QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 1, 2004
doi: 10.1242/10.1242/dev.01311

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, P. Z. Articles by Kaufman, T. C. Search for Related Content PubMed PubMed Citation Articles by Liu, P. Z. Articles by Kaufman, T. C. Social Bookmarking                         What's this?

Krüppel is a gap gene in the intermediate germband insect Oncopeltus fasciatus and is required for development of both blastoderm and germband-derived segments

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

View larger version (90K): [in a new window]   Fig. 1. Oncopeltus embryogenesis. (A) Blastoderm embryo shortly after cellularization stained with SYTOX, a fluorescent DNA dye. (B-D1) Since germband invagination and other embryonic movements can be potentially confusing, blastoderm images are oriented so that anterior is to the left, and the future dorsal region of the embryo at hatching is up. Note that dorsal/ventral fates of blastoderm cells may not correspond to final position of the embryo at hatching. See text for details. (B) Late blastoderm embryo undergoing early germband invagination, stained with SYTOX. Arrow marks site of invagination. `Lateral plates' can be seen as areas of higher cell density. Also note that segmentation is now apparent. (C) Embryo at approximately the same stage as in B, hybridized with probe made to Oncopeltus engrailed (en) to mark segmental boundaries. At this stage, six en stripes corresponding to the mandibular through third thoracic segment can be seen (MN, mandibular; T3, third thoracic en stripes). Arrow marks site of invagination. (D1) Embryo undergoing germband invagination and at a later stage than in C stained for en. Note that only four en stripes can be seen. (D2) Same individual embryo as in D1, but rotated to view ventral aspect with yolk partially removed to reveal underlying early germband. Note that the two most posterior en stripes now appear on the germband. (E-H) Germband stage embryos stained for engrailed. The arrow marks the third thoracic segment and the arrowhead marks the most newly formed abdominal en stripe. GZ, growth zone. Notice that the size of the growth zone decreases as more abdominal segments are added. Embryos oriented such that anterior is up. Scale bars: 200 µm (A,B and E-H).

View larger version (21K): [in a new window]   Fig. 2. Predicted amino acid sequence for Oncopeltus Krüppel aligned with homologous regions of Drosophila Krüppel. Locations of putative zinc fingers, A-box, and B-box are marked. Filled arrowhead denotes beginning of large 3' fragment used in RNAi and for synthesis of in situ probes. Solid arrows demarcate the short 150 bp 3' fragment used in RNAi. Open arrowhead shows the end of the 5' fragment used in RNAi.

View larger version (70K): [in a new window]   Fig. 3. Oncopeltus fasciatus Krüppel blastoderm expression. (A1) View of dorsal aspect of 28-32 hour blastoderm stained for Kr RNA. Kr transcript accumulates in the posterior 33.5% of the blastoderm surface. (A2) Ventral aspect of same embryo as in A1. Note lack of expression on ventral surface. (B) 28-32 hour blastoderm stained for hunchback (purple) and Kr (orange). Very little, if any, overlap can be seen for these two genes. (C) Kr in situ hybridization of 32-36 hour blastoderm. Kr transcript clears from posterior pole (arrow). (D1,D2) Lateral and ventral aspects, respectively, of 36-40 hour embryo undergoing germband invagination. Blastoderm cells expressing the Kr gap domain gene are migrating towards the site of invagination, marked by arrow in D1, and are contributing to the germband (visible beneath the yolk). Arrowhead in D2 marks an anterior patch of Kr expression. (E1,E2) Lateral and ventral aspects, respectively of 36-40 hour embryo during germband invagination. This embryo is at a later stage than in (D1,D2). Kr gap domain cells have now fully invaginated and made their contribution to the germband. Arrowhead in E2 marks position of the same anterior Kr patch indicated in D1. These Kr-expressing cells have now migrated to a more central position in this later embryo. (F) Montage consisting of two different embryos stained for engrailed (top) and Krüppel (bottom) aligned to show that Krüppel is expressed approximately from pT1-T3. Scale bars: 200 µm.

View larger version (132K): [in a new window]   Fig. 4. Krüppel germband expression. (A-G) Developmental series of successively older germband stage embryos stained for Krüppel. Kr expression begins in an ectodermal domain in the central portion of the germband. Expression in the mesoderm and in neural domains then develops in older germbands. (A1,B1) Kr expression in the germband begins as a continuation of the previous blastoderm domain and accumulates in the central region of the germband. (A2,B2) Higher magnification of boxed regions in A1 and B1 respectively. Note that ectodermal expression fades, but mesodermal expression remains strong. (D2,E2) Higher magnification of boxed regions in D1 and E1, respectively. Abdominal expression appears mesodermal and not ectodermal. (E1-G) In addition to the mesodermal expression, dots of neural expression also accumulate. Note the anterior to posterior maturation of the neural pattern. (F2) Higher magnification image of boxed region in F1 showing neural expression. (H-K) Embryos double stained for Kr (purple) and en (orange). Anterior limit of Kr expression is marked by arrowheads, posterior limit by arrows. By comparing the boundaries of mesodermal expression in successively later staged embryos, it is apparent that Kr mesodermal expression expands both anteriorly and posteriorly. Scale bars: 200 µm (A-G,H-K); 100 µm. (A2-F2) LB, labial segment; MN, mandibular segment; MX, maxillary segment; T1-T3, thoracic segments; A1-A6, abdominal segments.

