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Proc Natl Acad Sci U S A. Feb 3, 2009; 106(5): 1468–1472.
Published online Jan 15, 2009. doi:  10.1073/pnas.0811150106
PMCID: PMC2635816
From the Cover
Developmental Biology

Dynamic gene expression is required for anterior regionalization in a spider

Abstract

Patterning of a multicellular embryo requires precise spatiotemporal control of gene expression during development. The gradient of the morphogen bicoid regulates anterior regionalization in the syncytial blastoderm of Drosophila. However many arthropod embryos develop from a cellular blastoderm that does not allow the formation of transcription factor gradients. Here we show that correct anterior development of the cellularized embryo of the spider Achaearanea tepidariorum requires an anterior-to-posterior wave of dynamic gene expression for positioning the stripes of hairy, hedgehog, and orthodenticle expression. Surprisingly, this dynamic repositioning of the expression of these segmentation genes is blocked in orthodenticlepRNAi embryos and no anterior structures are specified in those embryos. Our data suggest that dynamic gene expression across a field of cells is required for anterior regionalization in spiders and provides an explanation for the problem of how positional values for anterior segmentation genes are specified via a morphogen-independent mechanism across a field of cells.

Keywords: Achaearanea tepidariorum, evolution and development, hairy, orthodenticle, segmentation

The establishment of positional information to regulate spatiotemporal gene expression patterns is key to metazoan development. The well-characterized segmentation gene cascade realizes this in the Drosophila embryo (1). A key feature of the Drosophila segmentation gene cascade is the successive refinement of gene expression patterns that ultimately defines the segments along the anterior–posterior body axis. At the top level of the cascade, the transcription factor bicoid (Bcd) forms an anterior-to-posterior gradient that is required for patterning of the anterior of Drosophila embryos (25). The syncytial blastoderm of the Drosophila embryo allows long-range transcription factor gradients, like the Bcd gradient, to directly determine positional values for the expression of target genes that determine where segmental boundaries eventually will arise (1, 6).

This gradient of the morphogen bicoid in Drosophila has long been a model for anterior regionalization in arthropods. However, there are 2 problems. First, bicoid is unique to higher dipterans and is not found in other insects or arthropods. Recently a solution has been proposed that an anterior-to-posterior orthodenticle (Otd) gradient may play a Bcd-like role in anterior segmentation in other holometabolous insects (7, 8). RNAi knockdown of otd in Nasonia vitripennis (a wasp) and Tribolium castaneum (a beetle) resulted in loss of anterior segments, presumably as a consequence of a shift of target gene expression toward the anterior pole (8). The fast evolving gene bcd therefore may have usurped a possible ancestral patterning system involving otd in the lineage leading to higher dipterans (79).

The second problem is that many arthropods pattern their anterior segments in a cellular environment (10, 11). The gradients of Bcd or Otd acting in holometabolous insects depend on the lack of cell membranes in the syncytial blastoderm embryo that allows diffusion of these transcription factors to form gradients (24, 79). The blastoderm stages of many other arthropod embryos, however, are cellularized rather than syncytial, which does not allow the formation of transcription factor diffusion gradients (1216). Very little is known about the mechanisms that account for regionalization of gene expression patterns during anterior patterning in these cellularized embryos.

Here we show that anterior regionalization in the spider Achaearanea tepidariorum requires a wave of dynamic gene expression of the segmentation genes hairy (h), hedgehog (hh), and otd. These genes are initially expressed at the anterior rim of the embryo, but subsequently a single wave of each moves away from the anterior rim toward the posterior. The dynamic gene expression depends on otd and is required for anterior regionalization because in otd RNAi embryos the expression of these genes does not move posteriorly, but instead persists at the anterior and no anterior structures are specified. Our data suggest that dynamic gene expression across a field of cells is required for anterior regionalization in spiders and thus transcription factor morphogen gradients in insects may be a newly acquired mechanism associated with the switch to syncytial blastoderm development.

Results

Dynamic Anterior Expression of hairy, hedgehog, and orthodenticle in the Spider.

Embryos of the spider Achaearanea cellularize at the 16-cell stage, before a blastoderm forms (12, 16). To understand anterior regionalization in the cellularized spider embryo we first analyzed the expression of the segmentation genes hairy (h), hedgehog (hh), and orthodenticle (otd). The early anterior expression of these genes exhibits remarkable dynamics.

