Expression patterns of “segmentation cascade” genes
We followed the expression patterns of 12 genes, mostly orthologues of the pair-rule and segment-polarity genes, during the formation of the abdominal segments of Oncopeltus fasciatus. Some of the genes discussed herein (eve, Dl, cad, inv) have previously been described and will only be mentioned briefly, adding details that have not been reported before. We present them roughly in the order of their appearance, from posterior to anterior. To facilitate inter-species comparison, we only report on genes that are orthologs of genes involved in the segmentation cascade of Drosophila, realizing that this does not give the full picture, as there may be other genes involved in segmentation in Oncopeltus that do not have such a role in Drosophila [18, 19]. Note that we use the terms pair-rule genes and segment-polarity genes as convenient shorthand for orthologs of genes that have a pair-rule / segment-polarity role in Drosophila, and this does not a-priori imply a similar role in Oncopeltus.
Even-skipped (eve)
The expression pattern of eve [11, 20] includes a domain of solid expression in the posterior growth zone, and a striped expression domain in the anterior growth zone (Fig. 1a-a’). The number of eve stripes early in abdominal segmentation can be as high as five or six, while by the end of the segmentation process, there is only a single eve stripe anterior to the solid expression domain. In some stained embryos, the posterior-most eve stripe is in contact with the solid expression domain in its medial portion, giving the impression of a stripe “peeling off” from the solid domain [20].
Odd-skipped (odd) and sister of odd-and bowel (sob)
The expression patterns of odd (Fig. 1b-b’) and its paralog sob (Fig. 1c-c’) are nearly identical to each other, and both are remarkably similar to that of eve. They also have a solid expression domain in the posterior growth zone, and a striped expression in the anterior growth zone. However, unlike eve, the expression of odd and sob in the posterior growth zone is graded, with highest expression in the anterior margin of the posterior growth zone, tapering off posteriorly, and ending before the posteriormost end of the embryo. The striped expression of odd and sob extends into the segmented germband slightly more than that of eve.
Runt (run)
Like odd and eve, run (Fig. 1d-d’) is defined as a “primary pair-rule gene” in Drosophila. In Tribolium, these three genes were found to work together in a pair rule regulatory circuit generating the repeating pattern of the segmentation process [21]. The probe for run gave very weak signal in our hands, so we could not analyze it at the level of detail we could for other genes. In the Oncopeltus germband, run does not display a striped expression pattern in the growth zone or in the segmented germband, in contrast with all other “segmentation cascade” genes in this study. It is mainly expressed in two to three broad graduated domains within the growth zone. This pattern is highly dynamic and variable among embryos, but we were unable to correlate this dynamic activity with that of the other genes we have looked at. In addition to its expression in the growth zone, in late stages of segmentation, run is expressed in paired domains in the germband near the ventral midline, probably representing neural precursor tissue. The aforementioned four genes are the only pair-rule gene orthologs that are expressed in the posterior growth zone.
Odd-paired (opa) and sloppy-paired (slp)
These two genes are often defined as “secondary pair-rule genes” in Drosophila. In the Oncopeltus anterior growth zone, opa is expressed in a striped pattern (Fig. 2a-a’), resembling that of eve, odd and sob. Unlike these genes, opa is not expressed in the posterior growth zone at any stage. The number of stripes in the growth zone varies from 2 to 3 stripes early in the segmentation process to a single stripe at later stages. These stripes are more anteriorly located than the eve and odd/sob stripes. Expression of opa continues into the segmented germband and expression is maintained in narrow stripes in the posterior of each segment throughout the germband stage.
The expression of slp (Fig. 2b-b’) is similar to that of opa with two main differences: the expression stripes are broader in the germband and are found in a more anterior-medial position in each segment. A more subtle distinction is that slp has a weak posterior-anterior expression gradient in each stripe, both in the anterior growth zone and in the germband.
Hairy (h)
Expression of h (Fig. 2c-c’) is weakly noticeable in the posterior growth zone of early germband embryos. In the anterior growth zone, it is expressed in two faint stripes, and in a narrow stripe in the posterior of every mature segment. Like run, it also shows expression in the mesodermal cells of the growth zone. Segmental expression fades in mature segments later in development.
Hedgehog (hh)
Known from Drosophila as a segment polarity gene, hh is expressed not only in every germband segment, but also in stripes in the anterior growth zone, similar to the pair-rule gene orthologs (Fig. 3a-a’). It is visible in the posterior region of the anterior growth zone as a wide stripe, not fully resolved and not always clearly separated from the next anterior, better defined stripe. The third hh stripe is fully separated from the two prior stripes, as are the more anterior stripes. The segmental stripes are situated in the posterior of each segment. In addition to the striped expression pattern, hh is expressed in a single patch at the very posterior of the embryo.
