Hox Gene Expression Patterns in Lethenteron Japonicum Embryos—Insights Into the Evolution of the Vertebrate Hox Code

Developmental Biology 308 (2007) 606–620 www.elsevier.com/developmentalbiology

Genomes & Developmental Control Hox gene expression patterns in Lethenteron japonicum embryos—Insights into the evolution of the vertebrate Hox code

Yoko Takio a, Shigehiro Kuraku a, Yasunori Murakami b, Massimo Pasqualetti c, Filippo M. Rijli d, ⁎ Yuichi Narita a, Shigeru Kuratani a, Rie Kusakabe a,

a RIKEN Center for Developmental Biology, Evolutionary Morphology Research Group, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan b Department of Biology, Faculty of Science, Ehime University, Matsuyama, Japan c Unità di Biologia Cellulare e dello Sviluppo, Università di Pisa, Pisa, Italy d Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch Cedex, CU de Strasbourg, France Received for publication 24 March 2007; revised 9 May 2007; accepted 9 May 2007 Available online 17 May 2007

Abstract

The Hox code of jawed vertebrates is characterized by the colinear and rostrocaudally nested expression of Hox genes in pharyngeal arches, hindbrain, somites, and limb/fin buds. To gain insights into the evolutionary path leading to the gnathostome Hox code, we have systematically analyzed the expression pattern of the Hox gene complement in an agnathan species, Lethenteron japonicum (Lj). We have isolated 15 LjHox genes and assigned them to paralogue groups (PG) 1–11, based on their deduced amino acid sequences. LjHox expression during development displayed gnathostome-like spatial patterns with respect to the PG numbers. Specifically, lamprey PG1–3 showed homologous expression patterns in the rostral hindbrain and pharyngeal arches to their gnathostome counterparts. Moreover, PG9–11 genes were expressed specifically in the tailbud, implying its posteriorizing activity as those in gnathostomes. We conclude that these gnathostome-like colinear spatial patterns of LjHox gene expression can be regarded as one of the features already established in the common ancestor of living vertebrates. In contrast, we did not find evidence for temporal colinearity in the onset of LjHox expression. The genomic and developmental characteristics of Hox genes from different chordate species are also compared, focusing on evolution of the complex body plan of vertebrates. © 2007 Elsevier Inc. All rights reserved.

Keywords: Evolution; Hindbrain; Rhombomere; Pharyngeal arches; Neural crest; Lamprey; Hox; Tail bud

Introduction Duboule and Morata, 1994; and by Carroll et al., 2005). In vertebrates, a colinear tendency has also been observed in the The expression patterns of the Hox genes, or Hox code, are temporal order of Hox expression (temporal colinearity; recognized as a manifestation of the molecular toolkit with Duboule, 1994), in which the onset of expression is earliest which the basic body plan of the bilateria was constructed for Hox genes located at the 3′ end of the cluster. Both spatial (Carroll et al., 2005). Homeobox-containing Hox genes are and temporal colinearities appear to be based upon the clustered tandemly on the chromosome. In the different animal structures of the clusters per se, but the mechanisms of which phyla, the Hox genes located closer to the 3′ end of the cluster are not well understood. Although only one Hox cluster are expressed along the embryonic anteroposterior (AP) axis generally occurs in invertebrate genomes, at least four are more anteriorly than those closer to the 5′ end, thus showing found in many gnathostome vertebrate groups, as a result of two rostrocaudally nested, spatially colinear expression patterns rounds of genome duplications (Dehal and Boore, 2005). (reviewed by McGinnis and Krumlauf, 1992; Slack et al., 1993; Genetic experiments in model organisms such as the mouse and Drosophila have so far shown that Hox genes can function as a molecular system providing ‘positional values’ at each ⁎ Corresponding author. Fax: +81 78 306 3370. rostrocaudal level of the animal body. Disruption of this system E-mail address: [email protected] (R. Kusakabe). leads to the homeotic transformation of body parts into anterior

