Identification of Four Engrailed Genes in the Japanese Lamprey

DEVELOPMENTAL DYNAMICS 237:1581–1589, 2008

RESEARCH ARTICLE

Identiﬁcation of Four Engrailed Genes in the Japanese Lamprey, Lethenteron japonicum

Manami Matsuura,1 Hidenori Nishihara,2 Koh Onimaru,1 Nobuhiro Kokubo,1 Shigehiro Kuraku,3† Rie Kusakabe,3‡ Norihiro Okada,2 Shigeru Kuratani,3 and Mikiko Tanaka1*

We have isolated four homologs of Engrailed genes from the Japanese lamprey, Lethenteron japonicum,an agnathan that occupies a critical phylogenic position between cephalochordates and gnathostomes. We named these four genes LjEngrailedA, LjEngrailedB, LjEngrailedC, and LjEngrailedD. LjEngrailedA, LjEngrailedB, and LjEngrailedD share a major expression domain in the presumptive midbrain–hindbrain boundary region of the central nervous system, although their levels and timing of expression differed. On the other hand, LjEngrailedC transcripts were in the pharyngeal ectoderm and the ventral ectoderm of the body wall. In addition, LjEngrailedA was expressed in the ventral side of the epibranchial muscle precursors. LjEngrailedD transcripts were seen in the mesodermal cells of the mandibular arch and later in a group of cells responsible for the formation of the upper lip, lower lip, and velum. Our results provide clues to the evolution of these structures as well as a possible scenario for duplication events of Engrailed genes. Developmental Dynamics 237:1581–1589, 2008. © 2008 Wiley-Liss, Inc.

Key words: Engrailed; lamprey; homeodomains; midbrain; hindbrain; epibranchial muscle; mandibular arch

Accepted 24 March 2008

INTRODUCTION 1976). En homologs have been cloned teleosts produced four En homologs from several protochordates (Holland in zebrafish: eng1a (renamed eng1), The Engrailed (En) genes encode a et al., 1997; Imai et al., 2002) and eng1b, eng2a (renamed eng2), and highly conserved homeodomain-con- many vertebrate species (Joyner et eng2b (renamed eng3) (Ekker et al., taining transcription factor and play pivotal roles in morphogenesis in in- al., 1985). The cephalochordate Bran- 1992; Force et al., 1999). Four En vertebrate and vertebrate develop- chiostoma floridae and urochordate paralogues, En-1A, En-1B, En-2A, and ment (Gibert, 2002). In Drosophila, Ciona intestinalis are reported to have En-2B have also been identified in the engrailed is known as a selector gene, only a single En gene, whereas chick, tetraploid Xenopus laevis (Holland as it is involved in the development of mouse, and humans contain two mem- and Williams, 1990; Hemmati-Brivan- appendages and in the establishment bers of the En family, En-1 and En-2 lou et al., 1991). In cephalochordates of segments, whereby it specifies the (Joyner et al., 1985; Joyner and Mar- and vertebrates, segmentally reiter- posterior identity of each compart- tin, 1987; Poole et al., 1989; Holland et ated En expression has been observed ment (Garcia-Bellido and Santama- al., 1997; Imai et al., 2002). Whole ge- in the paraxial mesoderm (Davidson ria, 1972; Lawrence and Morata, nome duplication prior to the origin of et al., 1988; Ekker et al., 1992; Hol-

1Laboratory for Developmental Biology, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan 2Laboratory for Molecular Evolution, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan 3Laboratory for Evolutionary Morphology, Center for Developmental Biology, RIKEN, Kobe, Japan Grant sponsor: Ministry of Education, Science, Sports and Culture of Japan; Grant sponsor: Hayashi Memorial Foundation of Female Natural Scientists. †Shigehiro Kuraku’s present address is Laboratory of Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Universitaetsstrasse 10, 78457 Konstanz, Germany. ‡Rie Kusakabe’s present address is Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan. *Correspondence to: Mikiko Tanaka, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-17, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. E-mail: [email protected] DOI 10.1002/dvdy.21552 Published online 9 May 2008 in Wiley InterScience (www.interscience.wiley.com).

