giant is a bona fide gap gene in the intermediate germband insect, Oncopeltus fasciatus

Abstract

Drosophila undergoes a form of development termed long germ segmentation, where all segments are specified nearly simultaneously so that by the blastoderm stage, the entire body plan has been determined. This mode of segmentation is evolutionarily derived. Most insects undergo short or intermediate germ segmentation, where only anterior segments are specified early, and posterior segments are sequentially specified during germband elongation. These embryological differences imply that anterior and posterior segments might rely upon different molecular mechanisms. In Drosophila, embryos mutant for giant show a gap in the anterior as well fusions of several abdominal segments. In Tribolium, a short germ beetle, giant is required for segmental identity, but not formation, in gnathal segments and also for segmentation of the entire abdomen. This raises the possibility that giant might not act as a gap gene in short and intermediate germ insects. Oncopeltus fasciatus is an intermediate germ insect that is an outgroup to the clade containing Drosophila and Tribolium. We cloned the Oncopeltus homolog of giant and determined its expression and function during segmentation. We find that Oncopeltus giant is a canonical gap gene in the maxillary and labial segments and also plays a gap-like role in the first four abdominal segments. Our results suggest that giant was a bona fide gap gene in the ancestor of these insects with this role being lost in the lineage leading towards Tribolium. This highlights the conservation of anterior patterning and evolutionary plasticity of the genetic regulation controlling posterior segmentation, even in short and intermediate germ insects.

Fig. 1.

Oncopeltus fasciatus giant. (A) Alignment of the binding domain of the co-repressor, CtBP, from the Oncopeltus, Drosophila, Tribolium and Nasonia orthologs of Giant. (B) The conserved leucine zipper domain of Giant from the four insects. (C) Cartoon diagram showing approximate locations of the CtBP binding and leucine zipper domains of Oncopeltus Giant. Boxed region is the predicted open reading frame, predicted to encode a 210 amino acid protein. Single lines show approximate relative lengths of the 5′ (100 bp) and 3′ (800 bp) untranslated regions. Lines below the cartoon diagram show approximate lengths and positions of the fragments used to synthesize in situ probes and double-stranded RNA. Fragment A was used for probe synthesis, fragment B to synthesize both probe and double-stranded RNA, fragment C was used only to synthesize double-stranded RNA, and fragment D, located in the 3′ untranslated region, was also only used to synthesize double-stranded RNA. Figure not to scale.

Fig. 2.

Wild-type Of'gt RNA expression. (A) Blastoderm stage embryo at 28-32 hours after egg lay (AEL). Of'gt transcript is in the anterior two-thirds of the blastoderm. (B) By 32 hours AEL, the anterior band of Of'gt has now split into two broad stripes. A new domain of expression has now arisen (arrow). (C) Blastoderm stage embryos around 36 hours AEL stained for Of'gt (top) and Of'en (bottom). By juxtaposing the images of the two embryos, it is possible to gain a rough estimate of the segmental register of Of'gt expression. The anterior bands span the head and maxillary segment and the maxillary and labial segments. The posterior domain has now expanded to cover the second and third thoracic segments. (D-I) Germband stage embryos stained for Of'gt (purple) and Of'en (orange). (D) An early germband stage embryo, during germband invagination. The head tissues would have still been on the outside of the yolk ball but have been removed in this preparation. Anterior Of'gt expression can be seen in the mandibular, maxillary and part of the labial segments. The posterior expression seen during the blastoderm stage continues as strong thoracic expression. (E) By the time two abdominal Of'gt stripes have appeared, gnathal expression has faded, leaving strong head expression and punctate dots in the mandibular and maxillary segments. Of'gt expression has also expanded into the second abdominal segment. (F) Anterior expression remains the same but thoracic and anterior abdominal expression forms weak stripes. A stripe of Of'gt arises in the growth zone (arrow). (G) By the time five abdominal stripes of Of'en have formed, thoracic and anterior abdominal expression has faded. The growth zone stripe is still strong (arrow). (H) An embryo where the seventh abdominal Of'en stripe has formed. The growth zone stripe remains. (I) By the time the ninth abdominal Of'en stripe has formed, the growth zone stripe of Of'gt has faded. (J-M) Germband stage embryos doubly stained for Of'gt transcript (purple) and Of'eve transcript (orange). These images show that the growth zone stripe of Of'gt is both co-expressed with the Of'eve stripes (M), as well as in the interstripe region (K,L). Panel J shows an early germband stage embryo before the appearance of Of'eve stripes. Arrowheads mark the position of Of'eve stripes. a2, second abdominal segment; a5, fifth abdominal segment; a7, seventh abdominal segment; a9, ninth abdominal segment; mn, mandibular segment; mx, maxillary segment; t1, first thoracic segment; t3, third thoracic segment.