0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.05.009 Y. Takio et al. / Developmental Biology 308 (2007) 606–620 607 identities (Lewis, 1978; Kaufman et al., 1990; Morgan et al., In the jawless (agnathan) lamprey, the rostralmost phar- 1992; Guthrie et al., 1992; Chisaka et al., 1992; Condie and yngeal arch can be regarded as the mandibular arch (Koltzoff, Capecci, 1993; Rijli et al., 1993). Thus, the Hox genes are 1901; Damas, 1944; Johnels, 1948; Horigome et al., 1999; generally regarded as the fundamental molecular players Kuratani, 2005; Kuratani et al., 1997, 2001) as it constitutes part underlying the morphological transformations observed during of the complex lamprey oral apparatus together with more the evolution of the body plans of bilaterians. rostrally located ectomesenchyme (Kuratani, 2005), although it As has been observed in insects, vertebrate Hox genes are does not differentiate into a jaw. Importantly, recent studies also involved in various aspects of development (Pearson et al., indicated that the lamprey branchial Hox code is partially 2005). For instance, they perform important functions in the conserved, including a Hox-free first arch (Takio et al., 2004; skeletal and nervous system development of the head and trunk, but also see Cohn, 2002; Murakami et al., 2004). both of which show clear metamerical patterns in develop- However, several differences in the hindbrain Hox expression mental organization. In the trunk, for example, Hox genes are patterns have also been observed between agnathans and responsible for the position-dependent specification of the gnathostomes, correlating with distinct rhombomeric patterns vertebrae and spinal nerves at each level of the body axis of reticular and motor neurons (Murakami et al., 2004, 2005; also (McGinnis and Krumlauf, 1992; Kessel et al., 1990; Kessel, see Gilland and Baker, 2005). Similarly, in the lamprey pharyn- 1992; Rijli et al., 1995; Tiret et al., 1998; Dasen et al., 2005). In geal arches only PG2 and PG3 cognates showed gnathostome- the gnathostome head, Hox PG1–4 display offset rostral like expression patterns (Takio et al., 2004). Furthermore, the boundaries of expression coinciding with the borders between expression patterns of PG1 and posterior PG9–12 genes have not hindbrain segments, or rhombomeres (r), and extending been reported in lamprey. posteriorly into the spinal cord (Hunt et al., 1991; Lumsden Thus, it is still not clear whether a similar basic Hox code was and Krumlauf, 1996). The rhombomeres are developmentally already established in the common ancestor of gnathostomes and specified by the combination of Hox transcripts expressed in agnathans (Kuratani, 2005), and which aspects of Hox gene each segment (reviewed by Nieto et al., 1992; Trainor and regulation have been modified in the transition from agnathans Krumlauf, 2000; Briscoe and Wilkinson, 2004). Interestingly, to gnathostomes. Here, we provide a systematic description of the expression of Hoxb1 is peculiar in that its expression is the spatiotemporal expression patterns of 15 lamprey Hox genes, specifically restricted to r4 during hindbrain segmentation, encompassing PG1–11. This study complements and extends while being downregulated in the posterior hindbrain (Frohman our previous analysis of the lamprey Hox code (Takio et al., et al., 1990; Murphy and Hill, 1991). Moreover, the PG2 genes 2004), and allows us to discuss the evolution of the Hox code by Hoxa2 and Hoxb2 display expression boundaries at the r1/r2 comparing it with that of the other vertebrates. We also briefly and r2/3 borders, respectively, thus more rostrally than PG1 summarize here the comparison of genomic organization and genes and in apparent violation of the spatial colinearity rule developmental characteristics of Hox genes of chordates, (Hunt et al., 1991). focusing on the evolution of the vertebrate central nervous Nested Hox expression patterns are also seen in the neural system and pharyngeal arches. crest-derived ectomesenchyme of the pharyngeal arches. Specifically, PG2 genes are expressed in the second (hyoid) arch and posterior to it, PG3 in the third and more posterior Materials and methods arches, and so forth. In mouse and Xenopus, Hoxa2 is the only Embryos PG2 gene functional in the patterning of the hyoid arch, as its inactivation, though not that of Hoxb2, resulted in hyoid to Adult male and female lampreys, Lethenteron japonicum (Figs. 1A–C), mandibular homeotic transformation (Barrow and Capecchi, were collected from either the Miomote River, Niigata, or the Ishikari River, 1996; Santagati et al., 2005; Baltzinger et al., 2005; Rijli et al., Hokkaido, Japan. The eggs were artificially fertilized and incubated in 10% 1993; Gendron-Maguire et al., 1993). However, in zebrafish Steinberg solution (Steinberg, 1957) at 20 °C. Embryonic stages were assessed both Hoxa2 and Hoxb2 share redundant functions in hyoid morphologically according to the table for L. reissneri published by Tahara (1988; Figs. 1D–J). For in situ hybridization and immunostaining, the embryos arch patterning due to their co-expression in second arch were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; postmigratory neural crest cells (Hunter and Prince, 2002). In PFA/PBS), dehydrated in a methanol dilution series, and stored in 100% contrast, the first (mandibular) arch does not express any of the methanol at −20 °C. Hox genes while overexpression of Hoxa2 in first arch leads to homeotic transformation of mandibular into hyoid arch identity cDNA isolation (Grammatopoulos et al., 2000; Pasqualetti et al., 2000). Thus, Unidentified cDNAs for lamprey Hox genes were amplified by polymerase the mandibular arch can be seen to represent a ground pattern chain reaction (PCR) using the sense oligonucleotide primer 5′-CG GAA TTC or default state, developmentally specified by the absence of YTN GAR YTN GAR AAR GAR TT-3′and the antisense primer 5′-CG GGA Hox expression. In this respect, the absence of Hox expression TCC NCK NCK RTT YTN RAA CCA DAT YTT-3′ corresponding to amino from the rostralmost arch could have been a prerequisite for acid stretches LELEKEF and KIWFQNRR in the homeodomain, respectively. the development of the jaw, the synapomorphy of the The cDNA for the LjGbxA gene was also amplified using the above set of primers. Template cDNA was prepared from total RNA extracted from whole gnathostomes (Rijli et al., 1993; Couly et al., 1998 also see embryos of stage 24–26 L. japonicum using the First-Strand cDNA Synthesis Matsuo et al., 1995, for the patterning of the first pharyngeal Kit (Amarsham, Buckinghamshire, UK). PCR products were purified with the arch). QIAquick PCR Purification Kit (Qiagen) and then cloned into the pCRII-TOPO 608 Y. Takio et al. / Developmental Biology 308 (2007) 606–620

Fig. 1. Morphology of Lethenteron japonicum.(A–C) Adult morphology. (A) Left lateral view of a male. (B) Ventral view of the oral disc. This species has not evolved a mouth with an articulated jaw. (C) Enlargement of the head. Seven gill pores are visible behind the eye. (D–J) Normal developmental stages of L. japonicum staged according to the description of a closely related species, L. reissneri,byTahara (1988). Left lateral views. This animal hatches from the egg at stage 25 (H). (D) Early neurula. (E–G) Early to mid-pharyngula. (H) A newly hatched larva corresponding to late pharyngula in amniote embryos. (I) Early larva. (J) fully formed larva. (K) Peripheral nervous system of a stage 26 larva, immunostained with anti-acetylated tubulin antibody. The hindbrain is segmented along the anteroposterior axis into bulges called rhombomeres (r1–6), as in other vertebrate embryos. Several major cephalic motor neurons are indicated (V, VII, IX and X). Numbers (pp1, 2–8) indicate the location of the pharyngeal pouches (pp). (L) Graphical representation of the changing spatial relationships between rhombomeres (r1–6) and oropharyngeal structures at stages 24.5, 25, and 26. mo, mouth; pp1–4, pharyngeal pouches; vel, velum. (M) Dorsal view of a stage 26 L. japonicum hindbrain with retrogradely labeled interneurons and transcripts of LjGbxA, a marker of the hindbrain, detected by whole-mount in situ hybridization. r4 develops a large Mauthner neuron (Mth) and a cluster of smaller B neurons (B). See Murakami et al. (2004) for details. I1, isthmic reticular neuron; mhb, mid-hindbrain boundary. (N) Lateral view of another stage 26 embryo similarly prepared as in panel M. (O and P) Lateral and dorsal views, respectively, of a stage 25 whole-mount embryo hybridized with an LjEnA probe. LjEnA is expressed in the mid-hindbrain boundary (mhb). Scale bars: 10 mm for panels A–C; 0.5 mm for panels D–G; 1.0 mm for panels H–J; 0.1 mm for panels M and N.

vector (Invitrogen). The cDNA inserts from more than three independent clones 3′-RACE using oligonucleotide primers 5′-CTCGCCCAGGAACTTCAATT-3′ per gene were sequenced with the 3130 Sequence Analyzer (Applied and 5′-GGAACTTCAATTGAACGAGGC-3′, designed based on the nucleotide Biosystems). The flanking sequences at the 5′- and 3′-ends of the isolated sequence of the EngA gene of Petromyzon marinus (AF129401; Force et al., cDNAs were determined by the rapid amplification of cDNA end (RACE) 1999). The cDNA sequences identified in this study have been deposited in (Frohman et al., 1988). The cDNA for the LjEnA gene was amplified by nested GenBank with accession numbers AB286671–AB286676. Y. Takio et al. / Developmental Biology 308 (2007) 606–620 609

Whole-mount in situ hybridization rhombomeric levels (Figs. 1K and L; Tahara, 1988). Each rhombomere develops specific sets of interneurons (Murakami Digoxigenin (DIG)-labeled antisense and sense riboprobes were transcribed et al., 2004) and even-numbered rhombomeres carry branchio- using DIG-11-UTP (Roche) according to the manufacturer's instructions. Fixed embryos were treated overnight with a mixture of hydrogen peroxide and meric nerve roots (Kuratani et al., 1997), as visualized by methanol (1:5) and were rehydrated in PBS containing 0.1% Tween 20 (PBT). immunochemical staining of the nervous system at stage 26 After treatment with 0.2 N HCl in PBT for 10 min at room temperature (RT), the (Figs. 1K and L). As molecular markers, we made use of samples were digested with 10 mg/mL proteinase K (Sigma). They were post- L. japonicum cDNAs encoding specific transcription factors fixed for 20 min with PFA/PBT containing 0.2% glutaraldehyde, then washed displaying rhombomere-specific expression patterns in gnathos- with PBT, and prehybridized in hybridization buffer (50% formamide, 5× SSC, 1% SDS, 0.05 mg/mL total yeast RNA, 50 mg/mL heparin sulfate, 5 mM tomes (not shown; Murakami et al., 2001, 2004). Moreover, we ethylene diaminetetraacetic acid (EDTA)-Na2, 0.1% CHAPS) for 1 h at 65 °C. describe here two novel L. japonicum genes that show high The specimens were then incubated in hybridization buffer with 0.1 mg/mL similarity to gastrulation brain homeobox (Gbx; Bulfone et al., DIG-labeled RNA probe for 48 h at 65 °C. After hybridization, the specimens 1993; Shamim and Mason, 1998; Su and Meng, 2002; Rhinn et were washed twice in 50% formamide, 5× SSC, 1% SDS for 30 min at 65 °C. al., 2003, 2004; and data not shown) and engrailed (en or eng; Then the solution was substituted gradually with 10 mM Tris–HCl (pH 7.5) containing 0.5 M NaCl and 0.1% Tween 20 (TST). RNase Awas added to a final Araki and Nakamura, 1999; see Supplementary Fig. 1). concentration of 0.05 mg/mL, and the specimens were reacted for 30 min at RT. Specifically, the expression of the lamprey Gbx gene, The samples were washed twice with 2× SSC in 50% formamide for 30 min at LjGbxA, defines the rostral end of the lamprey hindbrain 65 °C, twice in 2× SSC containing 0.3% CHAPS for 30 min at 65 °C, and twice (Figs. 1M and N; Murakami et al., 2001, 2004), while the in 0.2× SSC containing 0.3% CHAPS for 30 min at 65 °C. The embryos were lamprey mid-hindbrain boundary (MHB) is marked by the blocked with TST containing 0.5% blocking reagent (Roche) for 1 h, and reacted at 4 °C for overnight with alkaline phosphatase (AP)-conjugated anti- specific expression of LjEnA (Figs. 1O and P), which is the digoxigenin Fab fragments (diluted in 1:4000; Roche). The specimens were orthologue of the previously reported EnA gene of P. marinus washed five times for 1 h each in TST at RT. Alkaline phosphatase activity was (Force et al., 1999). The location of lamprey MHB was also detected with NBT/BCIP. Stained specimens were fixed in 4% PFA and 1% confirmed by the retrograde labeling of the Mauthner neurons in methanol in PBS (PFAM/PBS), then rehydrated with a methanol series, and whole-mount embryos hybridized with the LjGbxA probe (Figs. clarified with a 1:2 mixture of benzyl alcohol and benzyl benzoate. 1M and N; Murakami et al., 2004). Thus, the expressions of Whole-mount immunostaining LjGbxA and LjEnA support a conserved mechanism of developmental regionalization of the hindbrain in lampreys The fixed embryos stored in 100% methanol were placed in dimethylsulf- and gnathostomes (Murakami et al., 2001, 2004). oxide (DMSO) and methanol (1:1). After they were washed with TST containing 5% DMSO (TSTd), the embryos were blocked with aqueous 1% periodic acid Isolation of LjHox genes and 5% nonfat dry milk in TSTd (TSTM). They were then reacted for 4 days at RT with the antibody directed against acetylated tubulin (Sigma) diluted in TSTM (1:1000). After the samples were washed with TST, they were reacted In addition to the 11 LjHox genes already described in our with HRP-conjugated anti-mouse IgG (diluted 1:200 in TSTM; Zymed). After previous report (Takio et al., 2004), we isolated four novel the final wash in TSTd, the embryos were reacted with the peroxidase substrate LjHox cDNAs namely LjHox1w, LjHox10s, LjHoxW10a, and 3,3′-diaminobenzidine (DAB; 100 mg/mL) in TST for 1 h, and then reacted in LjHox11t. All of these 15 genes were classified into PG1–11 TST with the same concentration of DAB and 0.01% hydrogen peroxide. using their sequence alignments at the amino acid level (Fig. 2; Takio et al., 2004), as indicated by the number in the name of Neuronal labeling in combination with in situ hybridization each gene. This assignment is mostly consistent with the After the specimens were subjected to neuronal labeling with biotinylated categorization based on the inference from a molecular phy- dextran (Fritzsch and Northcutt, 1993), whole-mount in situ hybridizations were logenetic tree (data not shown). In addition, 7 out of 15 performed as described by Murakami et al. (2004). Stained embryos were fixed genes had orthologues characterized in another lamprey in PFAM/PBS, and reacted at 4 °C for overnight in streptavidin-HRP (Vector) species P. marinus (Amores et al., 1998; Carr et al., 1998; diluted with TST (1:500). The specimens were washed five times for 1 h each in TST at RT, and then the HRP activity was detected using 100 mg/mL DAB in Force et al., 2002; Irvine et al., 2002), based on the low degree of TST with 0.01% hydrogen peroxide. accumulated synonymous substitutions (Kuraku and Kuratani, 2006) and the strong similarity of the nucleotide sequences in untranslated regions. Therefore, we gave them the same gene Results names as those identified in other lamprey species: LjHox1w, LjHox4x, LjHox4w, LjHox5w, LjHox6w, LjHoxQ8,and Molecular and morphological analysis of the developing LjHoxW10a (Takio et al., 2004; see Supplementary Fig. 1). hindbrain in lamprey In the following sections, we describe the patterns of expression of the LjHox genes, both novel and already known, In the developing lamprey, the locations of the rhombomere with emphasis on the several important evolutionary implica- boundaries can be estimated from their topographical relation- tions obtained by the comparison of specific aspects of their ships with the pharyngeal pouches (Figs. 1K and L). However, expression, namely: (1) the absence of Hox transcripts, the anteroposterior levels of the pharyngeal arches and axial including that of novel LjHox1w, in the first pharyngeal arch; structures deviate from each other during development (Damas, (2) the expression of posterior Hox genes in the putative tailbud 1944). In this study, we adopted the stage 26 embryonic of the lampreys; (3) the spatial and temporal colinearity of their morphology to set the coordinates with which to determine the expression. 610 Y. Takio et al. / Developmental Biology 308 (2007) 606–620

