Animals

The spider Achaearanea tepidariorum was used in our experiments. Embryos were obtained as described previously (Akiyama-Oda and Oda, 2003). Stages 1 to 8 of embryonic development have been defined in the previous study (Fig. 1; Akiyama-Oda and Oda, 2003). Stage 9 is the period during which the nascent limb buds extend distally. Stage 10 is the period during which the extending limbs become segmented and the germ band splits to shift laterally.

cDNA cloning

To isolate Achaearanea homologs of the Drosophila twi and sna genes, we initially performed polymerase chain reactions (PCR) using degenerate primers. The following primers were used: twi forward primer, 5′ tngcnaa(t/c)gtn(a/c)gnga(a/g)(a/c)g 3′ (for the amino acid sequence (M/L)ANVRER); twi forward primer for nested PCR, 5′ gtn(a/c)gnga(a/g)(a/c)gnca(a/g)(a/c)gnac 3′ (for the amino acid sequence VRERQRT); twi reverse primer for the first and nested PCR, 5′ a(a/g)(t/c)ttnt(a/g)ngt(t/c)tg(a/g/t)at(t/c)tt 3′ (for the amino acid sequence LKLTQIK); sna forward primer, 5′ aa(a/g)atgca(t/c)at(a/t/c)(a/c)gnacnca 3′ (for the amino acid sequence KMHIRTH); sna reverse primer, 5′ aangg(t/c)tt(t/c)tcnccngt(a/g)tg 3′ (for the amino acid sequence FPKEGTH); sna forward primer for nested PCR, 5′ gnacnca(t/c)acn(t/c)tnccntg 3′ (for the amino acid sequence RTHTLPC); sna reverse primer for nested PCR, 5′ gt(a/g)tgngtnc(g/t)(a/t/g)at(a/g)tgncc 3′ (for the amino acid sequence THTRIHG). cDNA prepared from embryos and larvae were used as the template.

To isolate cDNA clones, digoxigenin (DIG)-labeled probes for the amplified fragments were prepared using a PCR DIG Probe Synthesis Kit (Roche). Two cDNA libraries of embryos and hatched prelarvae (Akiyama-Oda and Oda, 2003) were screened with the probes. Three clones for At.twi and one for At.sna were obtained. Both strands of the representative At.twi and At.sna clones were sequenced, revealing that these clones cover the entire or nearly entire coding regions of the mRNAs. However, the translation start site for At.twi mRNA was not determined with confidence since no in-frame stop codon was identified upstream of the first methionine in the isolated cDNA clones. In the At.sna clone, a potentially artifactual deletion of one nucleotide was detected. The DNA fragments covering the site were amplified by RT-PCR to determine the authentic sequence of At.sna. The At.twi and At.sna sequences are available from the DNA data bank of Japan (DDBJ) with the following Accession numbers: At.twi, AB167807; At.sna, AB167392.

Molecular phylogenetic analysis

The deduced amino acid sequences of the bHLH domains of At.twi and other twi-related genes as well as the zinc finger-containing regions of At.sna and other sna-related genes were aligned manually. Phylogenetic trees were constructed by the neighbor-joining method (Saitou and Nei, 1987) using PHYLIP (Felsenstein, 1993). Confidence in the phylogenies was assessed by bootstrap resampling of the data sets.

Whole-mount in situ hybridization

For whole-mount in situ hybridization, spider embryos were fixed as described previously (Akiyama-Oda and Oda, 2003). RNA probes were prepared using T7 RNA polymerase (Stratagene) and DIG RNA Labeling Mix (Roche). The first 916 bp of At.twi cDNA including the entire bHLH domain, and the 1692 bp region of At.sna cDNA (nt180-1871), which contains all the zinc finger motifs, were used as templates. Hybridization, washes and detection were performed as described previously (Akiyama-Oda and Oda, 2003). Some embryos were counterstained with the anti-pMAD antibody PS1(Persson et al., 1998).

Sectioning

Stained embryos were dehydrated in a graded ethanol-xylene series, embedded in TissuePrep (Fisher Scientific), and then serially sectioned at 5 μm thickness.

