© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev181453. doi:10.1242/dev.181453 RESEARCH ARTICLE Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus Katie Reding, Mengyao Chen*, Yong Lu‡, Alys M. Cheatle Jarvela and Leslie Pick§ ABSTRACT example, even-skipped (eve) and fushi tarazu ( ftz) are expressed in The discovery of pair-rule genes (PRGs) in Drosophila revealed the complementary seven-stripe patterns, each in the primordia of the existence of an underlying two-segment-wide prepattern directing alternate parasegments missing in eve or ftz mutants (Lawrence embryogenesis. The milkweed bug Oncopeltus fasciatus,a and Johnston, 1989). Other PRGs are expressed in similar hemimetabolous insect, is a more representative arthropod: most of complementary patterns, with the combined, staggered expression ‘ its segments form sequentially after gastrulation. Here, we report the of the full set of seven-striped PRGs generating unique double- ’ expression and function of orthologs of the complete set of nine segment codes to direct the formation of body segments (Gergen Drosophila PRGs in Oncopeltus. Seven Of-PRG-orthologs are et al., 1986; Graham et al., 2019; Scott and Carroll, 1987). Many of the expressed in stripes in the primordia of every segment, rather than Drosophila PRGs transition to segmental expression as development every other segment; Of-runt is PR-like and several orthologs are also proceeds but mutant phenotypes reveal the earliest roles of these genes expressed in the segment addition zone. RNAi-mediated knockdown in PR patterning: roughly half-sized mutant embryos missing alternate of Of-odd-skipped, paired and sloppy-paired impacted all segments, segments (reviewed by Wieschaus and Nüsslein-Volhard, 2016). with no indication of PR-like register. We confirm that Of-E75A is As all insects are segmented, the gene regulatory logic underlying expressed in PR-like stripes, although it is not expressed in this way in segmentation might be wholly conserved. However, Drosophila are Drosophila, demonstrating the existence of an underlying PR-like long-germ insects with all parasegments patterned more or less prepattern in Oncopeltus. These findings reveal that a switch simultaneously at blastoderm. This mode of development is derived occurred in regulatory circuits, leading to segment formation: and found only among holometabolous insects, where it while several holometabolous insects are ‘Drosophila-like’, using independently arose multiple times (Davis and Patel, 2002; Liu PRG orthologs for PR patterning, most Of-PRGs are expressed and Kaufman, 2005b). In contrast, most insect groups add segments ‘ ’ segmentally in Oncopeltus, a more basally branching insect. Thus, an sequentially after the blastoderm stage ( sequential segmentation ), evolutionarily stable phenotype – segment formation – is directed by from the posterior end of the germband, a region known as the alternate regulatory pathways in diverse species. growth zone or segment addition zone (SAZ) (reviewed by Davis and Patel, 2002; Liu and Kaufman, 2005b). Thus, the Drosophila- KEY WORDS: Pair-rule gene, Segmentation, Oncopeltus fasciatus, like PR-patterning of a double-segment unit might be restricted to Intermediate germ, E75A, Evo-devo, Milkweed bug, Hemiptera, simultaneously segmenting species. However, PR-like expression A-P patterning patterns – defined as stripes of gene expression in the primordia of alternate (every-other) segmental units – have been observed in one INTRODUCTION or more sequentially segmenting species for orthologs of each of the Mechanisms directing the formation of the basic segmented body nine Drosophila PRGs (PRG orthologs): ftz, fushi tarazu factor-1 plan have been unraveled for the model insect, Drosophila ( ftz-f1), eve, odd skipped (odd), runt (run), hairy (h), odd paired melanogaster (reviewed by Wieschaus and Nüsslein-Volhard, (opa), paired ( prd) and sloppy paired (slp). Within holometabolous 2016). This study identified a set of pair-rule mutants, insects, the expression and function of the complete set of orthologs characterized by absence of alternate body segments, revealing of Drosophila PRGs has been examined in two species, the that patterning of single segments is preceded by pre-patterning of a sequentially segmenting beetles Tribolium castaneum and Dermestes double-segment-wide unit that is repeated along the anterior- maculatus (Choe and Brown, 2007; Xiang et al., 2015, 2017). These posterior axis of the embryo at half the frequency of segment analyses, together with studies of selected PRG orthologs in a handful number. Most of the pair-rule genes (PRGs) responsible for this pre- of other holometabolous insects (Grbićand Strand, 1998; Kraft and pattern are expressed in seven stripes in the Drosophila blastoderm, Jäckle, 1994; Nakao, 2010, 2015; Rosenberg et al., 2014), suggest that with PRG expression foreshadowing the corresponding mutant a role for some PRG orthologs in Drosophila-style PR patterning is phenotype for individual PRGs (pair-rule stripes, Fig. 1). For shared among holometabolous insects, even those with sequential segmentation (Fig. 1). However, other members of this gene set have changed in expression and/or function within Holometabola (Choe Department of Entomology, 4291 Fieldhouse Drive, University of Maryland, College Park, MD 20742, USA. et al., 2017; Clark and Peel, 2018; Heffer et al., 2013a,b). For example, *Present address: Institute of Insect Science, Zhejiang University, Hangzhou ftz-f1 is expressed ubiquitously in Drosophila but in stripes in beetles 310058, P.R. China. (Heffer et al., 2013b; Xiang et al., 2017) and several PRG-orthologs are ‡Present address: Department of Anesthesiology, Stony Brook Medicine, Stony Brook, New York, NY 11794-8480, USA. expressed in the segment addition zone (SAZ) in sequentially segmenting species as components of a vertebrate-like clock-and- § Author for correspondence ([email protected]) wave mechanism, in addition to being expressed in PR stripes (El- L.P., 0000-0002-4505-5107 Sherif et al., 2012; Sarrazin et al., 2012). In contrast to studies in holometabolous insects, there has been Received 11 June 2019; Accepted 12 August 2019 less focus on PRG expression or function in hemimetabolous DEVELOPMENT 1 RESEARCH ARTICLE Development (2019) 146, dev181453. doi:10.1242/dev.181453 Fig. 1. Models for the ancestral origin of PR patterning. A simplified cladogram of arthropods with segmentation-related expression patterns of PRG orthologs in various insect and myriapod orders indicated as pair-rule-like, segmental (in every segment), SAZ (broad expression in the segment addition zone) or other. Non-segmentation-related expression patterns, such as expression in the nervous system, are not included. Oncopeltus is situated in the shaded region. Two hypotheses regarding the evolution of PR-like expression for the PRG orthologs are shown in red and blue on the tree. (Red) The ancestor of all arthropods exhibited PR expression of the PRG orthologs, which was subsequently lost in the lineage leading to Oncopeltus. (Blue) PRG orthologs were not expressed in a pair-rule manner in the arthropod ancestor, and PR-expression of these genes was gained independently in the lineages leading to myriapods and holometabolous insects. Numbered references for the expression patterns summarized here are detailed in Table S1. insects. PR-like expression of eve was observed in a cricket (Mito 2012). PR-like expression was observed in Oncopeltus embryos for et al., 2006, 2007), whereas in grasshoppers, eve and ftz have the gene E75A and RNAi resulted in fusion of neighboring distinctly non-PR-like expression, both being expressed in the SAZ segments, demonstrating the existence of an underlying PR-like pre- (Dawes et al., 1994; Patel et al., 1992). Also different from patterning mechanism in this species (Erezyilmaz et al., 2009). holometabolous species, h is expressed segmentally in a cockroach However, E75A does not have PR-like expression or function in (Pueyo et al., 2008). Interestingly, PR-like expression of PRG Drosophila (Bialecki et al., 2002; Buszczak et al., 1999; Segraves orthologs has been observed in evolutionarily distant, non-insect and Hogness, 1990). In Oncopeltus, eve is expressed in stripes in arthropods. For example, striped expression at half the frequency of every segment (‘segmental expression’) and in the SAZ (Liu and segmental stripes (sometimes referred to as ‘double segment Kaufman, 2005a). Here we have isolated and examined the periodicity’) has been observed for several PRG orthologs in a expression of all nine orthologs of the Drosophila PRGs in centipede (Chipman and Akam, 2008; Chipman et al., 2004; Green Oncopeltus (Of-PRG orthologs) and seven paralogs of these genes, and Akam, 2013) and the expression of prd in spider mites is and have compared their expression to the only known Of-PRG: suggestive of modulation by a PR-like regulator (Dearden et al., E75A. Despite the fact that Of-PRG orthologs are all expressed 2002). These findings suggest two evolutionary hypotheses: PR during the stages at which Oncopeltus specifies segments, only one expression of PRGs arose independently in holometabolous insects (Of-run) is expressed in a pattern reminiscent of Drosophila PRGs. and myriapods (Fig. 1, blue); or it was ancestral and lost in some Most others are expressed in segmentally reiterated patterns, either hemimetabolous species (Fig. 1, red).