© 2008 Wiley-Liss, Inc. 1582 MATSUURA ET AL. land et al., 1997). In amphioxus, midbrain and hindbrain of the neural they seem to have duplicated within metameric expression of AmphiEn in epithelium (Davis et al., 1991; Ekker the lineage (Holland and Williams, the forming somites was observed et al., 1992). Recent studies in mice 1990). En protein distribution has (Holland et al., 1997). Somitic En-1 showed that the development of the been studied in the lamprey using the expression was also observed in the tectum (midbrain) and cerebellum polyclonal antiserum aEnhb-1 raised muscle pioneer-like cells in the prim- (hindbrain) depends upon the dose of against mouse En protein (Holland et itive cartilaginous dogfish Scyliorhi- En proteins expressed in the mid- al., 1993). However, no En mRNA ex- nus canicula (Tanaka et al., 2002). In brain-hindbrain boundary (Sgaier et pression patterns have been described zebrafish, eng1a and eng2a expression al., 2007). In chick and mouse em- in lamprey during embryogenesis. was also observed in a subset of mus- bryos, ectodermal expression of En-1 In this study, we obtained 3 novel cle precursor cells in the myotomes, is seen in the ventral compartment of En sequences LjEnB, LjEnC, and such as muscle pioneers, during somi- the body epidermis at the pre-fin/limb LjEnD, in a Japanese lamprey, togenesis (Ekker et al., 1992). A small bud stage and then in the ventral ec- Lethenteron japonicum. Moreover, the cluster of mesodermal cells in the toderm and ventral apical ridge of nucleotide sequence of the previously mandibular arch also express eng2a limb buds where it positions limbs at reported partial LjEnA gene (Takio et and eng2b during zebrafish embryo- the dorso-ventral boundary (Loomis et al., 2007) was extended and its expres- genesis (Ekker et al., 1992). These al., 1996; Laufer et al., 1997; Rodri- sion was analayzed throughout em- cells seem to be the jaw muscle pre- guez-Esteban et al., 1997; Tanaka et bryogenesis. Phylogenetic and expres- cursor cells derived from the paraxial al., 1998). This limb-positioning mech- sion analyses provided valuable mesoderm (Hatta et al., 1990) and anism of En-1 appears to be also con- insights into the evolution of verte- similar mandibular En expression is served in the primitive cartilaginous brate novel morphologies, as well as a observed in Xenopus laevis (Hemmati- dogfish (Tanaka et al., 2002). possible model of duplication events Brivanlou et al., 1991), chick (Gardner Because the above-mentioned re- whereby En genes have undergone and Barald, 1992), and mouse (Logan gions of En expression, including mus- subfunctionalization of their roles. et al., 1993). En-expression has been cle of the oral apparatus, tectum, and observed in the somites of chick and cerebellum, and function are major mice specifically in the dermis precur- morphological innovations along the RESULTS sor cells (Davis et al., 1991; Gardner chordate lineage, examination of En Identification of En and Barald, 1992). expression in agnathan lampreys Sequences in the Lamprey En expression is also observed in should provide further understanding neurons of hemichordates and various of vertebrate evolution. As Verte- L. japonicum chordates (Davis et al., 1991; Holland brates acquired the jaw after the ori- To identify En genes in the lamprey et al., 1997; Imai et al., 2002; Lowe et gin of agnathan vertebrates, expres- L. japonicum, we used an RT-PCR al., 2003). In the hemichordate Sacco- sion pattern of lamprey En genes in approach with stage-21 to -30 em- glossus kowalevski, which lacks a cen- the pharyngeal regions should provide bryos using degenerate primers. tralized nervous system, En is significant insights into the acquisi- This strategy led to amplification of strongly expressed in the anterior me- tion of the jaw in vertebrate evolution. several fragments of En genes. sosome of the neurogenic ectoderm as The optic tectum and cerebellum are Three of them, which we named a narrow single band (Lowe et al., the novel morphological innovations LjEngrailedA (LjEnA; Takio et al., 2003). In chordates, the ectoderm be- in vertebrate evolution; thus delineat- 2007), LjEngrailedB (LjEnB), LjEn- comes partitioned into the neuroecto- ing the expression patterns of the lam- grailedC (LjEnC), were closely re- derm and the non-neural ectoderm prey En genes should provide further lated to gnathostome En genes and (also named general ectoderm; Hol- understanding for the evolution of the were further characterized. We ex- land, 2005), both of which appear to be subdivisions of the brain. Although tended their sequences using a combi- patterned along the anterior-posterior the lamprey lacks paired fins, some nation of 5Ј and 3Ј RACE to yield 873-, axis by a common mechanism (Hol- agnathan fossils seem to have one set 784-, and 167-bp fragments, corre- land, 2005). In amphioxus, AmphiEn of paired fins, and these ancestral ag- sponding, respectively, to each gene. is expressed in a small cluster of cells nathan fish might have already had The ortholog of LjEnC was then ex- in the dorsal nerve cord and the cere- dorso-ventral compartmentalization. tended 264 bp using primers against bral vesicle, and it is transiently ex- Thus, examination of En gene expres- the EnC sequences from sea lamprey pressed in a band of cells of the gen- sion patterns in the body ectoderm of (Petromyzon marinus) retrieved from eral ectoderm (Holland et al., 1997; lamprey embryos could provide in- the NCBI Trace Archive database Holland, 2005). In Ciona intestinalis, sight into the evolutionary process of (http://www.ncbi.nlm.nih.gov/Traces/). Ci-En is restricted to two domains of these appendages (Tanaka et al., The sequence of LjEnC was further the central nervous system during 2002). extended using 3Ј RACE to yield embryogenesis: the midbrain and the In lampreys, only a single En se- 661-bp fragments. An additional En midbrain-hindbrain boundary (Imai quence has been identified in Lam- gene, P. marinus EnD (PmEnD), was et al., 2002). In zebrafish, Xenopus, petra fluviatilis and Petromyzon mari- also found in this search. The ortholog chick, and mouse, En genes are ex- nus (Holland and Williams, 1990; of PmEnD in L. japonicum, LjEnD pressed in a single broad stripe in the Force et al., 1999). Indeed, the hagfish (402 bp), was isolated by genomic PCR boundary between the presumptive also has two En homologs, although using PmEnD-specific primers. Se- FOUR ENGRAILED GENES IN THE LAMPREY 1583