Cloning of A. tepidariorum genes related to twi and sna

A. tepidariorum cDNAs related to twi and sna were cloned. The details of each cDNA sample are described below and in the Materials and Methods.

At.twi

The isolated cDNA clone encoded a polypeptide of 221 or more amino acids with a bHLH domain and a WR motif characteristic of Twist proteins (Fig. 2A). The bHLH domain displayed 61–69% amino acid identity with those in the Twist proteins of Drosophila, jellyfish, mollusk, leech, lancelet and mouse, compared to the 54%, 47% and 43% identity that it shared with the bHLH domain of the Drosophila non-Twist-class bHLH proteins Hand, Fer1 and Dimm, respectively. Based on the topology of a phylogenetic tree constructed using the bHLH domain sequences (Fig. 2C), the isolated gene was found to belong to the twist family. Accordingly, the gene was designated At.twist (At.twi).

Fig. 2.

(A, B) Alignment of the amino acid residues of the bHLH domains and the WR motifs of At.twi and other Twi-related proteins (A) and of the zinc finger II -V domains of At.sna and other Sna-related proteins (B). Identical residues are highlighted. Gaps were introduced to optimize the alignments (indicated by dashes). Numbers in parentheses represent the number of residues omitted at the indicated sites. Asterisks indicate the cysteine and histidine residues characteristic of zinc fingers. (C, D) Molecular phylogenetic trees constructed by the neighbor-joining method based on the alignment of the bHLH domain (C) and zinc finger II-V domains (D). The numbers at the internal branches are bootstrap values (%). Accession numbers of the proteins used are as follows: At.twi (AB167807), Dm.twi (S00995), Pc.twi (CAC12667), Pv.twi (AAL15167), Hr.twi (AAL05567), Bb.twi (O96642), Dm. Hand (NP_609370), Dm. Fer1 (NP_524251), Dm.dimm (NP_523611), At.sna (AB167392), Cs.sna (CAE00182), Dm.sna (NP_476732), Dm.esg (NP_476600), Dm.wor (AAF12733), Pv.sna1 (AAL06240), Hr.sna1 (AAL05564), Lv.sna (AAB67715), Dm.scrt (AAA91035), Dm.gl (P13360), Dr.par (NP_571047), Dr.twi1 (NP_571059), Gg.dHand (Q90690), Gg.par (AAC60208), Mm.dHand (AAC52338), Mm.par (Q60756), Mm.twi1 (NP_035788), Xl.twi (P13903), Xl.dHand (P57101), Ag.ENSANGP12782 (XP_308953), Ag.ENSANGP18305 (XP_317196), Ag.ENSANGP20876 (XP_315399), Bf.sna (AAC35351), Dm.CG12605 (NP_647845), Dm.CG17181 (NP_612040), Gg.slug (I50738), Gg.sna (CAA71033), Hs.scrt1 (NP_112599), Mm.scrt (NP_570963), Mm.slug (NP_035545), Mm. Sna (NP_035557), Xl.slug (Q91924), Xl.sna (P19382). Abbreviations of species are as follows: Ag, Anopheles gambiae; At, Achaearanea tepidariorum; Bb, Branchiostoma belcheri; Bf, Branchiostoma floridae; Cs, Cupiennius salei; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hr, Helobdella robusta; Hs, Homo sapiens; Lv, Lytechinus variegates; Mm, Mus musculus; Pc, Podocoryne carnea; Pv, Patella vulgata; Xl, Xenopus laevis.

At.sna

Analysis of the isolated cDNA clone and amplified cDNA fragments revealed a polypeptide of 303 amino acids with five zinc finger motifs. The amino acid sequences of these motifs were highly similar to those found in Drosophila Sna, Escargot and Worniu and other animal Snail/Slug-class proteins (Fig. 2B). The isolated gene was classified into the snail/slug family, based on phylogenetic analyses using the sequences of the regions encompassing zinc finger II-V motifs (Fig. 2D). Accordingly, the gene was designated At.snail (At.sna). At.sna was most closely related to Cs.sna, a snail homolog in Cupienius salei, another spider species (Weller and Tautz, 2003).

