We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus. We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Small open reading frames (smORFs) encoding ‘micropeptides’ exhibit remarkable evolutionary complexity. Conserved peptides encoded by mille-pattes (mlpt)/polished rice (pri)/tarsal less (tal) are essential for embryo segmentation in Tribolium but, in Drosophila, function in terminal epidermal differentiation and patterning of adult legs. Here, we show that a molecular complex identified in Drosophila epidermal differentiation, comprising Mlpt peptides, ubiquitin-ligase Ubr3 and transcription factor Shavenbaby (Svb), represents an ancient developmental module required for early insect embryo patterning. We find that loss of segmentation function for this module in flies evolved concomitantly with restriction of Svb expression in early Drosophila embryos. Consistent with this observation, artificially restoring early Svb expression in flies causes segmentation defects that depend on mlpt function, demonstrating enduring potency of an ancestral developmental switch despite evolving embryonic patterning modes. These results highlight the evolutionary plasticity of conserved molecular complexes under the constraints of essential genetic networks.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

BackgroundOne of the best studied developmental processes is the Drosophila segmentation cascade. However, this cascade is generally considered to be highly derived and unusual, with segments being patterned simultaneously, rather than the ancestral sequential segmentation mode. We present a detailed analysis of the segmentation cascade of the milkweed bug Oncopletus fasciatus, an insect with a more primitive segmentation mode, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation modes.ResultsWe document the expression of 12 genes, representing different phases in the segmentation process. Using double staining we reconstruct the spatio-temporal relationships among these genes. We then show knock-down phenotypes of representative genes in order to uncover their roles and position in the cascade.ConclusionsWe conclude that sequential segmentation in the Oncopeltus germband includes three slightly overlapping phases: Primary pair-rule genes generate the first segmental gene expression in the anterior growth zone. This pattern is carried anteriorly by a series of secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband with conserved relationships. Unlike most holometabolous insects, this process generates a single-segment periodicity, and does not have a double-segment pattern at any stage. We suggest that the evolutionary transition to double-segment patterning lies in mutually exclusive expression patterns of secondary pair-rule genes. The fact that many aspects of the putative Oncopeltus segmentation network are similar to those of Drosophila, is consistent with a simple transition between sequential and simultaneous segmentation.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1293-z) contains supplementary material, which is available to authorized users.

Our understanding of the genetics of arthropod body plan development originally stems from work on Drosophila melanogaster from the late 1970s and onward. In Drosophila, there is a relatively detailed model for the network of gene interactions that proceeds in a sequential-hierarchical fashion to define the main features of the body plan. Over the years, we have a growing understanding of the networks involved in defining the body plan in an increasing number of arthropod species. It is now becoming possible to tease out the conserved aspects of these networks and to try to reconstruct their evolution. In this contribution, we focus on several key nodes of these networks, starting from early patterning in which the main axes are determined and the broad morphological domains of the embryo are defined, and on to later stage wherein the growth zone network is active in sequential addition of posterior segments. The pattern of conservation of networks is very patchy, with some key aspects being highly conserved in all arthropods and others being very labile. Many aspects of early axis patterning are highly conserved, as are some aspects of sequential segment generation. In contrast, regional patterning varies among different taxa, and some networks, such as the terminal patterning network, are only found in a limited range of taxa. The growth zone segmentation network is ancient and is probably plesiomorphic to all arthropods. In some insects, it has undergone significant modification to give rise to a more hardwired network that generates individual segments separately. In other insects and in most arthropods, the sequential segmentation network has undergone a significant amount of systems drift, wherein many of the genes have changed. However, it maintains a conserved underlying logic and function.

One of the best studied developmental processes is the Drosophila segmentation cascade.However, this cascade is generally considered to be highly derived and unusual. We present a detailed analysis of the sequential segmentation cascade of the milkweed bug Oncopletus fasciatus, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation. We analyzed the expression of 12 genes, representing different phases during segmentation. We reconstruct the spatio-temporal relationships among these genes And their roles and position in the cascade. We conclude that sequential segmentation in the Oncopeltus germband includes three phases: Primary pair-rule genes generate segmental gene expression in the anterior growth zone, followed by secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband. This process generates a single-segment periodicity, and does not have a double-segment pattern at any stage.Introduction: