regions of 15- to 20-somite embryos were sagittally somitic mesoderm and the last few somites) were sagittally divided divided into two halves after DiI labeling of a small group of cells into two halves. One half (left side) was immediately fixed, and the at the same anteroposterior level in the left and right PSM. One half other half (right side) was incubated on top of a millipore filter. Both (left side) was immediately fixed, and the other half (right side) was halves were hybridized with c-hairy1 probe. incubated on top of a millipore filter for 30 min. Both halves were (A) Experimental half-embryo cultured for 30 min. A different expres- hybridized with c-hairy1 probe after photoconversion of the DiI. In sion pattern is observed between the two halves, indicating the (A), the c-hairy1 expression pattern changes from stage I to stage extremely dynamic nature of c-hairy1 expression. II1 while in (B), it progresses from stage II to stage III. Cells labeled (B) Experimental half-embryo cultured for 90 min (the time required with DiI appear brown owing to the formation of DAB precipitate for the formation of one somite). The same expression pattern is and are indicated by white arrows. In both examples, the cells are found in both halves, indicating that c-hairy1 expression pattern located at exactly the same anteroposterior level after a 30 min cycles over a period exactly corresponding to somite formation. culture periodwhile the c-hairy1expression patternhas progressed, Open arrowhead, somite 0; arrowheads, segmented somites. Ros- indicating that expression dynamics are not due to cell movements tral to the top. Bar 5 350 mm. in the PSM. Asterisk marks the last formed somite. Bar 5 150 mm. was surgically ablated (n 5 8). The same expression their circuitry involves unstable components that are pattern is observed in ablated and unoperated halves, subject to negative autoregulation (reviewed in Sas￾even after extended culture (Figures 6A–6C). Therefore, sone-Corsi, 1994; Dunlap, 1996). The dynamic pattern cycling of c-hairy1 expression in the rostral PSM is inde- of c-hairy1 expression and the likelihood that c-hairy1 pendent of the presence of a caudal PSM, and the pro- is a transcriptional repressor led us to ask whether gression of the c-hairy1-expressing wavefront during c-hairy1 is itself a central component of the clock mech￾somite formation is not related to the spreading of a anism or if its cyclical transcription reflects an output signal originating in the posterior part of the embryo and from the clock. To address these questions, we exam￾travelling anteriorly along the cells in the PSM. ined the effects of blocking protein synthesis on c-hairy1 These experiments suggest that the dynamic c-hairy1 expression. expression sequence reflects an autonomous property Half-embryo explants were incubated in cyclohexi￾of the PSM. We therefore studied the c-hairy1 expres- mide for up to 90 min while the contralateral half was sion pattern in explant cultures of presomitic mesoderm fixed immediately. When explants are cultured for less isolated from all the surrounding tissues that might be than 75 min, the fixed and incubated halves show differ￾providing extrinsic signals. The presomitic mesoderm ent patterns, indicating that inhibiting protein synthesis of one-half of 15- to 25-somite embryos was separated does not block c-hairy1 oscillations (n 5 4/4; Figure from ectoderm, endoderm, neural tube, notochord, lat- 7A). We confirmed this result by studying half-embryos eral plate, and tail bud while the other half remained cultured for equal times in the presence or absence of intact. The two halves were cultured separately for peri- cycloheximide. For the first 60 min of culture, treated ods between 30 and 180 min (n 5 31). c-hairy1 expres- and untreated halves showthe same patterns of c-hairy1 sion patterns are similar in both types of explant (Figures expression (n 5 11/11; Figures 7C and 7D), suggesting 6D–6F), suggesting that the kinetics of c-hairy1 expres- that the periodicity of c-hairy1 pulsing is initially inde￾sion are independent of surrounding tissues, and derive pendent of de novo protein synthesis. autonomously from the PSM. Moreover, these cultures Nevertheless, protein synthesis may be required for cycle normally although Hensens node is absent, show- continued periodicity of c-hairy1 expression. Explants ing that cycling in the caudal PSM does not depend on cultured in cycloheximide for 90 min (one somite equiva￾a signal from the node (Figure 6). lent) usually show a different pattern of expression from halves fixed immediately (n 5 6/9; Figure 7B). Also, Periodic Oscillations of c-hairy1 Are Independent c-hairy1 expression in half-embryos cultured for 90 min of Protein Synthesis or more in the presence of cycloheximide often differs The above results show that c-hairy1 mRNA is ex- from that in the matched half-embryos incubated with￾pressed cyclically in cells of the PSM and are consistent out the drug (n 5 8/16; Figures 7E and 7F). Thus, a 90 with clock models for somitogenesis (see Discussion). min periodicity is not maintained in such longer term Studies of other clock control mechanisms indicate that cultures