View larger version (112K): [in a new window]   Fig. 5. Krüppel RNAi phenotype. (A-C) Uninjected embryos just prior to hatching. (A) Dorsal view. (B) Lateral view. (C) Ventral view. Antenna (ANT) and thoracic legs (T1-T3) are marked. Arrow in C indicates mandibular stylet; arrowhead indicates maxillary stylet; LB, labium. (D1-D3) Representative class I embryo. (D1) Dorsal view, abdomen is visibly shortened. (D2) Lateral view showing defective segmentation in the abdomen (arrow), and fusion of second and third thoracic segments. (D3) Higher magnification image of same embryo, showing fusion of second and third thoracic legs. (E1,E2) Class II embryo with deletion of third thoracic segment and more deleted abdominal segments resulting in shorter abdomen. Also note that second thoracic segment is present, but with a reduced leg. (F1-F4) Class III embryo. Second and third thoracic segments are deleted and the first thoracic leg is highly reduced (arrow in F2 and F3). Labium is unaffected (arrowhead in F3). (F4) High magnification image showing presence of mandibular (arrows) and maxillary (arrowheads) stylets. Scale bars: 200 µm in all images except F4, which is 100 µm.

View larger version (90K): [in a new window]   Fig. 6. Wild-type (A) and Krüppel RNAi (B-E) embryos stained for engrailed. (B) Putative class I RNAi embryo with a small deletion of anterior abdomen, possibly of the first two abdominal segments. All thoracic segments are present, but second and third thoracic segments show defective engrailed expression. Also note `skipping' of defect, indicated by an arrow. (C) Putative class II embryo, with larger abdominal deletion and stronger thoracic segmentation defects. Second and third thoracic segments are strongly affected, but first thoracic segment appears normal. (D) Class III embryo, with full Kr deletion of mesothoracic through fourth abdominal segments. (E) Class III embryo, with `skipping' of the sixth abdominal en stripe, indicates by an arrow. T1, first thoracic segment; A1-A10, abdominal segments. Scale bars: 200 µm.

View larger version (109K): [in a new window]   Fig. 7. Hox gene expression in uninjected and Kr RNAi embryos. (A) Uninjected embryo stained for Deformed (Dfd). Dfd is expressed in the mandibular and maxillary segments and appendages. (B) Kr RNAi embryo stained for Dfd. Normal domain of Dfd is unaffected, but note ectopic expression in first thoracic segment. (C) Wild-type expression of proboscipedia (pb) in the labium. (D) Kr RNAi embryo shows no alteration of pb expression. (E) Wild-type Sex combs reduced (Scr) expression in labium and mesoderm of first thoracic leg. (F) Kr RNAi embryo stained for Scr. Labial expression is normal but is reduced and spotty in first thoracic leg. (G) Wild-type expression of Ultrabithorax (Ubx). Expression is strong throughout the first abdominal segment, and weak in a neural pattern in the remainder of the abdomen. (H) Kr RNAi embryo stained for Ubx shows loss of A1 segmental staining, but neural expression is still present. (I) Wild-type expression of abdominal-A (abd-A) starting in A2 and extending to approximately A8. (J,K) Kr RNAi embryos with ectopic expression of abd-A in the labium and first thoracic segments. Scale bars: 200 µm.

View larger version (14K): [in a new window]   Fig. 8. Summary of Krüppel expression (red bars) and function (blue bars) in Drosophila, Tribolium and Oncopeltus. Solid blue bars denote deletions of affected segments while broken blue bars denote defects other than deletions. Black and gray bars denote body regions specified during blastoderm stage and from growth of the posterior growth zone, respectively. Drosophila Kr null mutations result in a deletion that spans the first thoracic through the fourth abdominal segment, with the fifth abdominal segment partially deleted, and with an enlargement of the sixth abdominal segment. The Drosophila Krüppel protein is expressed in a gradient from the mesothoracic through fourth abdominal segments. Tribolium jaws is probably the Krüppel homolog (see text) and mutants show a transformation of the thorax and first abdominal segment towards gnathal identity along with a deletion of most of the remaining abdomen. Tribolium Krüppel expression accumulates in the thorax. Oncopeltus RNAi results in a deletion of the mesothoracic through fourth abdominal segments with reduction, but not deletion of, the prothoracic appendage. Oncopeltus Kr transcript accumulates from the posterior of the first through third thoracic segments.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter