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First published online 23 March 2005
doi: 10.1242/dev.01807

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even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect

Department of Biology, Indiana University, 1001 East Third Street, Bloomington, IN 47405, USA

View larger version (21K): [in a new window]   Fig. 1. Predicted structure and sequence analysis of Oncopeltus even-skipped. (A) Cartoon of predicted Of'eve protein structure with conserved N-terminal domain (N), homeodomain (HD) and C-terminal domain (C). Underlined are the regions used for synthesizing dsRNA and in situ probes. The 3' fragment is approximately 600 bp long and spans a small region of the homeodomain. The 5' fragment is approximately 260 bp long and does not include any of the homeodomain. (B) Conserved N-terminal domain of even-skipped from Drosophila melanogaster, Tribolium castaneum, Oncopeltus fasciatus and Schistocerca americana. (C) Conserved C-terminal domain from the same insect species. The Groucho co-repressor interaction domain is underlined. (D) Conserved homeodomain region from the four insect species.

View larger version (89K): [in a new window]   Fig. 2. Oncopeltus even-skipped blastoderm expression. (A-F,J1-K3) stained with probe made to Of'eve. (A) Blastoderm at 20-24 hours. Of'eve transcript appears on the posterior two-thirds of the blastoderm surface. At this stage, transcript is evenly distributed around the blastoderm, without dorsal or ventral differences. (B) Blastoderm at 32-36 hours; dorsal aspect. The anterior boundary has refined to become the first stripe. (C) Embryo at a slightly later stage than in B; dorsal aspect. The first three stripes of Of'eve have already formed with the fourth stripe just becoming visible (arrowhead). (D) Embryo with four stripes visible. (E) Embryo with five stripes visible. Arrowheads bracket the remainder of the initial broad domain. (F) Lateral view of 36- to 40-hour-old embryo with all six Of'eve stripes visible on blastoderm surface. Posterior patch of transcript remains during germband invagination. Arrow indicates invagination site. (G) Blastoderm at 36-40 hour stained for Oncopeltus engrailed. Six vertical stripes of engrailed, corresponding to the mandibular (Mn) through third thoracic segments (T3) are present on blastoderm surface. Arrow indicates site of germband invagination. (H) Embryo at 36-40 hours stained for both hunchback (orange) and even-skipped (purple). Central band of Of'hb expression spans first three Of'eve stripes. Arrow indicates germband invagination. (I) Embryo at 36-40 hours stained for both Krüppel (orange) and even-skipped (purple). Of'Kr expression spans Of'eve stripes 4-6. Arrow indicates germband invagination. (J1-J3) The same 40-44 hour embryo as in I undergoing germband invagination stained for eve. (J1) Lateral aspect showing that first Of'eve stripe has faded, while stripe 6 is no longer visible on blastoderm surface. (J2) Ventral aspect. Stripe 6 in process of migrating to contribute to the germband. (J3) Higher magnification of embryo shown in J2. (K1-K3) Embryo during germband invagination, at a later stage than in J. (K1) Lateral aspect of blastoderm surface, showing that all Of'eve stripes on the blastoderm have faded. (K2) Embryo with ventral region of yolk removed to show underlying early germband. (K3) Higher magnification of same embryo. Expression of Of'eve can be seen in early germband and in three stripes (arrows). Scale bars: 200 µm.

View larger version (109K): [in a new window]   Fig. 3. even-skipped expression in the posterior growth zone and germband. Germband stage embryos are stained for even-skipped (purple) and engrailed (orange). (A1) Early germband with Of'eve accumulation in growth zone. Third thoracic segment is marked (T3). The growth zone is large. (A2) Higher magnification of embryo shown in A1, showing appearance of first abdominal Of'en stripe, coincident with a stripe of Of'eve. (B) Mid-germband stage embryo stained only for Of'eve. The midline region of the early stripe is still contiguous with the growth zone patch, indicating that this stripe is still in the process of leaving the growth zone. (C1) Mid-germband stage embryo. Of'eve expression is maintained in the growth zone and in three stripes just anterior to growth zone expression. Growth zone is now smaller than in A1. (C2) Higher magnification of embryo shown in B1, showing coincident expression of Of'eve and Of'en in three early segments. Of'eve is most strongly expressed in the younger (more posterior) segments, while Of'en is expressed more strongly in older (more anterior) segments. Thus, Of'eve expression is fading just as Of'en expression initiates. (D1) Embryo at end of germband elongation. Growth zone expression of Of'eve fades. (D2) Higher magnification of embryo shown in D1. Final Of'eve stripe is coincident with tenth abdominal Of'en stripe. Remaining posterior dot of Of'eve may represent anal ring expression. (E) Cartoon showing birth and progressive maturation of Of'eve germband stripes. Early stripes seem to `peel' away and are often contiguous with growth zone patch. Scale bars: 200 µm for A1,C1,D1; 100 µm for A2,B,C2,D2.

View larger version (91K): [in a new window]   Fig. 4. Oncopeltus even-skipped RNAi phenotype. (A1-A3) Uninjected hatching-staged animals. (A1) Lateral view. Thoracic and abdominal regions are marked. (A2) Dorsal aspect. (A3) Ventral view. (B1-B3) Class III (weak) phenotype. (B1) Lateral view of class III animal. Thorax weakly affected, all legs are present. The abdominal region is shortened. (B2) Dorsal view. Abdominal segments marked by arrowheads. (B3) Ventral view. (C) Fully formed germband of uninjected embryo, stained for engrailed. (D1,D2) Putative class III germband stained for engrailed. (D1) Lateral view. Head and thorax are at most weakly affected, but abdominal region is shortened and shows strong segmental defects. As the germbands were extremely curved and ruffled, and therefore impossible to mount under a cover slip, they were photographed unmounted. (D2) Dorsal aspect, showing segmentation defects in abdomen. (E1,E2) Class II (moderate) hatching stage embryo. (E1) Lateral view showing presence of first thoracic leg. Second thoracic leg is also present but difficult to see in this photograph. Third thoracic leg is missing. Abdomen is now severely shortened. (E2) Dorsal aspect. There are segmental defects in head and thorax. (F) Putative class II germband stained for engrailed. Body now highly reduced, and second and third thoracic legs are missing. Gnathal and first thoracic appendages still present. (G) Class I (strong) embryo. Only anterior structures such as labrum, eye and antenna are formed. (H1-H2) Class I embryo stained for engrailed (H1) and DNA (H2). All segments posterior of the head are missing. ANT, antenna; MN, mandible; MX, maxillary; LB, labium; T1-T3, thoracic segments; A, abdomen. Scale bars: 200 µm in A1-G; 100 µm in H1 and H2.

View larger version (49K): [in a new window]   Fig. 5. hunchback and Krüppel expression in Of'eve RNAi blastoderms. (A,C,E) Uninjected animals. (B,D1-2,F1-2) Of'eve RNAi animals. (A) Lateral view of uninjected 44-48 hour embryo stained for DNA. Germband has fully invaginated, leaving behind head lobes on outside posterior of yolk ball (densely staining region). Arrow indicates site of invagination. (B) Lateral view of 44-48 hour Of'eve RNAi embryo stained for DNA. Odd wrinkles and putatively mislocalized invagination site (arrow) can be seen. (C) Dorsal aspect of uninjected 36-40 hour blastoderm stained for Oncopeltus hunchback showing that Of'hb normally accumulates in two broad bands. Weaker anterior band corresponds to anterior head, while central band corresponds to posterior mandible through anterior first thoracic segments. (D1) Dorsal view of 36-40 hour Of'eve RNAi blastoderm, showing that Of'hb is now expressed in a single uniform domain in the posterior half of the blastoderm. (D2) Same embryo as in (D1), but rotated to show lateral view. Of'hb expression is restricted to the dorsal part of the blastoderm and is typical in late stage blastoderms. (E) Dorsal view of uninjected 36-40 hour embryo stained for Oncopeltus Krüppel. Of'Kr normally accumulates in posterior one-third of blastoderm. (F1) Of'eve RNAi blastoderm at 36-40 hour. Of'Kr expression is largely absent, with only a very small posterior patch remaining. (F2) Same embryo as in F1, end-on view of posterior tip of blastoderm, showing remaining Of'Kr patch. Scale bars: 200 µm.

View larger version (46K): [in a new window]   Fig. 6. Summary of Oncopeltus even-skipped evolution, expression and function. (A) Summary of even-skipped expression within the insects. Species where functional data for eve is known are listed in red. Some species with derived modes of embryogenesis such as polyembryony or complete early cleavage, have altered eve expression and may therefore represent secondarily derived conditions. The early broad domain of eve expression has been reported in several insects, but no function has ever been attributed to it. +, expression present in this species; –, expression not present in species;?, neither presence nor absence of expression was reported. Coleopteran, hymenopteran and dipteran phylogenies based on previous work (Lawrence, 1982; Dowton, 1994; Yeates, 1999). (B) Oncopeltus eve may occupy both upstream and downstream positions in the segmentation hierarchy. The early broad blastoderm expression (red) is required for activation of the blastoderm expression of the central hunchback and Krüppel domains. These gap genes may then in turn regulate striped Of'eve expression. (C) Summary of putative roles for Of'eve. The early broad blastoderm domain (red) is most likely responsible for regulation of hunchback and Krüppel. The eve stripes (blue) seen on blastoderms and germbands may regulate engrailed. The growth zone expression (red) may be required for proper growth of the posterior. The deleted region in Of'eve RNAi embryos is shown in purple.

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