Fig. 1. Phylogenetic analysis of L. japonicum and P. marinus En genes. The NJ phylogenetic trees of the En genes constructed with the (a) amino acid (JTT model) and (b) nucleotide (first and second codon positions, Tamura-Nei model) sequences (see Experimental Procedures section). Only the conserved EH1–5 regions were used for both analyses. Numbers above branches represent bootstrap values with 10,000 replicates. quence analysis of these fragments vious study, Ks values have been cal- when faint expression first appeared and comparison with the En gene culated for several other orthologous in the neural tube at the presumptive from a variety of species showed that genes between the two genera, and border between the future midbrain these four clones are orthologs of en- they ranges from 0.01 to 0.36 (Kuraku and hindbrain (Takio et al., 2007; ar- grailed (Fig. 1a). Phylogenetic analy- and Kuratani, 2006). Because the Ks rowheads in Fig. 2a,b). By stage 25, sis was performed using four En gene values for En genes are similar to expression in the neural tube was sequences of L. japonicum with those those of other genes, PmEnA, PmEnB, more intensified (arrowheads in Fig. of various chordates and sea urchins. PmEnC, and PmEnD are probably or- 2c,d). LjEnA was expressed in this re- The En sequences of sea lamprey and thologs of LjEnA, LjEnB, LjEnC, and gion until at least stage 28 (Fig. 2e–n, P. marinus retrieved from the NCBI LjEnD, respectively. q), the last embryonic stage examined. Trace Archive database were also The tree topology within the lam- Additional expression was first ob- used for the analysis. NJ (neighbor- prey clade is the same for the amino served in a group of rostral myotomes joining) trees were constructed with acid (Fig. 1a) and nucleotide (Fig. 1b) at stage 25 (arrows in Fig. 2c,d). By both amino acid and nucleotide (first datasets. Among them, PmEnA, stage 25.5, the number of labelled and second codon positions) datasets. PmEnB, PmEnC, and PmEnD in P. myotomes increased and extended Interestingly, all the lamprey En marinus form sister relationships of rostrally (arrows in Fig. 2e–g, o). By genes are grouped together in both LjEnA, LjEnB, LjEnC, and LjEnD in stage 27, LjEnA was expressed in the phylogenetic trees (Fig. 1). L. japonica, respectively. These re- rostral end of the extended infraoptic In addition, to confirm the orthology sults suggest that the four En genes muscle precursor cells (arrows in Fig. among the eight En sequences in the were duplicated in the lamprey lin- 2h–j, r) and remained expressed at lampreys, we performed a maximum- eage before the divergence of L. ja- least until stage 28 (arrows in Fig. likelihood estimation of Ks (synony- ponicum and P. marinus. 2k–m). mous substitution rate) for the EH1-5 Expression of LjEnB first appeared regions of the four En genes using the Expression of Lamprey LjEn at stage 22 in the neural tube at the PAML 3.14 program. The Ks values future midbrain-hindbrain boundary Genes calculated between P. marinus and L. (arrowheads in Fig. 3a,b). Expression japonicum are as follows: Ks (EnA) ϭ Expression of LjEnA was examined in in the neural tube persisted during 0.08, Ks (EnB) ϭ 0.45, Ks (EnC) ϭ lamprey developmental stages 21–28. subsequent stages, and became more 0.35, and Ks (EnD) ϭ 0.18. In the pre- Staining was absent until stage 22, intensified (arrowheads in Fig. 3c,d). 1584 MATSUURA ET AL.