Fig. 3.

Of'gt RNAi embryos at hatching. (A-A″) Uninjected embryo at hatchling stage showing the lateral aspect (A), dorsal aspect (A′) and ventral aspect (A″). The labium is marked with an arrowhead. (B-B″) Of'gt RNAi embryo with `strong' phenotype. Note that the abdomen is shortened and the labium is missing (arrowhead). (C-C″) Of'gt RNAi embryo with `weak' phenotype. Note that the abdomen is short but less defective than in the strongly affected embryos. Arrowhead marks the loss of the labium. Asterisks mark thoracic legs. a, abdomen; e, eye; r, labrum; t, thorax.

Fig. 4.

Of'gt RNAi embryos at the germband stage stained for Of'en. (A) Uninjected late germband stage embryo. (B) Late germband stage RNAi embryo with the `strong' phenotype. Note the loss of the maxillary and labial segments and the loss of the first four abdominal segments. (C) Late germband stage RNAi embryo with the `weak' phenotype. Note the loss of the maxillary and labial segments and the defective Of'en stripes in the anterior abdomen. a5, fifth abdominal segment; a10, tenth abdominal segment; lb, labium; mn, mandible; mx, maxillae.

Fig. 5.

Hox gene expression in uninjected and Of'gt RNAi embryos. (A,C,E,G,I,K,M) Uninjected embryos. (B,D,F,H,J,L,N) Of'gt RNAi embryos. (A) Of'Dfd in a normal embryo is expressed in the mandible and maxillae. (B) Of'gt RNAi embryo stained for Of'Dfd. As the maxillary segment is deleted, only the mandible expresses Of'Dfd. (C) Of'pb is normally expressed in the labium. (D) In an Of'gt RNAi embryo, the labium is deleted. Occasionally, we detect misexpression of Of'pb in the second thoracic leg (arrowhead). (E) Of'Scr is normally expressed strongly in the labium and weakly in the mesoderm of all the legs. Note the T1 leg patch (arrowhead). (F) Of'gt RNAi embryo. Note the labium is deleted and the ectopic expression of Of'Scr in a T3 leg patch (white arrowhead). Expression of the T1 leg patch is unaffected (black arrowhead). (G) Of'Antp expression is strong in the thorax and extends weakly throughout the abdomen. (H) Of'Antp expression in Of'gt RNAi embryos is not affected in the remaining segments. (I) Of'Ubx expression is strongest in the posterior of the third thoracic segment and the first abdominal segment. Of'Ubx is also expressed in a ring in the third thoracic leg (arrowhead). (J) In Of'gt RNAi embryos, the strong abdominal expression is missing, reflecting the deletion of that segment. The ring of expression in the third thoracic leg is still present (arrowhead). (K) Of'abd-A in normal embryos is expressed in the posterior of the first abdominal segment and strongly in the second through seventh abdominal segments. (L) Of'gt RNAi embryos stained for Of'abd-A. (M) Of'Abd-B expression in a normal embryo. (N) Of'Abd-B expression in an Of'gt RNAi embryo. a1, first abdominal segment; a5, fifth abdominal segment; a9, ninth abdominal segment; lb, labium, mn, mandible; mx, maxillae; t1, prothorax; t3, metathorax; t3^*, transformed metathorax.

Fig. 6.

