A SoxB gene acts as an anterior gap gene and regulates posterior segment addition in the spider Parasteatoda tepidariorum

The Sox gene family encode a set of highly conserved HMG domain transcription factors that regulate many key processes during metazoan embryogenesis. In insects, the SoxB gene Dichaete is the only Sox gene known to be involved in embryonic segmentation. To determine if similar mechanisms are used in other arthropods, we investigated the role of Sox genes during segmentation in the spider Parasteatoda tepidariorum. While Dichaete does not appear to be involved in spider segmentation, RNAi knockdown of the closely related Sox21b-1 gene results in a gap like phenotype in the developing prosoma and also perturbs the sequential addition of opisthosomal segments. We show that this is in part due to a role for Sox21b-1 in regulating the expression of Wnt8 and influencing Delta-Notch signalling during the formation of the segment addition zone. Thus, we have found that two different mechanisms for segmentation in a non-mandibulate arthropod are regulated by a Group B Sox gene. Our work provides new insights into the function of an important and conserved gene family across arthropods, and the evolution of the regulation of segmentation in these animals.

1 0 endodermal layers in particular, we assayed the expression of hh and fkh in 2 0 6 class I and II Sox21b-1 knockdown embryos. (31) and remains unaffected by Sox21b-1 knockdown (Fig. 3H). fkh is also 2 0 9 expressed in cells around the rim, as well as in the centre of the germ disc in 2 1 0 mesendodermal cells (Fig. 3C). In Sox21b-1 knockdown embryos both of 2 1 1 these fkh expression domains are lost (Fig. 3G) and it therefore appears that the controls (Fig. S3H).

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In both spiders and flies, the twist (twi) gene is involved in mesoderm 2 1 9 specification (32) and we therefore examined the expression of this gene after buds from L1 to L4, in the opisthosomal segments O1 to O4 and in an anterior 2 2 3 mesodermal patch in the central part of the developing head (32) (Fig. S3E).

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While the head expression persists in Sox21b-1 class I embryos, expression 2 2 5 in all the limb and opisthosomal segments is lower or absent (Fig. S3F). In knockdown embryos the formation of these layers is perturbed (Fig. S3J). These data suggest that the ectodermal segmentation in the prosomal region 2 3 0 occurs even upon a reduction of the internal layers of the embryo. In P. tepidariorum, formation of the SAZ and production of posterior segments Dl is expressed at stages 5 and 6 in the forming SAZ, in the region of 2 4 0 the L4 primordia, and in the presumptive head (33) (Fig. 4A). Subsequently at apparently perturbed in the presumptive L1 segment (Fig. 4D). This suggests 2 4 8 that the ectoderm up to the L1 segment differentiates normally, but the 2 4 9 development of the SAZ and posterior segment addition controlled by Dl is 2 5 0 lost upon Sox21b-1 knockdown.

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Wnt8 is initially expressed at stage 5 in the centre and at the rim of the 2 5 2 germ disc (Fig. 4E). At stage 9, striped expression of Wnt8 is seen from the  Knockdown of Sox21b-1 results in the loss of Wnt8 expression in late stage 5 2 5 5 embryos ( Fig. 4F). At stage 9, Wnt8 expression is observed in the cheliceral, 2 5 6 pedipalpal and first walking limb segments of Sox21b-1 knockdown embryos, 2 5 7 but no expression is detected in the remaining posterior cells (Fig. 4H).

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Consistent with the loss of Dl and Wnt8, cad expression is also lost in stage 5 2 5 9 and stage 9 Sox21b-1 knockdown embryos ( Fig. 4I-L). These observations  produced by RNAi knockdown of either Dl or Wnt8 (Fig. 5).

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The spider orthologue of h is expressed in the presumptive L2-L4 Sox21b-1 knockdown embryos, the expression of h is lost throughout the 2 6 8 entire germ disc (Fig. 4N). In addition, in Class I phenotype embryos at stage  To look at the effect of Sox21b-1 knockdown on segmentation in more  Here we show that, while Dichaete is not involved in spider Sox21b-1 has a gap gene role and is required for the specification of L1-L4  In the posterior, Sox21b-1 knockdown perturbs SAZ formation and 3 1 7 consequently results in truncated embryos missing all opisthosomal 3 1 8 segments. Therefore, Sox21b-1 regulates development of the SAZ, and our 3 1 9 observations indicate this is at least in part through roles in organising the 3 2 0 germ layers and specification of mesendodermal cells during stages 5 and 6.

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This is supported by the loss of fkh expression upon Sox21b-1 knockdown, which is required for mesoderm and endoderm formation in both spiders and Sox21b-1 in the SAZ after stage 6 is suggestive of a role in segment addition.

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Our work on Sox21b-1 provides an important new insight into the gene sequential addition of segments from this tissue. We show that Sox21b-1 acts 3 2 8 upstream of Wnt8 and Delta-Notch signalling in this GRN and is required for the activation of these important signalling pathways during posterior development (4,8,9,14,15,42). Interestingly, SoxB genes also cooperate 3 3 7 with Wnt and Delta-Notch signalling in various aspects of vertebrate of the stem state in the neuroepithelium (35,43,44,45). Our study shows that Sox21b-1 is not only involved in segmentation but is 3 4 4 maternally supplied and regulates cell division in the early germ disc, as well 3 4 5 as the transition from radial to axial symmetry during germ band formation.

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Further experiments with Sox21b-1 are required to fully elucidate the  The evolution of Sox21b-1 3 5 7 The evolution and diversification of Group B Sox genes in insects is not fully  believe it is highly significant that two very closely related SoxB genes are 3 6 7 involved in segmentation in both the spider P. tepidariorum and in insects, pointing to an ancient role for this subfamily of Sox genes in invertebrates.

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Given the close similarity between the HMG domains of Sox21b and Dichaete, it is possible that in some lineages the Dichaete orthologue 3 7 1 assumed the segmentation role, whereas in others it was Sox21b. In spiders, Wnt8 is involved in posterior development while in other arthropods this role is  The spider contains an additional related SoxB gene, Sox21b-2, that and/or function of this gene in this sister group to the arthropods. removed and a small number of embryos were immersed in halocarbon oil for 3 9 2 staging according to (26). To identify the phylogenetic relationship of P. tepidariorum Sox genes the Fragment of genes were amplified using PCR and cloned into pCR4-TOPO 4 3 0 (Invitrogen, Life Technologies). Oligonucleotide sequences are listed in Table   4 3 1 S1. Immunostaining was carried out following (51) with minor modifications: Triton was increased to 0.1%. The following primary antibodies were used: Millipore -06-570). For detection the following secondary antibodies were  (720 bp) as used previously (27), were transcribed using the same method. females every two days, with a total of five injections (n = 7 for each dsRNA; The authors declare no competing interests. part by a BBSRC grant (BB/N007069/1) to SR.   Commun 2:500.    oocytes of a common cellar spider, Pholcus phalangioides (Araneae: Pholcidae). Arthropod Struct Dev, 36: 317-326.