Fig. 2.

At stage 25, cells expressing LjEnB were detectable at the midbrain-hindbrain boundary (arrowheads in Fig. 3e–g). Weak LjEnB expression remained at the border between the midbrain and hindbrain at least until stage 28 (arrowheads in Fig. 3h–m). Faint expression of LjEnC ﬁrst appeared at stage 25 on the ventral side of the pharyngeal ectoderm (arrows in Fig. 4a–c). Expression of LjEnC was detected in the ectoderm over the pharyngeal arches at stage 26 (arrows in Fig. 4d–f). By stage 27, LjEnC expression intensiﬁed in the pharyngeal ectoderm (arrows in Fig. 4g–i, k) and was maintained until stage 28.5 (data not shown). By stage 29, LjEnC expression in the pharyngeal ectoderm was much lower (data not shown). On the ventral side of the body ectoderm,

Fig. 3. FOUR ENGRAILED GENES IN THE LAMPREY 1585 very faint LjEnC expression was first quently another duplication produced mouse En protein in L. japonicum seen at stage 26 (Fig. 4d). These sig- LjEnB. The sister relationship of (Holland et al., 1993). Thus, En ex- nals were seen from the heart level to LjEnA and LjEnD is strongly sup- pression in the central nervous system the cloaca level (arrows in Fig. 4j, l,m), ported. Therefore, at least three dupli- appears to be one of shared features of but no signals were detected in the cation events of LjEn genes seem to all the vertebrates (Takio et at., 2007; ectoderm caudal to the cloaca (Fig. have occurred after divergence of the see Fig. 6). Redundancy in the func- 4n). The LjEnC expression boundary lamprey lineage from other verte- tion of En genes in the midbrain-hind- is almost at the dorsal margin of so- brates. It remains unclear whether brain boundary has been demon- matopleure in the body ectoderm (ar- the LjEn genes originated from chro- strated in mice by the complete rows in Fig. 4j, l,m) and its expression mosomal-scale duplications only from absence of the tectum and cerebellum persisted through stage 28 (data not our results. However, given that mul- in En-1;En-2 double knockout mice shown). tiple copies of Hox and related genes (Liu and Joyner, 2001). Although En-1 LjEnD transcripts were first de- such as Dlx were found in the lamprey and En-2 have overlapping expression tected at stage 23 in a few cells of the genome (Force et al., 1999; Neidert et patterns, their levels and timing of ex- mandibular arch and in the border of al., 2001; Irvine et al., 2002; Takio et pression differ in various organisms the presumptive midbrain-hindbrain al., 2007), it is possible that the LjEn (Ekker et al., 1992; Wurst et al., region (Fig. 5a,b). At stage 25, the me- genes diverged by duplications involv- 1994). For example, in mice, En-1 is soderm of the mandibular arch orga- ing those Hox cluster. Similar to pre- first expressed in the midbrain-hind- nizes a band of cells on both sides of vious analyses of other lamprey genes brain boundary 12 hr earlier than embryos (arrows in Fig. 5c,d) that per- (Ueki et al., 1998; Ogasawara et al., En-2 and this earlier expression of sists through stage 25.5 (arrows in 2000; Myojin et al., 2001), the phylo- En-1 is required for the initial estab- Fig. 5f–i). An increase in LjEnD ex- genetic tree of lamprey En genes does lishment of the midbrain-hindbrain pression was also observed in the wall not provide enough resolution, espe- region (Wurst et al., 1994). Further- of the prospective midbrain-hindbrain cially when, and in which animal lin- more, recent studies in En conditional boundary at stage 25 (arrowheads in eages, the duplication events of the knock-in and knock-out mice showed Fig. 5c–e) and was seen in the same analyzed genes took place. In order to that En proteins subdivide the tectum domain until stage 25.5 (arrowheads resolve this, further genomic analyses and the cerebellum into distinct do- in Fig. 5f,g). By stage 28, LjEnD ex- by comparison of various gene cog- mains in a dose-dependent manner pression was seen in the upper and nates as well as synteny analyses are (Sgaier et al., 2007). Lamprey En lower lip mesoderm and in the velum, required in the future. genes LjEnA and LjEnB became de- but expression in the neural tube was Neural expression of an En homolog tectable in the midbrain-hindbrain down-regulated (Fig. 5j,k). The weak has been observed as two distinct boundary at stage 22 and continued to staining observed in the caudal part of stripes in the central nervous system be expressed in the same region until the body at stages 25 and 28 (Fig. 5c, j) of Ciona intestinalis, in a few cells of at least stage 28, whereas LjEnD appeared to be artefacts, as the sense the cerebral vesicle of amphioxus, and transcripts were first detected in a few probes were also trapped in the same at the midbrain-hindbrain boundary cells of neuroepithelium at stage 23 region (data not shown). of zebrafish, Xenopus, chick, and and disappeared by stage 26. Our re- mouse (Davis et al., 1991; Ekker et al., sults suggest that agnathan lampreys, 1992; Holland et al., 1997; Imai et al., which have multiple subdivided DISCUSSION 2002). The major neural expression brains, have already acquired the The present study is the first to dem- domain of LjEnA (Takio et al., 2007), same fundamental mechanisms of onstrate that the lamprey genome LjEnB, and LjEnD is the midbrain- partitioning the midbrain and the contains at least four En genes. Based hindbrain boundary, as previously hindbrain as gnathostomes. on the phylogenetic results, it is likely shown by using a polyclonal anti- Despite conserved expression of En that LjEnC diverged first, and subse- serum (␣Enhb-1) raised against in the midbrain-hindbrain boundary among chordates (Holland, 2005), LjEnC, a homolog of an En gene identified in Lampetra planeri (Holland and Williams, 1990), was not ex- Fig. 2. a, c, e, h, k: Expression pattern of LjEnA during embryonic development. Lateral view of pressed in the central nervous system, stage 22, 25, 25.5, 27, and 28 embryos, respectively. b, d, f, i, l: Higher magnification of the head region of the photographs shown in a, c, e, h, and k, respectively. g, j, m: Dorsal view of the head but rather was expressed in the ecto- region of the photographs shown in e, h, and k. n, o: Transverse sections of a stage-25.5 embryo derm of pharyngeal arches and the at the levels indicated by the lines in g. p: A horizontal section of a stage-27 embryo at the level body wall (Fig. 4). In chordates, the indicated by the line in i. q, r: Transverse sections of a stage-27 embryo at the levels indicated by ectoderm is partitioned into the neu- the lines in j. The hybridization signals are seen in the midbrain-hindbrain boundary (arrowheads) and epibranchial muscles (arrows). mhb, midbrain-hindbrain boundary; ov, otic vesicle. Scale roectoderm and the general ectoderm bars ϭ 1 mm (a), 500 ␮m (b–m), 100 ␮m (n–r). during gastrulation (Holland, 2005). Although the number of neurons Fig. 3. Expression pattern of LjEnB during embryonic development. a, c, e, h, k: Lateral view of seems to have decreased gradually in stage 22, 24.5, 25, 26.5, and 28 embryos, respectively. b, d, f, i, l: Higher magnification of the head region of the photographs shown in a, c, e, h, and k, respectively. g, j, m: Dorsal view of the head the general ectoderm during chordate region of the photographs shown in e, h, and k. The hybridization signals are seen in the evolution, several genes known to be midbrain-hindbrain boundary (arrowheads). Scale bars ϭ 1 mm (a), 500 ␮m (b–m). involved in the patterning of the cen- 1586 MATSUURA ET AL.