Wingless (wg) and invected (inv)
We have previously described the expression of wg in the Oncopeltus blastoderm [4], but not in the germband. Expression of the segment polarity gene wg begins in the forming segment, initially as two lateral dots, later expanding and fusing to form a segmental stripe in the middle of each segment (Fig. 3b-b’). The segmental stripes are notably thinner medially. In addition to its segment-polarity pattern, wg is strongly expressed in the posterior growth zone. In the early stages of the germband it appears in the posterior pole of the embryo, and as segmentation progresses, it gains a crescent like shape beginning at the medial part of the posterior growth zone, curving anteriorly. At later stages, expression moves slightly anteriorly and gains an M shape. Expression of inv (an engrailed paralog) has been described in several previous publications. It is expressed in the posterior of every segment in the germband.
Caudal (cad) and Delta (Dl)
We have previously described the expression of Dl and cad in the Oncopeltus germband [11]. Briefly – Dl Expression is seen in the anterior growth zone in 2–3 stripes, and in a punctate neural system related expression pattern in the segmented germband (Fig. 3c-c’). Expression of cad is restricted to the posterior growth zone through the germband stage.
Relative expression domains
To clarify the spatial relationships among the different genes, we carried out a series of double stainings (Fig. 4). Although not all combinations worked, we have sufficient pair-wise comparisons to be able to reconstruct the relative position of all of the genes studied, with the exception of run (summarized in Fig. 5).
Four of the genes we studied are expressed in solid domains throughout the posterior growth zone – cad, eve and odd/sob. We carried out double staining for cad and eve (Fig. 4a) to see whether they share an anterior border in the posterior growth zone (the border between the anterior and posterior domains of the growth zone). We could detect no difference in the anterior extent of these genes, indicating a single uniform border within the growth zone.
The expression patterns of eve and odd are similar (Fig. 4b), With full overlap in the posterior growth zone. However, looking at their anterior expression shows that they overlap only partially, with eve expression being anterior to that of odd in any given stripe. We did not double-stain odd and sob, however, they both show the same relationship to eve (Fig. 4c) suggesting that their expression patterns fully overlap.
The two secondary pair-rule genes, opa and slp (Fig. 4d) are expressed in complementary patterns in the anterior growth zone, with slp forming the posteriormost stripe. As segmentation progresses, in the later stripes of the anterior growth zone, a gap appears anterior to the opa stripe and posterior to the slp one. In the segmented germband the stripes are fully separated and occupy distinct regions of the nascent segment. Comparing slp with eve (Fig. 4e) shows that they have a narrow domain in the anteriormost growth zone where both are expressed. Expression of slp appears as a faint narrow band in the lateral anterior growth zone, anterior and adjacent to the second eve stripe. The second slp stripe is already much stronger, but still shows a gap in the midline where eve is expressed, indicating that at this stage, these two genes are probably not co-expressed, but are both present in different areas of the same position along the anterior-posterior axis. The third slp stripe is completely resolved to the anterior of the final, most anterior eve stripe. As visible in the single stainings, slp is expressed in a graduated manner, strongly expressed in the posterior of the band, weakening towards the anterior but still with a well-defined anterior border, after which there is a gap where neither eve nor slp are expressed. The relative expression of opa and slp suggests that eve overlaps opa in the anterior of each stripe.
The expression of eve and inv (Fig. 4f) overlap exactly in the only region where they are co-expressed – the posteriormost segment (as previously shown by Liu and Kaufman [20]). The domains of eve and hh (Fig. 4g) also overlap, but this overlap extends through the entire anterior growth zone. Thus, we conclude that inv and hh also overlap. The third segment polarity gene we have looked at, wg abuts hh and sits anterior to it (Fig. 4h). Thus, the expression of hh can be seen as a combination of the posterior expression of eve and the anterior expression of inv.
Finally, the expression of Dl lies anterior to that of eve (Fig. 4i), perhaps with a slight overlap. Thus, when it is still expressed in stripes, Dl overlaps the expression domain of slp. The picture is completed by the anterior expression of h, which lies adjacent and posterior to inv, but is expressed earlier in any given segment (Fig. 4j).
Spatial dynamics of the segmentation genes
In order to gain a better understanding of the dynamic pattern of the segmentation genes over time, we have measured the expression levels of three representative genes, eve, odd and hh, along the anterior-posterior axis (Fig. 6a-c, a’-c′). We summed the pixel intensity for every point along the posterior-anterior axis on photographs of stained embryos. For hh and eve, we followed this up in a large sample of > 50 embryos, and plotted summed pixel intensity along the axis over developmental time on a three-dimensional graph (Fig. 6d-e. See methods), including relative developmental age (a value-less order based on germband length), position along the axis (using the posterior boundary of the third thoracic segment as the origin) and normalized expression level. The individual embryos used for this analysis covered a range of stages, but are distributed randomly across this range. The time axis is thus not linear, but rather a category axis of increasing size, which serves as a good proxy for developmental age. The plot for hh (Fig. 6d), which is expressed in every segment starting from the anterior growth zone, shows that indeed size serves as a good proxy for age, since we can see the hh segmental stripes appearing in correct sequence. These plots allow us to follow the development of the expression patterns of the two genes, and by extension, shed some light on the dynamics of the entire process.
