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First published online 3 March 2004
doi: 10.1242/dev.01046

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hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus

Howard Hughes Medical Institute, Department of Biology, Indiana University, 1001 East Third Street, Bloomington IN, 47405, USA

View larger version (49K): [in a new window]   Fig. 2. Analysis of the Oncopeltus fasicatus hunchback sequence. (A) Predicted Oncopeltus fasciatus hunchback (O.f.) zinc-finger domains aligned with homologous domains from Schistocerca americana (S.a.), Drosophila melanogaster (D.m.), and Tribolium castaneum (T.c.). NF-1 and NF-2 are the amino-terminal two zinc fingers, MF 1-4 are the middle four fingers, and CF-1 and CF-2 are the carboxy fingers. Arrowheads indicate the locations of the critical cysteine and histidine residues. (B) Alignments of the Oncopeltus A-, Basic, and C-boxes with other insect species. (C) The predicted Oncopeltus huncback structure showing the locations of the zinc fingers (black boxes) and other conserved motifs (gray boxes). The locations of the 300 bp, 500 bp, and 1.1 kb dsRNA fragments used in RNAi are mapped above the protein structure. An asterisk indicates the second 300 bp fragment. (D) The predicted structures of insect hunchback proteins mapped upon the insect phylogeny. As both Schistocerca and Oncopeltus hunchback proteins are predicted to contain a total of eight zinc fingers, this is probably the ancestral state for the eumetabola (arrowhead). As Tribolium and Drosophila hunchback have only six zinc fingers, hunchback appears to have lost its two N-terminal fingers in the lineage leading to holometabola.

View larger version (74K): [in a new window]   Fig. 1. Oncopeltus embryogenesis and engrailed expression. (A,B) Blastoderms stained with SYTOX to show nuclei. Anteroposterior axis and dorsoventral axis are indicated. (A) Early blastoderm at 24-28 hours. (B) Late blastoderm at 36-40 hours. Arrowhead indicates site of germband invagination. Scale bar: 200 µm. (C) Late blastoderm at 36-40 hours hybridized with engrailed probe (purple color). The six en stripes are labeled: MN, mandible; MX, maxillary; LB, labium; T1, first thoracic; T2, second thoracic; T3, third thoracic. Arrowhead indicates site of germband invagination. (D1) Embryo stained for engrailed at slightly later stage than in C. Note that only the four anterior en stripes are now visible on the blastoderm surface. (D2) Same embryo as in D1, with `dorsal' region of blastoderm dissected away to reveal developing germband and rotated to view dorsal aspect. Note that the en stripes corresponding to the second and third thoracic segments are now on the invaginated germband. (E-H) Dissected germbands stained for engrailed, showing growth of the posterior and the sequential addition of the abdominal segments. Anterior is towards the top. (E) Early germband just completing germband invagination. (F) Forty-four- to 48-hour-old germband. A1, first abdominal engrailed stripe; GZ, posterior growth zone. (G) Embryo with five abdominal segments. (H) Fully elongated germband with the tenth and final abdominal engrailed stripe. Scale bar: 100 µm in F,H.

View larger version (64K): [in a new window]   Fig. 3. hunchback is maternally expressed and loaded into developing oocytes. (A) Northern blot using a hunchback probe to ovarian (O) or embryonic (E) total RNA. A single band of 3.2 kb is detected. (B) In situ with hunchback probe on a single fixed ovariole. Arrowhead indicates zone III of tropharium, the area where the nurse cells are concentrated. Older oocyte (arrow) beginning to lay down chorion does not stain as strongly. (C) Ovariole in situ using sense control hunchback probe.

View larger version (55K): [in a new window]   Fig. 4. hunchback blastoderm in situs. Anterior is towards the left in all images. (A) Twelve- to 16-hour-old blastoderm. hb transcript is diffuse along length of egg but is concentrated in the central region of the embryo (arrowheads). (B) Twenty- to 24-hour-old blastoderm. Accumulation in the central region of blastoderm becomes more pronounced. (C1-C2) Twenty-eight- to 32-hour-old embryo. (C1) Ventral view. Two bands of hb accumulation are apparent. Anterior and central bands (white and black arrowheads, respectively, are indicated. Posterior hb patch is indicated by an arrow. (C2) Dorsal view of blastoderm. hb expression is weaker in dorsal region of blastoderm. (D1-D2) Thirty-six- to -40-hour-old embryo. Bands of expression are fading from blastoderm surface and the appearance of hb expression in the underlying yolk nuclei. (D1) Lateral view. Arrow indicates the beginnings of germband invagination. (D2) Dorsal view. (E) Composite image consisting of two 36- to 40-hour-old embryos stained for engrailed (top) and hunchback (bottom). The central band of hunchback expression spans the first three engrailed stripes that correspond to the posterior of the mandibular, maxillary, labial and anterior of the first thoracic segments. Scale bar: 200 µm.

View larger version (99K): [in a new window]   Fig. 5. hunchback transcript expression in germband embryos. Anterior is upwards in all images. (A) Very early germband undergoing invagination. At this stage, tissues fated to become head have not yet invaginated, and are therefore not represented in this early germband preparation. hb transcript in blocky chevron pattern in the early thorax. (B) Germband invagination complete. Labial (LB) and third thoracic segment (T3) are labeled. Note that hb does not accumulate in a gap-like pattern in the labium, but rather in a segmentally reiterated blocky chevron pattern. (C-F2) Germbands double stained for hunchback (purple) and engrailed (orange). (C) Germband beginning elongation. Labial (LB) and first abdominal (A1) segments are labeled. Arrows indicate two dots of hb expression in the posterior zone. (D) Slightly later embryo than in C, posterior dots of hb have now expanded to become a half-moon patch in the posterior (arrow). (E) hunchback expression persists throughout germband extension. The sixth abdominal segment (A6) is labeled. Also note that hunchback is now expressed in a neural pattern in the trunk of the germband. (F1) hunchback is expressed in the posterior until the formation of the tenth abdominal segment (arrowhead). (F2) Close up of posterior germband in (F1). Scale bars: 200 µm in A-F1.

View larger version (65K): [in a new window]   Fig. 6. Testing parental RNAi with Oncopeltus Sex combs reduced (Of'Scr). (A) Hatchling from uninjected mother. Arrow indicates labium. (B) Of'Scr pRNAi hatchling. Arrowhead indicates transformed labium. Arrow indicates normal stylet. For a full description of the Of'Scr phenotype, see Hughes and Kaufman (Hughes and Kaufman, 2000). (C) Time course for Of'Scr pRNAi. X-axis is number of days the clutch was laid after injection of the female. y-axis is percent that each phenotypic category contributed to the total. The RNAi effect peaks around ten days after injection and declines after that.

View larger version (100K): [in a new window]   Fig. 7. Class I-III (mild and moderate) hunchback RNAi phenotypes. (A1-A3) Uninjected embryos. Anterior towards the left. Thoracic and abdominal regions are delineated. (A1) Lateral view. (A2) Dorsal view. (A3) Ventral view. Arrowhead indicates the mandibular stylet, arrow indicates the maxillary stylet. AN, antenna; LB, labium; T1, first thoracic leg; T2, second thoracic leg; T3, third thoracic leg. (B1-B2) Class I embryo. (B1) Lateral view. (B2) Ventral view. Labium is a lump of undifferentiated tissue. T1 leg is reduced, while T2 and T3 legs are less strongly affected. (C1,C2,C4,C5) Class II embryo. (C1) Lateral view. Abdominal region indicated. (C2) Close-up of gnathal and anterior thorax of embryo in C1. Arrowheads indicate abdominal-like spiracles. (C3) Close-up of abdominal spiracles (arrowheads) on uninjected animal. (C4) Ventral view of embryo shown in C1, C2. Reduced second and third thoracic legs are labeled. (C5) Close-up of gnathal region of embryo shown in C4. Arrowhead indicates location of ventral fourth abdominal (A4) spiracle. (C6) Close up of A4 of uninjected animal. Spiracle indicated with arrowhead. Scale bar: 50 µm. (D1-D2) Class III embryo. (D1) Dorsal view. Note dorsal segmentation defect. Abdominal region indicated. (D2) Ventral view. Defective third thoracic leg (T3) is labeled. Note abdominal-like segmentation in gnathal region of animal. (E) Wild-type germband stained for abdominal-A. Note expression in second through eighth abdominal segments and lack of expression in labial segment. (F) Putative class I embryo stained for abd-A. Note reduced labium (LB). (G) Putative class III embryo stained for abd-A. (H) Putative class II embryo stained for engrailed. Scale bars: 200 µm in A-C1,C4,D; 50 µm in C6; 100 µm in E-H.

View larger version (81K): [in a new window]   Fig. 8. Class IV and V embryos. (A) Dorsal view of class IV embryo. This embryo did not complete dorsal closure. Note defective segmentation and shortened abdominal region. (B) Class IV embryo. Arrow indicates posterior leg. (C1) Class IV/V embryo. Lateral view. Arrow indicates posterior leg. (C2) Dorsal view of embryo in panel C1. Note shortened abdominal region. (D) Dorsal view of class IV/V embryo. Note extremely shortened abdominal region. Normal antenna (AN) is indicated. (E) Class V embryo. (F) Putative class IV embryo stained for engrailed. Third thoracic leg is indicated (T3). Note highly reduced labium and T1 and T2 legs and defective segmentation in abdomen. Note that segmentation in labium (LB) and thoracic region (T1-T3) appears normal. (G) Putative class IV/V embryo stained for en. T3 leg reduced. Note defective segmentation in abdomen and normal segmentation in labium (LB) and thoracic region (T1-T3). (H) Putative class V embryo stained for abd-A. abd-A expression extends from the labial segment to the posterior of the animal. Maxillary (MX), mandibular (MN) and labial (LB) segments appear relatively normal. Scale bars: 200 µm in A,E; 100 µm. in F-H.

View larger version (44K): [in a new window]   Fig. 9. Model for hunchback depletion in Oncopeltus fasciatus. Increasingly stronger hunchback knockdown results in two distinct effects. The first aspect of the depletion phenotype is transformation of the labial and anterior thoracic segments towards abdominal identity. In stronger depletion animals, this region of transformation expands, eventually encompassing the entire thorax. The second aspect of the hunchback phenotype is posterior compaction. Weak hb depletion results in mild compaction of the abdomen. In moderate and strong phenotypic classes, the abdomen is significantly shortened, with associated defects in segmentation. Therefore in the most severe cases (class V), these two aspects together result in a phenotype where the head is followed by an indeterminate number of segments, all with abdominal identity.