Fig. 4. Expression pattern of LjEnC during embryonic development. a, d, g: Lateral view of stage 25, 26, and 27 embryos, respectively. b, e, h: Higher magnification of the head region of the photographs shown in a, d, g. c, f, i: Ventral view of the head region of the photographs shown in a, d, and g. j: Lateral view of the trunk region of the photograph shown in g. k–n: Transverse sections of a stage-27 embryo at the levels indicated by the lines in g. The hybridization signals are seen in the pharyngeal ectoderm and the ectoderm of the ventral side of body (arrows). Scale bars ϭ 500 ␮m. tral nervous system are regionally expressed in the general ectoderm (Hol- land, 2005). In amphioxus, En as well as Distal-less and Pax6 are expressed in both the neuroectoderm and the general ectoderm (Holland et al., 1996, 1997; Glardon et al., 1998). In- terestingly, LjOtxA is expressed in the pharyngeal ectoderm of lamprey Lethenteron japonicum,asisLjEnC (Murakami et al., 2001). LjEnC expression was also observed in the ectoderm of the ventral side of the body wall (Fig. 4). In chick, mouse and dogfish embryos, En-1 is expressed in the ectoderm of the ventral side of the body from the forelimb/pectoral fin level to the hindlimb/pelvic fin/cloaca level (Loomis et al., 1996; Laufer et al., 1997; Rodriguez-Esteban et al., 1997; Tanaka et al., 1998, 2002). The ectodermal expression of En-1 in the body wall is critical for the positioning of the apical ectodermal ridge of the Fig. 5. FOUR ENGRAILED GENES IN THE LAMPREY 1587

bryos, and its developmental and morphological significance remains to be clarified. Transcripts of LjEnD were observed in the mesodermal component of the mandibular arch (Fig. 5), which was almost identical to the distribution of the cells in L. japonicum labelled with a polyclonal antiserum, ␣Enhb-1, raised against mouse En protein (Hol- land et al., 1993). En proteins have been found in the mandibular arch myoblasts of zebrafish, Xenopus, chick, and mouse during embryogenesis (Hatta et al., 1990; Davis et al., 1991; Hemmati-Brivanlou et al., 1991). Morphological homologies of these muscles, however, remain unclear (Lubosch, 1938). In the lamprey, the developmental fate of the mandibular arch mesoderm has previously Fig. 6. Hypothetical scenario for the evolution of En expression. Phylogenetic tree indicating the been described (Kuratani et al., 2004); probable timing of acquisition of the morphological characteristics in relation to different expression vital dye-labeling into the lamprey patterns for En during the developmental process (bold line). Hemichordates lack a central nervous mandibular mesoderm resulted in the system (CNS), but the entire ectoderm is neural. Expression of En is seen in the general ectoderm in hemichordate (Holland, 2005), which contains sensory cells. In chordates, En expression is seen labelling of upper lip, lower lip, and both in the CNS and in the general ectoderm. En transcripts are seen in certain varieties of velar muscles, as well as the trabecu- myotomes derived from the paraxial mesoderm. Expression of En is observed in the developing lar primordium (Kuratani et al., 2004) somites of the cephalochordates, amphioxus. In lampreys, En transcripts are observed in myo- and the expression pattern of the mus- blasts derived from the first somites. In jawed fishes, En-positive myoblasts are observed in the muscle pioneer-like cells, whereas, in tetrapods, En-positive cells are seen in the dermatomes. cle actin gene, LjMA2, suggested that Expression of En on the ventral side of the ectoderm of the body and limbs is conserved among myoblasts of the mandibular arch of jawed vertebrates, and similar ecotodermal En expression is observed in the ventral side of the lampreys migrated into the preman- body of lampreys. En-positive mesodermal cells are also observed within the mandibular arch of dibular domain (Kuratani et al., 2004; lampreys, as in gnathostomes; these cells differentiate into the muscles of the oral apparatus. Kusakabe and Kuratani, 2005). Al- though the En-expression in the mandibular arch muscles in gnathostomes limb buds (Loomis et al., 1996; Laufer expression domain supports the view and in lampreys functions in neuro- et al., 1997; Rodriguez-Esteban et al., that the body ectoderm of ancestral muscular target recognition (Hatta et 1997; Tanaka et al., 1998) and has agnathans may have been compart- only been observed in limbed verte- mentalized dorso-ventrally with En al., 1990; Holland et al., 1993), consis- brates. The ectodermal expression of expression ventrally (Tanaka et al., tent with the innervation by the max- LjEnC may not correspond to that of 2002). illomandibular portion of the trigemi- En-1 in limbed vertebrates because Transcripts of LjEnA were seen in a nal nerves (Kuratani et al., 2004), the LjEnC expression boundary is al- small cluster of myotomes in the first different subsets of the mandibular most at the margin of somatopleure somite and later in the elongated in- arch may express En genes; En-posi- (Fig. 4l); however, the fact that the fraoptic muscle precursors (Fig. 2). In tive myoblasts differentiate into the body ectoderm of the limbless ag- the lamprey head, there are two sets masseter, temporalis, and pterygoid nathan has already acquired the En of preotically located myotomes, such muscles in amniotes (Degenhardt and as supraoptic and infraoptic muscles. Sassoon, 2001), and into the levator Although these myotomes were first arcus palatini and the dilator operculi identified as derivatives of the ce- muscles in zebrafish (Hatta et al., phalic mesoderm, they arise from pos- 1990). Taken together, En expression Fig. 5. Expression pattern of LjEnD during em- totic myotomes that are secondarily in the mandibular mesoderm appears bryonic development. a, c, f, j: Lateral view of shifted rostrally into the preotic re- to function in the specification of the stage 23, 25, 25.5, and 28 embryos, respec- gion (Kuratani et al., 1999; Kusakabe mandibular arch muscle primordium b, d, e, g, h, k: tively. Higher magnifications of among the branchiomerie, especially the head region of the photographs shown in a, et al., 2004; Kusakabe and Kuratani, c, f, and j. b, d, g, k: Lateral view. e: Dorsal view. 2007). Expression of LjEnA supports at early stages of development. Thus, h: Ventral view. i: A horizontal section of a these previous conclusions that the in- consistent with the shared identity of stage-25.5 embryo at the level indicated by the fraoptic myotomes arise from the pos- the mandibular arch between gnatho- line in g. The hybridization signals are seen in stomes and lampreys (Takio et al., the midbrain-hindbrain boundary (arrowheads) totic somites (Fig. 2d–k). No similar and mandibular regions (arrows). Scale bars ϭ expression pattern of En cognates has 2004), expression of En in the mandib- 500 ␮m (a, c–k), 200 ␮m (b). been found in other vertebrate em- ular mesoderm appears to have been 1588 MATSUURA ET AL. established in the common ancestor of and the orthologs of LjEnA, LjEnB, in PBT. Embryos were then cleared in vertebrates (Fig. 6). and Lj EnC were obtained. This re- 50% glycerol in PBT. Selected em- search allowed us to extend the bryos were processed for frozen sec- sequence of LjEnC using primers tions by dehydration in 30% sucrose in EXPERIMENTAL in forward (GGGGGD) and reverse PBS followed by embedding in 7.5% PROCEDURES (AQGLYNH) orientations. We then gelatin in 15% sucrose for cryosections Ј ␮ Embryo Collection performed 3 RACE to obtain longer (15 m). fragments of LjEnC. An additional En Adult male and female lampreys gene (PmEnD) was also found in this (Lethenteron japonicum) were pur- search. The ortholog of PmEnD in L. ACKNOWLEDGMENTS chased from Ebetsu fishery coopera- japonicum (LjEnD) was isolated by We thank the Ebetsu fishery coopera- tive, Hokkaido, Japan, during the genomic PCR using primers in for- tive for providing adult male and fe- breeding season (early June). Spawn- ward (CRLRAEF) and reverse (AQG- male lampreys. This work was partly ing was induced, and embryos were LYNH) orientations. The LjEnA (873 supported by a Grant-in-Aid for reared to the desired developmental bp), LjEnB (784 bp), LjEnC (661 bp), Young Scientists Category A from the stages at 16°C in 10% Steinberg’s so- and LjEnD (402 bp) partial coding Ministry of Education, Science, Sports lution (Steinberg, 1957). Lamprey em- sequences have been submitted to and Culture of Japan and a research bryos were staged according to Taha- GenBank under accession numbers grant from the Hayashi Memorial ra’s staging of Lethenteron reissneri,a EF546817, EF546814, EF546815, Foundation of Female Natural Scien- species closely related to L. japonicum and EF546816, respectively. tists. (Tahara, 1988). For in situ hybridization, embryos were fixed overnight in 4% paraformaldehyde in 0.1 M phos- Phylogenetic Analysis REFERENCES phate-buffered saline (PBS), then de- In addition to lamprey, En genes from Davidson D, Graham E, Sime C, Hill R. hydrated in a graded methanol series sea urchins (Heliocidaris tuberculata 1988. 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