Fig. 2. Amino acid alignment of Hox gene products. Those of the newly identified LjHox genes are aligned with those of gnathostome Hox genes. The homeodomain is shaded in gray, and amino acid residues unique to each PG are highlighted in black. Gaps are indicated with hyphens. Human and shark sequences were added as gnathostome representatives. GenBank accession numbers for these sequences are; horn shark HoxA1, AF224262; human HoxA1, AAH32547; human HoxB1, EAW94746; horn shark HoxA10, AF224262; horn shark HoxD10, AF224263; human HoxA10, AAH13971; human HoxC10, AAH01293; human HoxD10, AAH69616; horn shark HoxA11, AF479755; horn shark HoxD11, AF224263; human HoxA11, AAH40948; human HoxC11, AAH01543; human HoxD11, AAI09395. See our previous paper for PG2–9(Takio et al., 2004).

Expression of the PG1 LjHox1w gene In the pharyngeal region, LjHox1w was expressed only in the caudal most pharyngeal arch endoderm (Figs. 3B and F). Similar to gnathostome PG1 genes, LjHox1w was highly As those of other LjHox genes reported so far, the expressed specifically in rhombomere 4 (r4), which was rather expression of LjHox1w is not detected in neither the easily recognized as this rhombomere lies just medial to the otic ectomesenchyme nor the endoderm of the first pharyngeal pit, which can be observed on the surface of the embryo at stage arch, confirming the Hox-free nature of this arch. The 26 and later (Figs. 3A, B, and D; Kuratani et al., 1998; specific expression in the 8th pharyngeal arch is similar to Horigome et al., 1999; see also von Kupffer, 1900). Weaker that reported for LjHox4w, LjHox5i, LjHox6w and LjHoxQ8 expression was also detected in the posterior part of the (Takio et al., 2004). hindbrain and in the neural tube (Fig. 3B). Somitic expression was observed throughout the trunk (Fig. 3G). This gene was PG9–11 LjHox genes are expressed in the tailbud also expressed in the posterior lateral line ganglion (Figs. 3B, E, and E′) and in the geniculate and petrosal placodes (Fig. 3C; We examined the expression pattern of four “posterior Kuratani et al., 1997). group” LjHox genes, LjHox9r, LjHoxW10a, LjHox10s, and

Fig. 3. Expression of the newly examined LjHox genes. (A) Expression of LjHox1w in a stage 26 larva. Arrowheads indicate the expression in the somites. (B) Enlargement of the head of the embryo in A. Expression in the pharyngeal arch ectomesenchyme is intensified in the posterior arches. Asterisk indicates the posterior lateral line ganglion. n, notochord; nt, neural tube; ot, otic vesicle; pa1–8, pharyngeal arches. (C–G) Transverse sections of the same embryo as in panel A, sectioned at the levels indicated by the lines in panels A and B. (C) LjHox1w expression was detected in the facial ganglion (glf). ph, pharynx. (D) This gene is specifically expressed in r4 (white arrowheads), which lies medial to the otocyst (ot), as in gnathostome embryos. (D′) Enlargement of panel D. Weak expression is detected in the ectoderm overlying the pharyngeal arch 3 (arrows). (E) Section at the level of the pharyngeal arch 4 (pa4). (E′) Enlargement of panel E. Expression in the posterior lateral line ganglion (pllg). (F) Transverse section showing the low level of expression in the neural tube, and in the endoderm (white arrowheads) and the overlying ectoderm (arrows) of the eighth pharyngeal pouch (pp8). (G) Transverse section at the level shown in panel A. LjHox1w is expressed in the somite (som). (H) Expression of LjHox9r at stages 26. Expression is detectable from the mid-trunk level and thence posteriorly along the neuraxis, as well as in the tail bud (white arrow). The rostral boundary of the neural tube expression is not clear. (I and J) Expression of LjHox9r at stage 26.5, enlarged views of the tail bud region. (I) Left lateral view. (J) Dorsal view. (K) Expression of LjHoxW10a in stage 27 larva. Transcripts were detected in the posterior neural tube and in the tail bud (white arrow). (L and M) Expression of LjHoxW10a at stage 28, enlarged views of the tail bud region. (L) Left lateral view. (M) Dorsal view. (N) Top; expression of LjHox10s at stage 26. Bottom; a stage-26 embryo stained with muscle-specific antibody MF20. Arrowheads are placed on every 10 myotomes. (O–T) Comparison of LjHox10s expression and myotome development at stage 26 (O and P), 27 (Q and R), and 28 (S and T). (O, Q, and S) Enlargement of the tailbud expressing LjHox10s. (P, R, and T) Myotomes were visualized with MF20 monoclonal antibody. Note that LjHox10s expression in the tail bud shifts posteriorly with respect to the myotomal numbers as the body axis extends posteriorly. (U) The structure of lamprey tail bud described by Nakao and Ishizawa (1984). C1 cells contain precursor cells for the somites. The rest of the C1 cells give rise to anteriorly located C2, the main constituents of which are the neural tube and notochord precursors. (Vand W) Histological observation of the tail bud in stage 28 larvae sectioned at the levels indicated by the lines in panel S. LjHox10s is upregulated strongly in the tail bud mesenchyme (arrow). (X–Z) Expression of LjHox11t in a stage 28 larva. The insert in panel X is a ventral view of the same larva, showing that the expression of the gene in the tail bud extends caudal to the anus (arrow). (Yand Z) Enlargement of the tail bud in stage 26 and stage 27 larvae. Note that the expression of the gene is restricted to the tail bud in these larvae, which have different numbers of myotomes. Scale bars: 0.5 mm for panels A, H–T, and X–Z; 0.1 mm for panel B; 50 μm for panels C–G, V, and W. Y. Takio et al. / Developmental Biology 308 (2007) 606–620 611 612 Y. Takio et al. / Developmental Biology 308 (2007) 606–620