Achaearanea tepidariorum has 2 otd genes, but only At-otd-1 is expressed during early development. At-otd-1 expression is first visible in a ring around the germ disk at stage 5 (Fig. 1A) (17). This is the anterior border of the embryo (16, 17). At stage 6, At-otd-1 remains expressed at the anterior rim as the radial symmetry of the germ disk is broken and the embryo opens at its dorsal side (12, 16, 17) (Fig. 1B). During stage 7 the germ disk transforms into a germ band and At-otd-1 expression moves from the anterior rim toward a more posterior position (Fig. 1C). The At-otd-1 stripe eventually is found in a head domain that corresponds to the ocular segment and that is clearly anterior to the first appendage-bearing segment of the cheliceres (Fig. 1D). Later there is also expression of At-otd-1 in the ventral midline (data not shown).

Fig. 1.
Dynamic expression of orthodenticle, hairy, and hedgehog during anterior pattering in the spider Achaearanea tepidariorum. (A–D) At-otd-1 is expressed in a ring at the rim of the germ disk at stage 5 (A); this is the future anterior of the embryo. ...

At-h and At-hh also show dynamic anterior expression patterns that are similar, but not identical to At-otd-1. Both At-h and At-hh are first coexpressed with At-otd-1 in a ring at the anterior rim of the germ disk in stage 5 embryos (Fig. 1 E and I). At stage 6, the At-hh stripe moves to a more posterior position (Fig. 1J), while both At-otd-1 and At-h remain at the rim (Fig. 1 B, F, and N). Double in situ hybridizations for At-h and At-hh confirm this (Fig. 1M). Later At-h and At-otd-1 expression is also found more posteriorly, but at different specific positions for each gene (Fig. 1 C, G, N, and O; Fig. 3 A and C). The anterior stripes of At-h and At-hh expression then both split into 2 stripes that eventually end up at the location where the cheliceral and pedipalpal segments respectively form (Fig. 1 H and L). At-h, At-hh, and At-otd-1 thus each display a single wave of expression across a field of cells in the anterior spider embryo with individual dynamics for each gene. This is indirectly shown for At-hh expression, which is initially coexpressed with At-h and At-otd-1 at the anterior rim (Fig. 1 A, E, and I), but is subsequently found in more posterior cells than At-h and At-otd-1 expression (Fig. 1 B, F, J, and M). In addition, At-h and At-otd-1 are coexpressed at the anterior rim until stage 6 (Fig. 1N), but eventually end up at different positions (Fig. 1O). Extensive cell divisions or cell movements at the anterior rim that “push” the expressing cells posteriorly are unlikely as a single explanation for the dynamics because the stripes of each gene move independently of each other. Furthermore, BrdU incorporation experiments did not show enhanced cell divisions at the anterior rim (supporting information (SI) Fig. S1).

Fig. 3.
At-otd-1 controls the dynamic movement of anterior At-h and At-hh expression. Expression of At-h (A–D) and At-hh (E–I) in control (A, C, E, and G) or At-otd-1pRNAi (B, D, F, H, and I) embryos. At stage 7 the At-h stripe neither moved from ...

orthodenticle pRNAi Embryos Lack all Anterior Structures.

Because otd is an early anterior patterning gene in insects (7, 8) and the early At-otd-1 expression is consistent with a role in anterior patterning, we next tested the function of At-otd-1 using parental RNAi (18). In embryos from mothers that had been injected with At-otd-1 dsRNA (otdpRNAi embryos) the anterior At-otd-1 expression is strongly reduced (Fig. S2 A–D). These otdpRNAi embryos show severe head defects (Fig. 2 A–E) and lack structures anterior to the pedipalpal segment, which is the second appendage-bearing segment in spiders (Fig. 2 C and E). Although in most otdpRNAi embryos this segment does not carry an appendage (the pedipalp) or only carries remnants of the appendage, expression of the Hox gene labial (At-lab) demonstrates that this segment retains pedipalpal identity. At-lab is expressed in this segment and extends to the anteriormost border of the otdpRNAi embryo (Fig. 2G; Fig. S3 C and D). In wild-type embryos, At-lab is strongly expressed in the pedipalpal segment, but not in more anterior structures (19, 20) (Fig. 2F; Fig. S3 A and B). Thus all structures anterior to the pedipalpal segment are missing in otdpRNAi embryos. This is confirmed by the lack of At-Pax6 and At-six3 expression in these embryos (Fig. S3 E–L). Deformed (At-Dfd) and dachshund (At-dac) expression shows that the walking leg segments (L1–L4) that are posterior to the pedipalpal segment form normally in otdpRNAi embryos (Fig. 2 H and I and Fig. S3 MT) even if the appendage on L1 is often broader than normal and oddly bent (e.g., Fig. 2B).