Using this visualization, we show that when eve stripes first peel off from the posterior growth zone’s solid expression domain (Fig. 6e), they remain stationary relative to the germband (represented by the third thoracic segment) but shift slightly in position relative to the solid expression domain of eve in the posterior growth zone. The stripes of hh expression, in contrast, remain in a constant position relative to the third thoracic segment after they are formed.
RNAi experiments
Following the detailed analysis of gene expression patterns, we went on to examine the function of representative genes in the segmentation process by knocking them down through RNAi. For each gene knocked down, we collected early germband stages and late germband stages of RNAi embryos and stained them for inv and eve. In addition, we collected a small number of pre-hatching larvae to identify morphological phenotypes.
RNAi experiments have previously been conducted for some of our genes of interest. Knocking down eve leads to a truncation of the embryo and a complete loss of all growth zone derived segments [20]. Knocking down the segment polarity genes, inv (previously identified as engrailed) and wg leads to malformed segmental boundaries, but does not lead to any specific truncation phenotypes [22]. We have knocked down the second important primary pair rule gene odd, the secondary pair rule gene slp, and the remaining segment polarity gene hh. Most of the eggs in these experiments were fixed during development, and only a small percentage were left to develop until hatching. Thus, we have a relatively small sample of larval phenotypes (Additional file 1). For all three genes, the larval phenotypes were fairly uniform and could not be broken down into relevant specific phenotypic classes. Thus, we treat them all together (but see difference between the two dsRNA fragments in slp below).
Early odd-RNAi germband embryos (Fig. 7b) exhibit a reduction in the distance between the anterior gnathal and thoracic segments, and a much broader expression of inv. The maxilla and labium are closer together with cells between them expressing inv ectopically. T1 and T2 are also wider and closer, and are fused in the midline. T3 seems to be normal, and so do the abdominal segments present at this stage. The growth zone exhibits no visible abnormalities.
In later, fully segmented germband embryos (Fig. 7b’), we see that this phenotype has progressed to limb fusion: T1 and T2 are fused. T3 remains mostly separated. In the abdominal segments, we see a similar effect of segment fusion and occasional ectopic expression of inv between segmental stripes. Notably, we see no evidence of segment deletion. The number and location of the segments is normal, but with defective segment boundaries. This is true both for the blastoderm derived anterior segments and for the growth zone derived posterior segments. The larval odd RNAi phenotypes are remarkably uniform, and nearly all show the same findings: segments are formed with irregular boundaries, appendages are fused and we see deformation in the head, mainly indicating abnormal midline closure. A very small number of hatchlings (around 10%) show a stronger phenotype with anterior or posterior segments missing (Additional file 1).
Similarly, slp RNAi embryos retain the normal number of segments (Fig. 7c’), but these are misshapen. Already in the early germband embryo (Fig. 7c) the embryos are noticeable wider, and we can see an expansion and ectopic expression of inv in the midline of the blastoderm derived segments. At later stages, the ventral (medial) region is much wider and thinner, with unusual secretions obscuring the cells (possibly indicating apoptosis). Expression of inv is lost from the ventral portions of these segments (Fig. 7c’). The first abdominal inv stripes are expressed relatively normally, and only in the later embryo (Fig. 7c’) do we see that the segment border is malformed in the mediolateral aspect of the abdomen, lacking normal expression of inv. The most striking outcome of slp RNAi is the loss of thoracic appendages. Some embryos are completely devoid of appendages, while some maintain residual stumps of limbs T1 and T2. This is also seen in the hatchlings (Fig. 7c”), where instead of limbs we find actual holes in the cuticle. In under 10% of the hatchlings, we found more severe phenotypes with possible failed dorsal closure. We repeated the RNAi experiment with a second fragment. The results were similar, but the phenotypes were generally weaker (Additional file 1), and we saw none of the extreme phenotypes. Interestingly, both in germband embryos and in hatchlings, we see that the T2 limb is lost before the other limbs.
RNAi for hh (Fig. 7d-d”) gave similar results to those previously reported for the other segment polarity genes [22]. All segments are present, and germband embryos look almost normal. However, hatchlings are compressed and show disrupted segmental boundaries. Malformations of the head are seen both in the germband embryos and in the hatchlings.
We looked at the expression of eve in RNAi embryos for all three genes. In all cases, the expression of eve in the growth zone is almost indistinguishable from wildtype expression (Additional file 2). However, in hh-RNAi embryos, we see ectopic expression of eve in a stripe in the head region, and in odd-RNAi embryos we see ectopic expression in the midline of the germband.