LjHox11t (Figs. 3H–Z). These genes were expressed in the myotomes formed rostral to its expression domain (Fig. 3N). posterior neural tube and/or the tailbud, an embryonic organ As shown in Figs. 3O–T, as the development proceeds, more which forms posteriorly to the anus at stage 25 (Figs. 3H–O, Q, somites are added posteriorly, but the most posterior somites S, and X–Z), consistently to the situation in the gnathostome are always located anteriorly to the expression domain of PG9–10 Hox genes ( Lance-Jones et al., 2001; Omelchenko and LjHox10s in the caudal mesenchyme. Thus, unlike typical Hox Lance-Jones, 2003). gene expressions, which tend to be fixed at certain axial levels, Specifically, LjHox9r, LjHoxW10a,andLjHox11t were the rostral boundary of the LjHox10s expression domain expressed in both the posterior neural tube and the putative moves dynamically in a posterior direction relative to the tailbud. In the neural tube, these genes were expressed in a elengating axis (Fig. 4B). nested pattern, in which the expression domain of the gene with According to Nakao and Ishizawa (1984), the lamprey the larger PG number is included in that of the smaller PG genes tailbud consists of undifferentiated mesenchyme called ‘C1 (compare Figs. 3H, K, and X). Interestingly, LjHox10s express- cells’, which later give rise to somite primordia, and another ion in the neural tube was restricted only within the tailbud (Fig. class of cells, called ‘C2’, the common precursors for the neural 3N). In the stage 27 tailbud, all four genes were expressed in tube and notochord (Fig. 3U). Taking into account that the both the mesenchymal cells and the neural tube, and later the notochordal portion is devoid of LjHox10s transcripts in the expression of these genes was especially strong in the dorsal rostral part of the bud (Fig. 3V), the strong expression domain mesenchymal cells (Figs. 3V and W; see Nakao and Ishizawa, observed in the dorsal component of the caudal bud (Fig. 3W), 1984 for tailbud development in the lamprey). There was no corresponding to C2, most likely represents the neural tube expression in the tailbud notochord (Fig. 3V). precursors. To confirm that the expression at the caudal end of the body is specific to the tailbud whose location is supposed to Spatial order of LjHox gene expression progress posteriorly as the embryonic body elongates (Fig. 4C), the relative position of the anterior limit of the caudal The spatial expression of the LjHox genes in the neural tube expression of LjHox10s was compared with the number of was in accordance with the spatial colinearity found in other

Fig. 4. Schematic comparison of the expression patterns of the posterior Hox genes between amniotes and lampreys. Hox gene expression domain is colored pink in tailbud, green in neural tube, and yellow in somites. In the lamprey, LjHoxW10a (A) expression is seen in both the tail bud and the neuraxis, whereas LjHox10s (B) is only expressed in the tail bud. During development, the tail bud expression follows the extension of the body axis and therefore, these expression domains are not fixed at any axial levels, unlike the neuraxial domain, which has a fixed expression boundary. In the chicken embryo (C), Hoxd10 expression resembles that of LjHoxW10a, implying that the tail bud-restricted regulation of LjHox10s is a lamprey-specific type of regulation. Abbreviation: nt, neural tube; som, somite; TB, tailbud. Y. Takio et al. / Developmental Biology 308 (2007) 606–620 613 vertebrates; genes with smaller PG numbers tended to have Spatial colinearity was also observed in the pharyngeal arch expression domains with a more anterior limit, and larger PG ectomesenchyme, as shown in Figs. 5B–E. LjHox2 and numbered genes were expressed more posteriorly (Takio et al., LjHox3d are the only lamprey Hox genes that so far show 2004). We confirmed this notion by a more precise determina- typical expression in the pharyngeal arch ectomesenchyme; tion of the location of each anterior limit. Fig. 5A shows an LjHox2 in the second and more posterior pharyngeal arches, and example of the comparison carried out on embryos simulta- LjHox3d in the third arch and posteriorly (Takio et al., 2004). neously marked by the expression of selected LjHox genes and Both genes present higher expression in the anterior arches by the reticulospinal neurons labeled retrogradely at stage 26 (Figs. 5B and D). In histological sections of stage 26 larvae, (Murakami et al., 2004). At this stage, there is a clear tendency LjHox2 (Fig. 5C) and LjHox3d (Takio et al., 2004) were for genes with greater PG numbers to be expressed more expressed predominantly in a lateral population of mesenchymal posteriorly in a nested pattern (Fig. 5A). cells, which has been shown to be composed of neural crest cells

Fig. 5. Spatial relationships of expression domains of LjHox genes. (A) Comparison of expression of LjHox4x,-4w, -5i, -Q8, and -8p and the location of reticulospinal neurons in the hindbrain. Arrowheads indicate the rostral boundary of the LjHox expression domains. Reticulospinal neurons were retrogradely labeled (brown). (B) At stage 26, LjHox2 is expressed in the second arch and those posterior to it. n, notochord; nt, neural tube; pa1–8, pharyngeal arches. (C) A transverse section of the same larva shown in panel B, sectioned at the level indicated by the line in panel B. Note that the expression of this gene in the pharyngeal arch is restricted to the ectomesenchyme. ph, pharynx. (D) Expression of LjHox3d in a stage 26 embryo. ot, otic vesicle. (E) Expression of LjHox4x in a stage 27 embryo. LjHox4x expression in the pharyngeal arch ectomesenchyme becomes stronger in the posterior direction, ending abruptly caudal to the eighth pharyngeal pouch. (F) A stage 26 embryos immunostained with MF20 monoclonal antibody, which recognizes tropomyosin. pp1, 2–8, pharyngeal pouches. (G) Graphic comparison of the spatial expression domains of the LjHox genes, as determined by the numbers of myotomes (som 1/2–8), rhombomeres (r2–6) and pharyngeal pouches (pp1–8). The thickness of the bars reflects the intensity of gene expression. Names of the genes that are suspected to be on a same chromosome (LjHox4w, LjHox6w, and LjHoxQ8) are shaded in red (Force et al., 2002). Nested pattern of expression of these genes is highlighted by red circles connected by dashed lines. 614 Y. Takio et al. / Developmental Biology 308 (2007) 606–620

