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Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

¹ Department of Organismal Biology and Anatomy and Howard Hughes Medical Institute, University of Chicago, 5841 S. Maryland Ave., MC1028, Chicago, IL 60637, USA
² Molecular Genetics and Evolution Group, Research School of Biological Sciences, PO Box 475, Canberra, A.C.T. 2601, Australia

View larger version (136K): [in a new window]   Fig. 1. Expression of Drosophila hb during embryogenesis. Immunostaining reveals the dynamic expression of Hb during D. melanogaster development, and provides a basis for interpretation of the grasshopper expression pattern. (A) At blastoderm stage, Hb is found in a large anterior domain (arrow) and a posterior domain around PS 13/14 (arrowhead). In a stage 12 germ band extended embryo (B), Hb is found in multiple tissues including the nervous system (arrow). In addition, it is expressed in extra-embryonic tissue and in nuclei around the yolk (arrowhead). Both domains are also seen in stage 13 germ band retracted embryos (C,D; arrow is neural expression, arrowhead is extra-embryonic expression), with the extra-embryonic domain shown in both a lateral (C) and dorsal view (D). Drosophila hb expression in a dissected stage 14 embryo (E) reveals a ‘bow-tie’-like pattern in each neuromere (arrow points to CNS) as well as in lateral domains (orange arrowheads) of mesodermal and possibly PNS expression. (F) Close-up view showing hb expression in the stage 15 CNS (arrow indicates the midline).

View larger version (84K): [in a new window]   Fig. 2. Alignment of the L. migratoria and S. americana hb sequences. Clustal alignment of the predicted full-length amino acid sequences of the two grasshopper Hb proteins reveals 76% identity. Labeled brackets indicate conserved domains, including two zinc fingers towards the N terminus of the proteins (NF-1 and 2), four central zinc fingers (MF-1 to 4), two C terminal fingers (CF-1 and 2), and the conserved A, C and basic boxes. L.m., Locusta migratoria, S.a., Schistocerca americana.

View larger version (69K): [in a new window]   Fig. 3. Alignments of hb zinc fingers and putative NREs from different species. Alignment of orthologous zinc fingers from different species reveals that the various fingers are highly related across species in the position of structural residues. Alignments are shown of (A) the two NF fingers, (B) the four MF fingers and (C) the two CF metal-binding fingers. Black arrowheads indicate the structural residues of the putative metal binding fingers. The spacing between cysteine and histidine residues within and between each finger in each cluster (N, M and C) is shown at the top of each section of the figure. (D) Alignment of ExF, an additional putative zinc-finger identified in the C. elegans and H. triserialis Hb sequences. These fingers are in analogous positions in their respective proteins, but have little structural similarity to each other. (E) DNA nucleotide alignment of predicted nanos response element (NRE) sequences from the 3'UTR of hb transcripts of various species. D.v. and D.m. 1 and 2 indicate the two NRE sequences found in the 3'UTR of both of these Drosophila species. (F) Overall structure of Hb, with regard to zinc fingers. The putative H. triserialis Hb-coding sequence was reconstructed from the published genomic sequence (Savage and Shankland, 1996) by comparison with the grasshopper and C. elegans hb sequences, and manual assignment of splice junctions. This process yields a predicted leech Hb of similar structure to C. elegans Hb. As the overall structure illustrated for L. migratoria and S. americana is conserved across phyla, it may be the ancestral insect structure, with the NF fingers being lost in the lineage leading to Tribolium and Drosophila. Whether or not the ExF fingers are part of an ancestral protostome structure is less clear, as the leech and C. elegans ExF fingers lack noticeable similarity. Species used are L.m., L. migratoria; S.a., S. americana; C.e., C. elegans; H.t., H. triserialis (leech); D.m., D. melanogaster; T.c., T. castaneum (flour beetle); M.d., M. domestica (house fly); and D.v., D. virilis.