Expression of segmentation genes in normal and Of'gt RNAi embryos. (A) Normal late blastoderm stage embryo stained for Of'eve. Six stripes Of'eve corresponding to the mandibular through third thoracic segment are apparent at this stage. (B) Of'gt RNAi embryo at the late blastoderm stage. The second and third Of'eve stripes are missing. Note that the spacing of the remaining stripes appears normal. (C) Normal late blastoderm stage embryo stained for Of'slp. Six stripes corresponding to the mandibular through third thoracic segments are labeled. Several head stripes are also visible. (D) Of'gt RNAi late-stage blastoderm embryo stained for Of'slp. The third and fourth Of'slp stripes are missing, replaced by a region of fuzzy expression. (E) Normal mid-germband stage embryo stained for Of'eve. Of'eve is normally expressed in the posterior growth zone and in three or four newly formed segments (dots). (F) Mid-germband stage Of'gt RNAi embryo stained for Of'eve. Of'eve expression appears normal in the growth zone but only one strong stripe is visible (dot). Also note the putative anterior abdomen appears fatter and shorter than in normal embryos. (G) Late germband stage Of'gt RNAi embryo stained for Of'eve. Of'eve expression in the growth zone is fading but the final stripes have formed (dots). (H) Normal mid-germband stage embryo stained for Of'slp. Of'slp is expressed in strong broad bands in the posterior of each segment. (I) Of'gt RNAi embryo at the mid-germband stage stained for Of'slp. Note the region of aberrant Of'slp expression in the anterior abdomen. (J) Of'gt RNAi embryo at the late germband stage stained for Of'slp. Note the aberrant Of'slp expression in the anterior abdomen and the normal stripes of expression in the posterior abdomen. (K) Normal mid-germband stage embryo stained for Of'en. Of'en is expressed in the posterior of each segment. (L) Of'gt RNAi embryo at the mid-germband stage stained for Of'en. Of'en expression is aberrant in the putative anterior abdomen. (M) Of'gt RNAi embryo at the late germband stage stained for Of'en. Defective Of'en expression can be seen in the putative anterior abdomen but stripes appear normal in the posterior abdomen. t3, third thoracic segment.

Fig. 7.

Homeotic transformation of the third thoracic leg in Of'gt RNAi embryos and hatchlings. (A-H) A and E show magnified images of the embryo shown in Fig. 5E. B and F show magnified images of the embryo shown in Fig. 5F. (A) T1 leg from a normal germband stage embryo stained for Of'Scr. Note the T1 leg patch (arrowhead). (B) Of'gt RNAi embryo. Of'Scr T1 leg patch appears normal. (C) Magnification of the tibial comb on a T1 leg from a newly hatched normal animal. (D) Magnification of the tibial comb on a T1 leg from an Of'gt RNAi embryo at the hatchling stage. The tibial comb is unaffected. (E) T3 leg from a normal germband stage embryo stained for Of'Scr. (F) T3 leg from an Of'gt RNAi germband stage embryo stained for Of'Scr. Note the ectopic patch of expression (arrowhead). (G) Magnification of the T3 distal tibia from a newly hatched normal animal. Note the morphology of distal tibial bristles. (H) Magnification of ectopic T1-like combs on a T3 leg of an Of'gt RNAi animal at the hatchling stage. Compare the morphology of the tibial comb to the WT T1 combs in C. RNAi, Of'gt RNAi embryo; T1, first thoracic leg; T3, third thoracic leg; WT, normal embryo.

Fig. 8.

Summary of giant expression and function in Oncopeltus and other insects. The evolutionary relationships of the insects are shown in the simplified phylogenetic tree on the left. Arrowhead marks the Neopteran ancestor. The expression domains for the different insects are shown in green. Regions that are deleted or fused in giant-depleted embryos are shown in orange and regions of homeosis are shown in blue. The precise boundaries of anterior Of'gt could not be determined, and so are shown as broken bars. The second thoracic leg was transformed in only a fraction of Of'gt RNAi embryos, denoted by the broken blue bars. In Tribolium, giant knockdown resulted in defects in a variable number of all of the posterior thoracic and all of the abdominal segments, denoted by the broken orange bars. a1-a10, first through tenth abdominal segments; lb, labium; mn, mandible; mx, maxillae; t1-t3, first through third thoracic segments.