(see Horigome et al., 1999; see also Kimmel et al., 2001; spatial colinearity in the neural tube and pharyngeal ectome- McCauley and Bronner-Fraser, 2006, for the position of neural senchyme (Fig. 5G; Takio et al., 2004). crest cells within a pharyngeal arch). LjHox4x was also ex- pressed in the pharyngeal ectomesenchyme (Fig. 5E; Takio et al., Temporal order of the onset of LjHox gene expression 2004). However, unlike LjHox2 and LjHox3d, LjHox4x was expressed comparatively later in development (stage 27), with a All of the identified LjHox genes were first expressed in the graded pattern that achieved its highest level in arch 9, the neural tube, with subsequent expression in other tissues posteriormost arch of the lamprey (Fig. 5E). (described below; Takio et al., 2004). Assuming that the LjHox Fig. 5G summarizes the spatial relationships among the genes are located in clusters as in other vertebrate model anterior limits of the neural tube expression domain for each animals, in an order suggested by their PG assignment, would LjHox gene. In this analysis, in addition to the rhombomeric the lamprey Hox genes be expressed with temporal colinearity? segmentation (Fig. 1K), somites served as additional morpho- To address this issue, we compared the onset of the expression of logical landmarks to determine the axial levels of gene LjHox genes during embryogenesis, focusing on the neural tube expression (Fig. 5F). The results show that LjHox genes display in which the colinear patterns of expression, both temporally and