Fig. 2.
At-otd-1 pRNAi results in loss of anterior structures. (A–C) Flat-mounted wild-type, weak and strong At-otd-1pRNAi embryos, respectively, stained with the nuclear dye DAPI. Structures anterior to the Pp are missing in the At-otd-1pRNAi embryos; ...

The dynamic hairy and hedgehog Expression Depends on orthodenticle.

To determine whether At-otd-1 is required for providing the positional information for At-h and At-hh gene expression, we investigated At-h and At-hh expression after At-otd-1 RNAi. We observed a severe effect on the anterior At-h and At-hh expression, which is no longer dynamic, but is stationary in otdpRNAi embryos. The 2 genes remain expressed at the anterior rim of the germ band and their expression does not move away from the anterior rim (Fig. 3B, D, F, H, and I). Also the At-otd-1 expression itself does not move toward the posterior in otdpRNAi embryos; this is obvious from the very low levels of residual At-otd-1 transcripts that can still be detected in stage 7 otdpRNAi embryos and that do not move from the anterior rim (Fig. S2). Thus, the dynamic repositioning but not the onset of anterior At-h and At-hh expression depends on otd. The posterior At-h and At-hh stripes are not affected; they are at the same position as in control embryos and show the same dynamics as seen in controls (Fig. 3 A, C, E, and G). The dynamics of anterior At-h, At-hh, and At-otd-1 expression thus requires At-otd-1.

Discussion

Our present data from the spider provide an explanation to the problem of how positional values for anterior segmentation genes are specified via a morphogen-independent mechanism in cellularized arthropod embryos. In Drosophila development the syncytial blastoderm allows transcription factor morphogen gradients, like the Bcd gradient, to directly determine positional values for the expression of target genes and where segmental boundaries eventually will arise (1, 6). In other holometabolous insects an Otd gradient may play a role like Bcd in anterior segmentation (79). However, the cellular blastoderm of many other arthropods does not allow the formation of diffusion gradients (10, 11) and thus requires a different mechanism for providing positional clues across a field of cells.

We showed that anterior regionalization of the spider requires dynamic spatiotemporal pattern formation that leads to the positioning of the At-h and At-hh stripes in the anterior spider embryo. The stationary At-h and At-hh expression at the anterior of the otdpRNAi embryos explains the morphological phenotype of these embryos that lack all structures anterior to the pedipalpal segment: In wild-type embryos the posteriormost position of the moving At-h and At-hh expression defines where the pedipalpal segment forms. However, as a consequence of the inhibition of their dynamic expression, At-h and At-hh remain at the anterior rim of otdpRNAi embryos. Consequently, the pedipalpal segment is incorrectly specified at the extreme anterior, and all more normally anterior structures are missing. The entire embryo up to L2 thus consists of only a pedipalpal and an L1 segment (summarized in Fig. 4). Both the pedipalpal and L1 segment seem to be bigger in otdpRNAi embryos (e.g., Fig. 2 A–C), which is consistent with their specification over a larger field of cells. L2 is the first segment that is completely normal in otdpRNAi embryos, which again is consistent with the normal appearance of At-h, At-hh, and other genes in L2 and more posterior segments. The lack of anterior structures in otdpRNAi embryos is not caused by increased cell death, because TUNEL experiments showed no additional apoptosis in otdpRNAi embryos (Fig. S4). The missing anterior structures thus are the result of mispatterning caused by the blocking of dynamic gene expression. The dynamic gene expression therefore is required for correct patterning of the field of cells anterior to L2.

