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that approximately 10-12 prospective somites are contained within the 2-day-old chick PSM(Packard, 1976; L P. et al., unpublished data) levelopment.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 showCell, 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