At.twi transcripts are expressed in mesoderm cells

We examined the expression patterns of At.twi transcripts in spider embryos by whole-mount in situ hybridization. At.twi transcripts were not detected before late stage 5, when the CM cells reached the periphery of the germ disc (Akiyama-Oda and Oda, 2003). At early stage 6, At.twi transcripts were detected in cells located at the polar and equatorial areas of the embryo (Fig. 3A). In the initial pattern of At.twi expression, no reproducible asymmetry could be recognized with respect to the embryonic-abembryonic axis. As the extra-embryonic area expanded, more cells expressed At.twi transcripts (Fig. 3B). The At.twi-expressing cells became distributed nearly uniformly in the embryonic area. At these initial stages, some At.twi-expressing cells were found in the surface epithelial layer but other At.twi-expressing cells were located a little deeper than at the surface (Fig. 3H, I).

Fig. 3.

Expression patterns of At.twi transcripts as revealed by whole-mount in situ hybridization. Signals of the At.twi transcripts are presented in purple. (A) An early stage 6 embryo viewed from the top of the germ disc. This embryo was also stained for pMAD (brown) to determine its orientation. The asterisk indicates the site of the germ disc rim at which the cumulus arrived. (B) A mid-stage 6 embryo viewed from the prospective caudal side. The emerging extra-embryonic area (Ex) shows little At.twi expression. (C) A stage 7 embryo viewed from the ventral side. Anterior is to the top. CL, caudal lobe; L1 to L4, first to fourth walking legs. (D–G) Flat preparations of embryos at stage 7 (D), stage 8 (E) and stage 9 (F, G). Anterior is to the left. One anterior domain and the following four stripes of At.twi expression are seen in the stage 7 embryo (D). The four stripes correspond to the segments bearing walking legs (L1–L4). The anterior domain was subdivided to give rise to two additional stripes (arrowheads) that correspond to the chelicerae (Ch) and pedipalps (Pp). In opisthosomal segments produced from the caudal lobe (CL), stripes or patches of At.twi expression are seen (arrows). (H, I) Close-up images showing the polar area of an embryo at early stage 6. The focus was adjusted to the surface epithelial layer for the first image (H), and shifted a little deeper into the interior of the embryo for the second image (I). Arrows indicate the At.twi-expressing cells in focus. y, yolk. (J, K) Longitudinal section of a stage 7 embryo. Anterior is to the left. The area boxed in J is magnified in K. At.twi-expressing cells are situated just beneath the surface ectoderm. (L, M) A stage 9 embryo viewed from the lateral side. The area boxed in L is magnified in M. Expression stripes that appeared in the opisthosomal region (arrows) are situated just beneath the surface ectoderm. Expression is not detectable at the caudal lobe (CL). Scale bar: 250 μm in AC; 100 μm in D–G, L, M; 25 μm in H–K.

The stage 7 embryo, which is transforming into a germ band, showed one anterior domain and four stripes of At.twi expression (Fig. 3C, D). Sectioning of stained stage 7 embryos revealed that all the At.twi-expressing cells were situated just beneath the ectoderm (Fig. 3J, K). The four stripes were persistently seen at later stages (Fig. 3D–F), and probably correspond to the segments bearing walking legs. The fourth stripe displayed higher levels of At.twi expression than the remaining three stripes (Fig. 3C–E). The anterior domain of At.twi expression was sequentially subdivided to produce two stripes (Fig. 3D–F). These two stripes probably correspond to the chelicerae and pedipalps. Each of the six, in total, At.twi expression stripes at the prosomal region was laterally split into two large patches. These changes in the shapes of the At.twi expression domains occurred in accordance with the formation of the limb buds (Fig. 3E, F), although the expression gradually declined during limb bud extension (Fig. 3G). In embryos whose limb buds were extending, relatively high levels of At.twi expression remained detectable at the anterior terminal region (Fig. 3G).

