DEVELOPMENT 1253 RESEARCH ARTICLE INTRODUCTION The development of the vertebrate hindbrain involves a transient segmentation process along the anteroposterior (AP) axis, leading to the formation of seven or eight transversal morphological units, called rhombomeres (r) (Vaage, 1969) (reviewed by Lumsden and Keynes, 1989; Lumsden and Krumlauf, 1996; Wingate and Lumsden, 1996). The rhombomeres behave as compartments, constituting units of cell lineage restriction (Fraser et al., 1990; Birgbauer and Fraser, 1994) and domains of specific gene expression (reviewed by Lumsden and Krumlauf, 1996; Rijli et al., 1998). This subdivision presages the metameric pattern of neuronal specification in the hindbrain (Lumsden and Keynes, 1989; Clarke et al., 1998), underlies the pathways of neural crest cell migration into the branchial arches and participates in their patterning (Lumsden et al., 1991; Serbedzija et al., 1992; Birgbauer et al., 1995; Kulesa and Fraser, 2000; Trainor and Krumlauf, 2000; Trainer et al., 2002; Ghislain et al., 2003), thus playing an essential role in craniofacial morphogenesis. Numerous genes have been implicated at different levels of the segmentation process, including the initial formation of segmental territories (Lufkin et al., 1991; Chisaka et al., 1992; Schneider- Maunoury et al., 1993; Frohman et al., 1993; McKay et al., 1994; Barrow et al., 2000; Waskiewicz et al., 2001; Deflorian et al., 2004; Choe and Sagerstrom, 2004; McNulty et al., 2005), the specification of their AP identities (Rijli et al., 1993; Studer et al., 1996; Seitanidou et al., 1997; Rossel and Capecchi, 1999; Bell et al., 1999) and their stabilisation by restriction of cell intermingling between adjacent rhombomeres (reviewed by Pasini and Wilkinson, 2002), and development of specific cell populations at boundaries (Cheng et al., 2004; Amoyel et al., 2005). Segment formation and specification are highly intricate processes in the hindbrain, with several genes participating in both aspects (Gavalas et al., 1998; Rossel and Capecchi, 1999; Voiculescu et al., 2001). Krox20, which encodes a zinc-finger transcription factor (Chavrier et al., 1988), plays a key role in coupling segmentation and specification of rhombomere identity (Voiculescu et al., 2001). Krox20 is one of the earliest genes expressed in a segmental pattern in the developing hindbrain, specifically in odd-numbered rhombomeres r3 and r5 (Wilkinson et al., 1989; Schneider- Maunoury et al., 1993). Krox20 mutation leads to loss of r3 and r5 (Schneider-Maunoury et al., 1993; Swiatek et al., 1993), owing to mis-specification of these territories, which acquire r2 and r4, and r6 identities, respectively (Voiculescu et al., 2001). Krox20 has been shown to exert its function by up- and downregulating the expression of numerous genes, including Hox genes of the paralogous groups 1 to 3, such as Hoxb1, Hoxa2, Hoxb2 and Hoxb3, which are also involved in the specification of segmental AP identity (Sham et al., 1993; Nonchev et al., 1996a; Nonchev et al., 1996b; Vesque et al., 1996; Seitanidou et al., 1997; Giudicelli et al., 2001; Manzanares et al., 2002) (M. Garcia-Dominguez, P. Gilardi and P.C., unpublished), and the EphA4 receptor gene, which is involved in the segregation of cells between odd- and even- numbered rhombomeres (Theil et al., 1998). Krox20 has also been shown to positively regulate its own expression, both cell- autonomously and non cell-autonomously, and these processes are Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements Diane Chomette 1 , Monique Frain 1 , Silvia Cereghini 2 , Patrick Charnay 1, * and Julien Ghislain 1 The vertebrate hindbrain is subject to a transient segmentation process leading to the formation of seven or eight metameric territories termed rhombomeres (r). This segmentation provides the basis for the subsequent establishment of hindbrain neuronal organization and participates in the patterning of the neural crest involved in craniofacial development. The zinc-finger gene Krox20 is expressed in r3 and r5, and encodes a transcription factor that plays a key role in hindbrain segmentation, coordinating segment formation, specification of odd- and even-numbered rhombomeres, and cell segregation between adjacent segments, through the regulation of numerous downstream genes. In order to further elucidate the genetic network underlying hindbrain segmentation, we have undertaken the analysis of the cis-regulatory sequences governing Krox20 expression. We have found that the control of Krox20 transcription relies on three very long-range (200 kb) enhancer elements (A, B and C) that are conserved between chick, mouse and human genomes. Elements B and C are activated at the earliest stage of Krox20 expression in r5 and r3- r5, respectively, and do not require the Krox20 protein. These elements are likely to function as initiators of Krox20 expression. Element B contains a binding site for the transcription factor vHNF1, the mutation of which abolishes its activity, suggesting that vHNF1 is a direct initiator of Krox20 expression in r5. Element A contains Krox20-binding sites, which are required, together with the Krox20 protein, for its activity. This element therefore allows the establishment of a direct positive autoregulatory loop, which takes the relay of the initiator elements and maintains Krox20 expression. Together, our studies provide a basis for a model of the molecular mechanisms controlling Krox20 expression in the developing hindbrain and neural crest. KEY WORDS: Hindbrain segmentation, Pattern formation, Transcriptional enhancers, vHnf1, Mouse, Chick Development 133, 1253-1262 (2006) doi:10.1242/dev.02289 1 INSERM, U784, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France. 2 UMR CNRS 7622, Université Pierre et Marie Curie, 9 quai Saint-Bernard, 75252 Paris Cedex 05, France. *Author for correspondence (e-mail: charnay@biologie.ens.fr) Accepted 18 January 2006