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A Segmentation Clock with Two-Segment Periodicity in Insects

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Science  20 Apr 2012:
Vol. 336, Issue 6079, pp. 338-341
DOI: 10.1126/science.1218256

Abstract

Vertebrate segmentation relies on a mechanism characterized by oscillating gene expression. Whether this mechanism is used by other segmented animals has been controversial. Rigorous proof of cyclic expression during arthropod segmentation has been lacking. We find that the segmentation gene odd-skipped (Tc-odd) oscillates with a two-segment periodicity in the beetle Tribolium castaneum. By bisecting embryos and culturing the two halves over different time intervals, we demonstrate that Tc-odd cycles with a period of about 95 minutes at 30°C. Using live imaging and cell tracking in green fluorescent protein–expressing embryos, we can exclude that cell movements explain this dynamic expression. Our results show that molecular oscillators represent a common feature of segmentation in divergent animals and help reconcile the contrasting paradigms of insect and vertebrate segmentation.

Many animals generate body segments sequentially from a posterior region known as the growth zone (1, 2). Whether there are common mechanisms underlying this process of segmentation in disparate segmented animals, such as vertebrates, annelids, and arthropods, has been intensely debated (38). A role for molecular oscillators in segmentation was initially proposed on theoretical grounds by Cooke and Zeeman (9). Their “clock and wavefront” model explained how the temporal periodicity of a clock could be translated into a repetitive spatial pattern during sequential segmentation. Subsequent studies showed that oscillating patterns of gene expression sweeping through the growth zone play a key role in vertebrate segmentation (1013).

A number of studies have indicated that an equivalent segmentation clock may operate in the presegmental zone of arthropods (35, 14, 15). These studies revealed changing patterns of gene expression in the presegmental zone of an insect, a centipede, and a spider. They also showed that disrupting Notch signaling, which is an important element of the vertebrate segmentation clock (13, 16), leads to defects in segmentation in some of these species. These results have been interpreted by some researchers as evidence that a common mechanism for segmentation was inherited by vertebrates and arthropods from a segmented common ancestor.

However, several doubts remain regarding this interpretation. First, Notch signaling is known to be involved in many other developmental processes, such as specification of the presegmental zone. These diverse functions may provide alternative explanations for segmentation defects (8, 17, 18). Second, cycling expression patterns have been inferred from in situ hybridization stainings on fixed embryos and comparison of similarly staged embryos. However, embryo-to-embryo variation and difficulty in accurately staging embryos (relative to the speed of segment formation) limits the reliability of this approach. Moreover, it has not yet been proven that these dynamic expression patterns reflect intracellular changes in gene expression, rather than cell movements. Thus, there is no rigorous demonstration that cyclic waves of expression are sweeping through the growth zone of arthropods. We use embryo culture and live imaging in the insect Tribolium castaneum to address these issues.

We focus on the expression of the pair-rule ortholog odd-skipped (Tc-odd) during early germband elongation. Tc-odd is expressed in primary “pair-rule” stripes, corresponding to alternate segments, emerging from the growth zone (19); secondary segmental stripes appear later. Knockdown of Tc-odd arrests segmentation (19). We noticed that Tc-odd expression in the growth zone and in the overlying amnion varies among similarly staged embryos (Fig. 1). In particular, embryos that have the same number of primary Tc-odd stripes in the segmented part of the embryo show differences in the extent and strength of Tc-odd expression within the posterior growth zone (e.g., compare panels A′ to F′ in Fig. 1). These differences are also evident when Tc-odd expression is compared to the expression of the Tribolium ortholog of even-skipped (Tc-eve) (fig. S3). Tc-odd expression appears dynamic both in the ectoderm, the mesoderm, and the amnion (fig. S4). Taking the extent of amnion closure, the emergence of the secondary (segmental) Tc-odd stripes, and the overall shape of the germband as reference points (Fig. 1, A to F ), we placed embryos in a putative temporal sequence and subdivided the period that leads to the production of each new Tc-odd stripe into three phases, named I, II, and III (see supporting online material and Fig. 1). By comparing these stainings to time-lapse recordings of live embryos (movie S1), using amnion closure as a reference, we roughly estimate that a new primary Tc-odd stripe is generated within 40 to 110 min at 30°C.