At the formed caudal lobe, little At.twi expression was observed (Fig. 3C–E). However, in the segments produced from the caudal lobe, expression stripes appeared (Fig. 3F, G, arrows). Like the prosomal stripes, these opisthosomal stripes were also situated just beneath the ectoderm (Fig. 3L, M). At later stages, each was laterally split into two patches (Fig. 3G).

We speculated that the cells expressing At.twi are mesoderm cells for four reasons, as follows. First, at least in stage 7 and older embryos, all the At.twi-expressing cells were situated just beneath the surface ectoderm. Second, the At.twi-expressing cells, unlike typical endoderm cells, became metamerically arranged. Third, in stage 9 embryos, most of the At.twi-expressing cells present in the prosomal segments were associated with the forming limb buds, where somatic and visceral mesoderm cells have been observed morphologically (Montgomery, 1909). Fourth, in Achaearanea, the central nervous system develops from the surface ectoderm at stage 9 and later in a way similar to that described in Cupienius (Stollewerk et al., 2001; Y. A. and H. O., unpublished observations). It is likely that the At.twi-expressing cells are distinct from the internalized neural cells. The distributions of the At.twi-expressing cells presented here are similar to those of cells that Montgomery has described as mesentoblasts and mesoblasts (Montgomery, 1909). We propose that At.twi can be used as a marker for analyzing mesoderm development in the spider. Moreover, our observations suggest that twi and At.twi play conserved roles in the mesoderm development of the fly and spider.

At.sna transcripts are expressed in part of the ectoderm

The expression patterns of At.sna transcripts were also examined by whole-mount in situ hybridization. At.sna transcripts were detected in a broad ring near the margin of the germ disc from late stage 4 or early stage 5, and similar patterns persisted up to the beginning of stage 6 (Fig. 4A–C). Sectioning of stained stage 5 embryos revealed that the At.sna-expressing cells were located only in the surface cell layer (Fig. 4E, F). As the extra-embryonic area expanded, this area displayed lower levels of expression compared to the embryonic area (Fig. 4C). At stage 7, At.sna expression was seen in stripes (Fig. 4D). Although this expression pattern was, at a glance, similar to that of At.twi at the corresponding stage, the At.sna expression stripes were located in the surface ectoderm with no expression in the inner layer (Fig. 4G, H). At later stages of embryogenesis, At.sna transcripts were detected in the central nervous system (data not shown), consistent with the expression pattern of Cs.sna (Stollewerk et al., 2001; Weller and Tautz, 2003). These observations indicate that At.sna is expressed in part of the ectoderm, but not in the twi-expressing mesoderm. Our data do not suggest that the role for snail in the mesoderm formation of the fly is conserved in the spider.

Fig. 4.

Expression patterns of At.sna transcripts as revealed by whole-mount in situ hybridization. Signals of the At.sna transcripts are presented in purple. Embryos shown in A–C and E were also stained for pMAD (brown). (A) An early stage 5 embryo viewed from the top of the germ disc. The asterisk indicates the center of the germ disc. (B) An early stage 6 embryo viewed from the top of the germ disc. At.sna expression is detected in a broad ring of the germ disc area. Around the polar area, lower levels of At.sna expression are observed. The asterisk indicates the site of the germ disc rim at which the cumulus arrived. (C) A mid-stage 6 embryo viewed from the prospective caudal side. The bracket indicates the extra-embryonic area, which displays little or no At.sna expression. (D) A stage 7 embryo viewed from the ventral side. Anterior is to the top. Metameric patterns of expression are observed. (E, F) Longitudinal section of a stage 5 embryo. The area boxed in E is magnified in F. (G, H) Longitudinal section of a stage 7 embryo. Anterior is to the left. The area boxed in G is magnified in H. At.sna expression is detectable in surface cells (arrowheads) but not in internal cells (arrows). Scale bar: 250 μm in A–D; 50 μm in E, G; 25 μm in F, H.