Fig. 4.
Schematic drawing of the model for anterior otd-dependent patterning in the spider A. tepidariorum. A single movement of the anterior expression domains of hairy (used as example in drawings), hedgehog, and orthodenticle is required for proper patterning ...

Anterior patterning in the spider, like posterior patterning (2124), thus involves dynamic gene expression. While posterior patterning involves several waves of dynamic gene expression (2124), anterior pattering involves only a single wave of gene expression for each gene. We showed that At-otd-1 is required for controlling these dynamics during anterior patterning but it is unlikely that the transcription factor Otd is the only component controlling these dynamics. It is possible that a signaling pathway is also involved, like the Notch signaling pathway in posterior segmentation (21), but it is unclear which signaling pathway because Notch-signaling does not influence anterior segments (25). In the mouse, the otd homolog Otx2 is also a key factor in the head developmental process (26, 27) and is required for proper anterior positioning of the expression of Dickkopf1 (Dkk1), which codes for a secreted protein that acts as a Wnt inhibitor (28, 29). Wnt signaling thus may be a candidate for the anterior patterning in the spider. Furthermore, a gene regulatory network that includes otx and Wnt8 controls a dynamic pattern of gene expression in the specification of endomesodermal territories in sea urchin (30, 31). The dynamic gene expression in the sea urchin is fully explained by positive and negative feedback loops that control wave front generation and decay via transcriptional cis-regulatory logic. None of the components involved here meets the definition of a morphogen and the model does not require a graded morphogen concentration distribution (30, 31). Although the precise underlying mechanism for the anterior wave of dynamic gene expression in the spider is not yet solved, similar machinery might be involved. Further research has to dissect the precise mechanisms by which otd controls the dynamic expression patterns in the spider.

Interestingly, anterior patterning in the spider is reminiscent of patterning of the eye-antennal imaginal disk in Drosophila. The morphogenetic furrow progresses anteriorly in the eye disk, and photoreceptors differentiate in the tissue posterior to the morphogenetic furrow (32, 33). The movement of the morphogenetic furrow is associated with gene expression that moves dynamically over the eye disk cells (e.g., atonal) (33). Thus patterning of both the fly eye disk and the anterior spider head requires a mechanism that involves a wave of dynamic gene expression across a field of cells.

Both insects and spiders require otd for the precise positioning of target gene expression required for the correct development of the anterior of the embryo. Therefore the last common ancestor of spiders and insects probably used otd as an ancestral organizer of anterior regionalization. However, there is a major difference. In the spider, a field of cells is patterned through dynamic gene expression in a cellularized environment, while in holometabolous insects, a field of nuclei is patterned in a syncytial environment by an Otd morphogen gradient (or Bcd gradient in higher dipterans) (24, 8). The transcription factor morphogen gradient as seen in the insects therefore may be a newly acquired mechanism that is associated with the switch to syncytial patterning in the lineage leading to insects.

Methods

Animals.

Animals of the common house spider Achaearanea tepidariorum (Chelicerata, Aranida, Theridiidae) are kept in a colony in Cologne (24). Embryos are obtained as described (17).

In Situ Hybridizations, TUNEL, BrdU Incorporation, and RNAi.

Whole mount in situ hybridizations and TUNEL were performed with modifications for spider embryos as described previously (18, 35, 36). Parental RNAi was performed as described previously (18). We used dsRNA against 3 different fragments of At-otd-1: A large 1042-bp fragment (nt 1–1042), covering almost the complete sequence, and 2 nonoverlapping fragments of 577 bp (nt 113–689) and 350 bp (nt 693-1042). dsRNA against all 3 fragments gave the same phenotypes. In the cell proliferation experiments BrdU labeling reagent (5-Bromo-2′deoxyuridine Labeling and Detection Kit II, Roche) was diluted 1:50 (200 μmol/L) and injected into the perivitelline space of the embryo. Embryos were incubated for 45–60 min and the incorporated BrdU was detected using an AP conjugated anti-BrdU antibody (37).

Supplementary Material

Supporting Information:

Acknowledgments.

This work was partially supported by the Deutsche Forschungsgemeinschaft (DFG) via SFB572 of the University of Cologne and the European Union via Research and Training Network Zoonet (MRTN-CT-2004–005624).

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