Fig. 6. Temporal order of LjHox gene expression in the neural tube. (A) Expression of LjHox4w, LjHox6w, and LjHoxQ8 in developing embryos of L. japonicum.In P. marinus, these gene cognates are assumed to be located in the same cluster. White arrows mark the gene expression in the neural tube detected by in situ hybridization. (B) Diagram showing the timetable of LjHox expression in the neural tube (blue bars). There is no apparent correlation between the onset of expression and the PG numbers. The genes shown in panel A are connected by a broken red line. Y. Takio et al. / Developmental Biology 308 (2007) 606–620 615 spatially, have been reported from many vertebrate Hox clusters unambiguously determined (Fig. 2), even though the mole- (Dollé and Duboule, 1989; Graham et al., 1989; McGinnis and cular phylogenetic analyses that included the LjHox genes did Krumlauf, 1992). not provide sufficient resolution to determine the cluster to In another lamprey species, P. marinus, the orthologues of which each gene belongs (data not shown), consistently with LjHox4w, LjHox6w, and LjHoxQ8 are linked to form a gene the above-mentioned difficulties that hindered immediate cluster (Force et al., 2002) a prerequisite for the establishment of cluster assignment. temporal colinearity in mammalian Hox genes (Kmita and The spatial colinearity in LjHox genes is plausible, since the Duboule, 2003). Thus, we first compared the chronological spatial expression pattern of the LjHox genes is largely similar to order of the expression among these 3 LjHox genes. LjHoxQ8 that of gnathostomes (see Takio et al., 2004), in which the started to be expressed earliest among these 3 genes at stage 20, anteroposterior expression domain of these genes depends on the followed by the expression of both LjHox4w and LjHox6w, order of the genes along the clusters (Burke et al., 1995). which started at stage 22 (Fig. 6A). We also compared the However, unlike in other vertebrates, there was no apparent timing of the expression of all the identified LjHox genes temporal colinear correlation of LjHox expression with the PG to (summarized in Fig. 6B). For example, LjHox8p showed the which each gene was assigned. Moreover, the precise mechan- earliest onset of overt expression in the neural tube (Takio et al., ism underlying Hox gene colinearity, both spatial and temporal, 2004) at stage 19, whereas the first transcript of LjHox2 (Takio is not yet fully understood (see Kmita and Duboule, 2003). It et al., 2004) was not detected until stage 21. Intriguingly, the should be noted that the fragmentation of Hox clusters with no data did not therefore suggest any sign of temporal colinearity in significant influence on the overall body plan is not restricted to the neural tube, unlike in gnathostomes in which the 3′-located Drosophila, and is also known in chordates such as tunicates Hox genes (the Hox genes with smaller PG numbers) are (Ikuta et al., 2004; Seo et al., 2004), although the chordate case is expressed earlier than the 5′-located Hox genes (those with associated with the partial disruption of spatial expression larger PG numbers) within the same gene cluster (Izpisúa- patterns. Further understanding of the regulation of lamprey Hox Belmonte et al., 1991; Dekker et al., 1993; van der Hoeven et al., gene colinearity will have to await precise genomic mapping and 1996). cluster characterization of LjHox genes. These studies will also provide information on the potential presence of PG12–14 Discussion genes, which, nonetheless, we were unable to isolate in this study. Hox gene repertoire in the lamprey Developmental evolution of the hindbrain and the jaw, inferred Because whole-genome sequences are not yet available for from the expression of LjHox1w this group of animals, including L. japonicum studied here, there is no complete information about the number of Hox In this paper, we have reported for the first time the expression clusters or the order of the genes in the clusters. The sea lamprey pattern of a PG1 Hox gene from a lamprey, LjHox1w. This gene P. marinus has been reported to possess at least three or four was clearly upregulated in the r4 of stage 26 embryos, with no Hox gene clusters (Irvine et al., 2002; Force et al., 2002). significant expression in the anterior pharyngeal ectomesench- This ambiguity results from the incomplete sequencing of the yme (Figs. 3A and B). The anterior and posterior boundaries of Hox gene clusters per se, and the technical limitations of LjHox1w expression were clearly mapping in r4, based on the molecular phylogenetic inference using genes with extremely position of the expression domain medial to the otocyst (see von short and similar amino acid stretches, such as the Hox genes. Kupffer, 1900; Horigome et al., 1999 for embryonic anatomy). Recent reports have also suggested that there has been an The overall expression pattern of this gene, including its weaker additional cluster duplication in the agnathan lineage (Fried expression in the posterior hindbrain and spinal cord, is highly et al., 2003; Stadler et al., 2004). From the partial genomic reminiscent of that of the gnathostome Hoxb1 (Frohman et al., linkages of the Hox genes reported for P. marinus (Irvine et al., 1990; Sundin and Eichele, 1990; Guthrie et al., 1992). This does 2002; Force et al., 2002), agnathans are thought to have a not necessarily imply an orthology between LjHox1w and similar structure of Hox gene clusters to that of the gnathos- gnathostome Hoxb1, even though we assume that duplications tomes, at least in terms of the organization of the PGs within of the Hox gene clusters predated the split between the lampreys clusters. Our inference of ‘colinearity’ in LjHox genes depends and gnathostomes. Instead, it seems more likely and more therefore on the assumption that the LjHox genes are also parsimonious to assume that the r4-restricted expression and arranged in clusters, in the order suggested by their PG function of this gene had already been established in the common assignment. While necessarily conjectural, such an assumption ancestor of the lamprey and gnathostomes. The identity of r4 is supported by the extremely small evolutionary distance appears to be very primitive and stable in the entire vertebrate between L. japonicum and P. marinus (Kuraku and Kuratani, lineage, as seen in the specific development of the Mauthner 2006). neuron in this rhombomere. In this study, we used RT-PCR to isolate cDNAs for four Combination of the data of our present and previous reports novel Hox genes from an agnathan species, L. japonicum, and (Takio et al., 2004; Murakami et al., 2004) shows that the presented a full description of the expression of the other expression of PG1 through PG3 Hox genes in the rostral LjHox genes identified so far. Assignment of PGs was hindbrain is highly conserved between the lamprey and the 616 Y. Takio et al. / Developmental Biology 308 (2007) 606–620 gnathostomes. If one takes additionally into account the tomes, some Hox genes have been reported to be expressed in conserved expression patterns of LjGbxA, LjEnA, LjKrox20, the tailbud, although their rostral limit of expression remains at and LjEphC (Figs. 1M–P; Murakami et al., 2004), it is likely specific axial levels throughout later development (Fig. 4C; that the expression of gene sets associated with the embryonic Burke et al., 1995; Omelchenko and Lance-Jones, 2003). mid-hindbrain development was already present in the common Moreover, initial transcription of many of the Hox genes takes ancestor of the lampreys and gnathostomes (close to the pan- place as part of the axis formation, and then is secondarily fixed vertebrate common ancestor). in each tissue at specific axial levels (Kessel, 1992; Hogan et al., In the pharyngeal arch region, LjHox1w was expressed only 1992; reviewed by Stern and Foley, 1998). In contrast, the in the endoderm of 8th pharyngeal arch (Fig. 3B), confirming tailbud expression domain of LjHox10s shows a characteristic that no LjHox gene is expressed in the mandibular arch, as in behavior; it moves posteriorly along the axis as the development gnathostomes (pa1; see Shigetani et al., 2005; Kuratani, 2005; proceeds (Fig. 4B). Takio et al., 2004). Among the LjHox genes expressed in the It is noteworthy that the neural expression of LjHox10s is pharyngeal arches, the most rostrally expressed gene is LjHox2 restricted to the putative tailbud, and not found in the more (Fig. 5B), the only cognate so far isolated as a PG2 gene in anterior neural tube. The other PG10 member, LjHoxW10a, has lampreys. The mandibular and the hyoid arch skeletons of a canonical expression pattern in the neural tube, with a rostral vertebrates, including lampreys, are morphologically distin- limit at a level compatible for this gene in terms of spatial guishable from the rest of the arches; in other words, there are colinearity (Figs. 3K and 4A). Given that the expression pattern three detectable morphological identities (mandibular, hyoid, of the LjHoxW10a mostly resembles that of the PG10 genes of and the more posterior respiratory arches) in the branchial other vertebrates (Fig. 4C), including that of Hox10w of skeletal systems of the vertebrate embryos (Kuratani, 2005). P. marinus (Freitas et al., 2006), the expression of LjHox10s Our data suggest that these three distinct morphological may represent a secondary loss of the typical neural tube- identities of the pharyngeal arches along the AP axis might be associated expression domain after cluster duplication, only determined, respectively, by the absence of Hox transcript in the retaining the tail bud-associated regulation. This differential mandibular arch, by the only expression of PG2 Hox genes in expression of PG10 genes in lampreys may thus represent a type the hyoid arch, and by the expression of both PG2 and PG3 in of subfunctionalization of the Hox genes, as found in zebrafish the rest of the morphologically similar pharyngeal arches. (Bruce et al., 2001). The Hox-free nature of the lamprey mandibular arch would argue against a primary involvement of Hox genes in the Evolution of the Hox code in chordate lineages evolution of the jaw apparatus, an evolutionary innovation considered to have occurred in the common ancestor of Recently a growing interest in the evolution of vertebrate gnathostomes (but see Cohn, 2002). Alternatively, the retreat body plan has urged the comparative study of Hox codes over of Hox expression from the first arch in an hypothetical different phylum, including invertebrate chordates (Galliot, common ancestor of agnathans and gnathostomes might have 2005). Representative protochordates studied so far have been been a prerequisite for the distinct evolution of the lamprey the urochordate ascidians (Katsuyama et al., 1995; Ikuta et al., complex oral apparatus or the jaw in the agnathan or 2004; Ikuta and Saiga, 2005; Keys et al., 2005) and the gnathostome lineages, respectively. amphioxus (Wada et al., 1999; Minguillón et al., 2005; Schubert et al., 2006). The recent report on another urochordate species, Tail bud and LjHox expression larvacean Oikopleura, also added important insights into the evolution of the Hox code (Seo et al., 2004), providing insights LjHox9r, LjHoxW10a, LjHox10s and LjHox11t were all into the complex path of evolutionary changes in the Hox gene expressed in the so-called tailbud (Figs. 3H–Z). In gnathos- clusters of the chordate lineages. As summarized in Table 1, the