Divergent aspects of mesoderm development in the fly and spider

Comparison of the expression patterns of twi and At.twi revealed divergent aspects of mesoderm development in Drosophila and Achaearanea (Fig. 5). In the fly embryo, all the mesoderm arises from the future ventral side (Fig. 5A). In contrast, in the spider embryo, the initial distribution of At.twi transcripts was not ventrally localized; instead, this distribution was rather symmetric with respect to the embryonic-abembryonic axis (Fig. 3A). The emerging asymmetry of At.twi expression during stage 6 (Figs 3B, 5B) appeared to be associated with the formation of the extra-embryonic area. The At.twi expression pattern within the embryonic area showed little unevenness along the emerging DV axis until the limb buds began to form. These observations indicate that the relationship between DV pattern formation and mesoderm formation differs in the fly and spider. In the Drosophila embryo, all prospective mesoderm cells are internalized from the ventral side within a short period, and this process can be observed (Sweeton et al., 1991; Kam et al., 1991; Oda and Tsukita, 2001). In the spider embryo, however, it was not so easy to determine from where and how the mesoderm was internalized. This difficulty may be at least partially due to asynchronous internalization of prospective mesoderm cells, as At.twi-expressing cells were found both at the surface and inner layers in the same germ discs (Fig. 3H, I). However, we could not reconstruct the dynamic processes of cell behavior and gene expression with any certainty. Nonetheless, the initial patterns of At.twi expression (Figs 3A, 5B) may suggest that the central and peripheral areas of the germ disc are the sites of mesoderm internalization in the Achaearanea embryo. Invagination of the individual cells that have stochastically started to express At.twi at the surface might explain the mesoderm internalization. The mechanism by which the At.twi expression is initiated is an interesting issue to be studied in the future, which will clarify the difference between the fly and spider.

Fig. 5.

Comparison of twi expression between Drosophila and Achaearanea. (A) Schematic illustrations showing twi expression patterns in the stage 5, 7 and 10 embryos of Drosophila (based on Bate, 1993). (B) Schematic illustrations showing At.twi expression patterns in the stage 6, 7, 8 and 9 embryos of Achaearanea. The embryonic area of each embryo is lightly shaded, and the cells (indicated by circles) or domains expressing twi or At.twi are darkly shaded. See the legend of Fig. 1 for abbreviations.

In the Drosophila embryo, twi transcription is initiated simultaneously in all prospective mesoderm cells, and mesoderm differentiation proceeds synchronously in all segments. Apparently, this is not the case in the Achaearanea embryo. The number of At.twi-expressing cells increased progressively during early and mid stage 6 (Fig. 3A, B). This increase may be due to initiation of At.twi transcription as well as to cell division. The four expression stripes of At.twi corresponding to the segments bearing walking legs emerged during the formation of the germ band, when At.sna and many other genes showed striped expression in the surface ectoderm (Fig. 4D; our unpublished data). Thus, it appears that the patterning of the prosomal mesoderm is accompanied with that of the surface ectoderm. The appearance of new At.twi expression stripes at the growing opisthosomal region during stages 8 and 9 (Fig. 5B) is the result of initiation of At.twi transcription rather than subdivision of cell populations with persistent At.twi expression, since we have not observed intermediate states of such subdivision. Interestingly, in contrast to the opisthosomal region, the At.twi expression stripes corresponding to the chelicerae and pedipalps appeared to be formed as a result of sequential subdivision (Fig. 3D–F). In the prosomal and opisthosomal regions, different mechanisms may regulate the initiation of At.twi transcription. What cellular and molecular mechanisms cause the segmentation of the mesoderm in the spider embryo remain to be elucidated.

We suggest that there are crucial differences in the way the mesoderm develops in the fly and spider. Investigation of the cellular and molecular mechanisms underlying these differences will contribute to a better understanding of the ancestral mode of mesoderm development in the phylum Arthropoda. Although possible variations in the mode of mesoderm development among spiders as well as chelicerates will be considered, we believe the twist family genes will be useful in comparative analyses that aim to reconstruct the evolution of the arthropod mesoderm.