Fig. 1

Expression of Tc-odd during early germband elongation. (A to F) In situ hybridization for Tc-odd in elongating germbands at stages 3.II to 5.I, in ventral view (for staging, see supporting online material). The growth zone is located posterior to the black arrowheads. White arrowheads mark the posterior edge of the amniotic fold. (A′ to F′) Intensity profiles of Tc-odd expression along the anterior-posterior axis of germbands. The intensity of staining was measured on the lateral part of the germbands shown in (A) to (F), encompassing the ectoderm and the amnion (for explanations, see fig. S1). Note the changing profile of Tc-odd expression within the posterior growth zone (gray area) during the emergence of new primary Tc-odd stripes from that zone. During phase I of stripe formation [(C) and (F)], the growth zone has low levels of Tc-odd, except for two lateral spots of expression that mark the initiation of a new cycle of Tc-odd expression. At phase II [(A) and (D)], Tc-odd expression extends through the growth zone, such that a posterior domain of high Tc-odd expression is established. At phase III [(B) and (E)], Tc-odd expression in the posterior-most part of the growth zone is reduced to low levels, whereas cells at the anterior of the growth zone still express high levels, establishing a broad primary stripe. In a typical collection of embryos, each phase is found in roughly equal numbers. P1 to P4, primary stripes; S1 to S4, intercalating secondary stripes; gz, growth zone.

To address the criticism that different expression patterns may reflect variation among embryos, rather than temporal changes, we need to observe gene expression in the same embryo during successive time points. We achieved this using the approach taken by Palmeirim et al. (10) to prove oscillations in the vertebrate presomitic zone. First, we developed a method for Tribolium embryo culture, which allows the developing germband (embryo proper) to be dissected away from the surrounding yolk and serosa and to be grown in cell culture medium (Fig. 2). Germbands taken during early segmental stages (phase 3.III) continue to form segments for up to 5 hours in culture, at normal speed, with no overt morphological defects (Fig. 2C). This enabled us to bisect embryos in culture and to sample the expression pattern of Tc-odd at different time points in each half-embryo: Immediately after dissection, we cut individual germbands along the midline into halves (Fig. 2B); one half was fixed immediately after bisection (t = 0), whereas the other was cultured over different time intervals (15 to 105 min) before fixation. Both halves were then simultaneously subjected to in situ hybridization for Tc-odd. By comparing expression in these half-embryos, we demonstrate that Tc-odd expression in the growth zone changes from high to low levels, and back to high levels again, during production of the third and fourth primary stripes (Fig. 2, D and E). These experiments allow us to define more precisely the cycling period of Tc-odd, which we estimate to be ~95 min at 30°C (fig. S5).

Fig. 2

Germband culture and bisection experiments examining Tc-odd expression at successive time points. (A) Side view of early Tribolium embryo expressing nuclear-localized GFP. At this stage, the germband has sunk into the yolk and is becoming enveloped by the serosa and the amnion. The asterisk marks the point of first incision for dissecting the germband. (A′) Same embryo, after the serosa and yolk have been partially removed. (B) Ventral view of fully dissected germband in culture. (B′) Same embryo shown after bisection along the midline. (C) Successive pictures of a germband in culture. The germband was mounted under a coverslip and cultured for more than 5 hours at 30°C. (D and D′) In situ hybridization for Tc-odd on two halves of the same embryo. One half was fixed immediately after dissection (D), whereas the other was cultured for 30 min at 30°C before fixation (D′). Tc-odd expression in each half-embryo is at phase 3.II and 3.III, respectively. (E and E′) Same experiment, with half-embryos at phases 4.I and 4.II. The lateral part of the growth zone, encompassing the ectoderm and amnion, is outlined with a dashed line; the medial part, which also includes mesoderm, is marked M (see fig. S1). In panel E′, the oblique stripe of Tc-odd expression (white arrowhead) is in the amnion. Anterior is toward the top in all panels.

Next, we asked whether the observed changes in Tc-odd expression might be explained by cell movements in the growth zone, rather than changes in expression levels within cells. To directly address this, we established live imaging in Tribolium embryos and dissected germbands. We generated a transgenic line expressing nuclear-localized green fluorescent protein (GFP) driven by a Tribolium ubiquitous promoter (see supporting online material) and developed methods for imaging live Tribolium embryos under a spinning-disc confocal microscope. Whole embryos could be kept alive for more than 5 hours, with no apparent defects in morphogenesis, allowing us to visualize morphogenetic processes such as embryo condensation, amnion formation, germband elongation, and segmentation (Fig. 3A and movies S1 and S2). Dissected germbands were viable under a coverslip and could be imaged for 2 to 3 hours, allowing us to track the movements of individual nuclei in the growth zone and elongating germband (movie S3). Although only a small number of nuclei can be tracked reliably over a period of 1 hour at 30°C, this is sufficient to provide a map of cell movements in the early Tribolium germband (Fig. 3B). Compared with Tc-odd expression in corresponding stages, this map shows that cell movements could not account for the changes in Tc-odd expression that we observe in the growth zone (Fig. 3, C to E). These changes must be due to changes in mRNA levels within cells.

Fig. 3

Live imaging and tracking cell movements in Tribolium germbands. (A) Live imaging of Tribolium embryo expressing nuclear-localized GFP, during early stages of germband elongation. White arrowheads mark the posterior edge of the amniotic fold as it extends over the germband (compare to Fig. 1). The frames shown were taken at 15-min intervals at 30°C; the corresponding time-lapse movie is shown in movie S1. (B) Tracks of individual ectodermal cells over a 60-min period at 30°C; black circles mark the initial position of cells and red circles mark their final position. (C to E) Cell positions at 0, 30, and 60 min are superimposed on Tc-odd stained embryos at the corresponding developmental stages (4.I, 4.II, and 4.III) (see Fig. 1, C to E). Cell populations that initially express very low levels of Tc-odd subsequently lie in areas that express the gene at high levels, and then low levels. All panels show ventral views.

We find that cells in the early extending germband tend to become separated along the anterior-posterior axis and converge toward the ventral midline, which indicates that they undergo convergent extension. This is likely to be a major force driving germband elongation. In contrast, mitosis does not appear to contribute significantly to early germband elongation (see fig. S6).

Overall, our analysis provides compelling evidence for a segmentation clock in the growth zone of arthropods. By exploiting methods for embryo culture, transgenic markers, live imaging, and cell tracking in Tribolium, we are able to demonstrate that oscillating expression is due to temporal changes in expression levels, proof of which was missing in previous studies. The clock involves Tc-odd, a pair-rule gene known to be essential for Tribolium germband elongation and segmentation (19). An odd-related gene is also expressed dynamically with a two-segment periodicity in the growth zone of a centipede (4), raising the possibility that a widely conserved segmentation clock may exist in the arthropods. These results are consistent with the hypothesis that pair-rule genes were ancestrally part of a segmentation clock and subsequently evolved regulation by gap genes, which underlies Drosophila segmentation (20).

In a wider context, our results support the idea that a clock-based mechanism underlies segmentation in animals as widely separated as arthropods and vertebrates. It will be interesting to discover whether this common feature reflects a common evolutionary origin of segmentation, or a design principle that was reinvented on separate occasions. In the latter case, the clock mechanism may have evolved independently but became integrated with a preexisting mechanism of posterior growth (1, 21, 22). Ultimately, this question might be resolved by comparing the gene regulatory networks underpinning the segmentation clock across phyla, as has already been attempted within the vertebrates (16). Tribolium, as an emerging model organism with an increasing array of genetic tools and resources (23), provides opportunities to investigate the arthropod clock mechanism by genetic means.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1218256/DC1

Materials and Methods

Figs. S1 to S6

Movies S1 to S3

References (2428)

References and Notes

  1. Acknowledgments: We thank J. Schinko and G. Bucher for teaching us how to handle Tribolium and for help in generating the GFP-expressing transgenic line; J. P. Couso and S. Bishop for advice on embryo culture; D. Kosman, W. McGinnis, and C. Delidakis for advice on fluorescence in situ; and M. Strigini for critical comments on the manuscript. Our work was funded by the Marie Curie programs CELLIMAGE and ZOONET (FP6, European Union).
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