Table 1 Comparison of the genomic structures and the colinearity in expression of Hox genes in chordates Tunicate Amphioxus (3) Lampreys (4) Gnathostomes (5) Larvaceans (1) Ascidians (2) Hox clusters in the genome × a ▵ b ○○○ Spatial colinearity in central nervous system ○○○ ○○ Hox1 expression in r4 ––× c ○○ Spatial colinearity in pharyngeal arches ––× ○○ Temporal colinearity × × ○ d × ○ ○, traits present; ×, traits not found; –, No equivalent tissue exists. Underlines indicate the features reported in this study. (1) Seo et al., 2004. (2) Ikuta and Saiga, 2005. (3) Schubert et al., 2006. (4) Fried et al., 2003; Takio et al., 2004; this study. (5) McGinnis and Krumlauf, 1992; Trainor and Krumlauf, 2000. a None of the larvacean Hox genes is clustered on the genome. b Some of the ascidian Hox genes are located closely on the genome. c Hox genes, including Hox1, are expressed in a nested pattern in the anterior neural tube of amphioxus. d Temporally collinear expression in the presumptive neural tube has been reported for Hox1, -3, and -4. Y. Takio et al. / Developmental Biology 308 (2007) 606–620 617 comparison of the characteristics of Hox genes of chordates the LjHox genes. It is possible that the lampreys are still on the revealed that the lamprey seems to have established the basic process of acquisition of temporal colinearity and showing functional traits of the Hox code required for the body plan of significant incompleteness in Hox gene temporal regulation, gnathostomes, and that many of these traits have not been found when compared with gnathostome Hox genes. Conversely, it in invertebrate chordates. can also be presumed that the common ancestor of vertebrates The genomic organization of Hox genes has been extensively would have already established a tight temporal colinearity in studied in both tunicates and amphioxus. It has been speculated Hox expression, which would be gradually lost in different that the ancestor of chordates probably possessed nearly vertebrate lineage, especially in lampreys. Elucidation of the complete, vertebrate-like set of Hox genes, consisting of several complete structure of lamprey Hox clusters should provide anteriorly, centrally, and posteriorly expressed genes (Garcia- insights to resolve this problem. Fernàndez and Holland, 1994; reviewed by Ikuta and Saiga, Pharyngeal arches are one of the synapomorphic traits of 2005). These genes were probably linked in a single chro- vertebrates. As shown by series of embryological and experimental mosomal cluster. At the radiation of chordates, in the tunicate analyses in vertebrates, including lampreys, the identity of each lineage, Hox gene clusters underwent splitting or disintegration, arch appears to be specified by anterior PG Hox genes expressed in which in larvaceans resulted in the complete dispersal of the Hox the neural crest derivatives. The amphioxus possesses a large genes into the separate genomic regions (Ikuta et al., 2004; Seo pharynx associated with pharyngeal slits separated by many et al., 2004). Such degradation of the clusters was associated vertical pharyngeal bars (Jollie, 1973). However, since this animal with the loss of many of the central Hox genes (PG4–8; Seo et lacks neural crest cells, amphioxus does not possess equivalent al., 2004). In contrast, clustered organization was conserved in tissues to the vertebrate pharyngeal skeleton nor the pharyngeal the amphioxus lineage (see Minguillón et al., 2005) and was also nerves. Therefore, the colinear expression pattern of Hox genes in maintained in vertebrates in which the Hox clusters underwent lamprey pharyngeal arch neural crest cells suggests that the Hox- several rounds of duplications (McLysaght et al., 2002). These dependent mechanism for patterning of cephalic neural crest duplication events might have occurred very early in vertebrate derivatives would probably have been established in close evolution, and caused the lampreys to possess more than 3 Hox association with the emergence of primitive neural crest cells in clusters (Force et al., 2002; Irvine et al., 2002), and the the vertebrate ancestor. gnathostomes to possess more than 4 clusters (McGinnis and In conclusion, our analysis in lamprey suggests that the early Krumlauf, 1992; Krumlauf, 1994, Amores et al., 1998). evolution of the vertebrate Hox code may have been tightly Although incomplete and in some cases only residual, the linked to the development of the vertebrate head. To further nested pattern of expression within the central nervous system understand the evolutionary development of the vertebrate can be recognized throughout the chordate lineages (Table 1). In head, it will be critical to examine the development and Hox the ascidian Ciona intestinalis, PG1 and 3 genes are expressed gene expression of the hagfish, another agnathan animal, which in the visceral ganglion, which locates anterior to the nerve may exhibit an even more basal state of developmental features cord, and PG5, 10, and 12 are expressed posteriorly in the nerve among vertebrates. cord (Ikuta et al., 2004). The expression of amphioxus PG1–6 genes shows rather clear spatial colinearity in the central Acknowledgments nervous system (Wada et al., 1999; Schubert et al., 2006). It should also be noted that a set of transcription factor encoding WearegratefultoDr.Shigekihirano at Niigata University for genes that specifies brain compartments, such as Pax2/5/8 supply of the animals. This work was supported by Grants-in-Aid (Wada et al., 1998) and Dmbx (Takahashi and Holland, 2004) from the Ministry of Education, Science, and Culture of Japan are expressed in the amphioxus brain in a corresponding pattern (Specially Promoted Research) to S.K. M.P. was supported by a to those of the vertebrate cognate genes. One remarkable dis- European Commission (EEC) fellowship. Work in F.M.R. crepancy between the amphioxus and lamprey is that laboratory is supported by the Agence Nationale pour la Recherche amphioxus PG1 gene simply follows the rule of spatial coline- (ANR), the Fondation pour la Recherche Medicale (FRM, « Equipe arity in amphioxus central nervous system (Schubert et al., Labelisée»), the Association pour la Recherche contre le Cancer 2006), whereas it is specifically expressed in the r4 in lamprey (ARC), the Association Française contre les Myopathies (AFM), (Fig. 3B), violating the spatial colinearity. This insight may add the Ministère pour le Recherche (ACI program), and by another evidence to the evolutionary timing of the establishment institutional funds from CNRS and INSERM. of the identity of r4, which would probably have occurred after chordate divergence but before the agnathan/gnathostome split Appendix A. Supplementary data and might correlate with the loss of PG1 expression from the rostral ‘hindbrain’. Supplementary data associated with this article can be found, Expression of amphioxus Hox genes is also remarkable in in the online version, at doi:10.1016/j.ydbio.2007.05.009. that PG1–4 genes are expressed in a temporally colinear pattern (Wada et al., 1999). This might represent the ancestral characteristics of vertebrate Hox clusters, associated with References tightly linked cluster organization of Hox genes. In this study Amores, A., Force, A., Yan, Y.-L., Amemiya, C., Fritz, A., Ho, R.K., Joly, L., we did not detect a temporally colinear order of expression of Langeland, J., Prince, V., Wang, Y.-L., et al., 1998. Genome duplications in 618 Y. Takio et al. / Developmental Biology 308 (2007) 606–620

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