View larger version (75K): [in a new window]   Fig. 4. Maternal hb expression in S. americana. Northern analysis, in situ hybridization and immunostaining reveal that hb is expressed during oogenesis. One transcript of 4.2 kb is expressed in the L. migratoria ovary (A), with an additional transcript of 6.0 kb appearing during embryogenesis. (B-E) In situ hybridization results using antisense (B-D) and sense (E) hb probes on S. americana ovarioles. Expression begins within the oocyte, as it leaves the germarium (B, arrow): there is no detectable expression in the germarium (left of arrow) or the somatic cells that surround the oocyte. By mid-oogenesis, hb transcript is abundant (C), but fades to background levels in older oocytes (D). (E) Control hybridization with a sense probe of oocytes at about the same stage as those seen in C. (F-J) Immunostaining of S. americana ovaries with monoclonal antibody PP 7C11. In F, the almost completed egg (*) is rotated relative to the rest of the ovariole and the presence of a vitelline membrane around it prevents the penetration of antibodies; (g) indicates the position of the germarium. Hb protein is expressed at high levels within developing oocytes and is localized to the germinal vesicle (oocyte nucleus). Translation begins around the time that transcript can first be detected (G), and continues through mid-oogenesis (H) until the germinal vesicle moves posteriorly in late oogenesis (arrowheads in F). As the nucleus moves posteriorly, Hb protein is still nuclear (I), but seems to exit the posteriorly located nucleus about the same time that the egg undergoes a phase of rapid expansion as it fills with yolk (J).

View larger version (94K): [in a new window]   Fig. 5. hb expression during early embryogenesis. Nomarski views of immunostaining (A,B,E,G,I,M) compared with DAPI staining (blue, C,D,F,H,J,L) reveal S. americana (A-I,L,M) and L. migratoria (J,K) hb localization from egg lay to condensation of cells to form the blastodisc. After egg lay, Hb protein is found in the posterior 15% or so of the egg (A), in a granular network on the surface of the egg (B). As nuclei begin to clump at the posterior end of the egg (C,D), Hb protein is still evident cytoplasmically (E, same field as D), but then enters nuclei of the posterior (G) region of the egg (matching DAPI shown in F). Nuclei in more lateral regions of the same egg (outside of the posterior 15% region) do not contain Hb protein (I, matching DAPI in H). Grasshopper hb transcript is first detectable in the condensed blastodisc but not extra-embryonically (K, matching DAPI in J). At about 34 hours AEL, the disc starts to become asymmetric (L, this embryos is not stained for Hb), and a short while later when gastrulation has begun, all embryonic nuclei still express hb at some level. Expression is not seen in extra-embryonic nuclei (M, arrows point towards the head lobes of the embryo).

View larger version (122K): [in a new window]   Fig. 6. hb expression in the developing germband. Immunostaining and paired DAPI images show the development of S. americana hb domains as the posterior growth zone forms and begins extension. (A,B) At 44 hours hb is expressed throughout the embryo, but starts to become less intense in more posterior regions of the embryo (arrowhead). In addition, strong Hb labeling is seen at this time in extra-embryonic nuclei of the serosa (arrow). By around 48 hours (C,D), Hb protein has disappeared from the posterior growth zone, but remains expressed at low levels in the headlobes (diamond). At this time the transcript pattern (E,F) matches that of the protein. (G,H) As posterior growth begins, hb is absent from the posterior growth zone (arrowhead), but is upregulated in an arc anterior to it. Strong expression continues in serosal nuclei (arrows). By about 15% (I, closer view in J), there are at least four levels of hb expression: weak in the headlobes (diamond), strong anteriorly (arrow), weaker posteriorly (arrowhead) in the gnathal/thoracic domain and absent in the more posterior part of the embryo. These expression domains are still evident at 17% (K-N), for both the protein (K, closer view in L) and the transcript (M, closer view in N) in S. americana (K,L) and L. migratoria (M,N). Red lines in J,L,N indicate the boundary between the strong and weak hb domains. Anterior is at the top in all cases.

View larger version (78K): [in a new window]   Fig. 7. hb expression during abdomen formation. Grasshopper Hb and engrailed double immunolabeling allow localization of hb domains during abdominal growth. At 17%, the posterior boundary of the hb weak domain (black) extends to the T1 engrailed stripe (brown, A, closer view in B). As the gnathal engrailed stripes appear (C, closer view in D), the anterior Hb domain (black, bracket) can be seen to extend from the posterior of Mn to anterior T1 (brown lettering indicates position of engrailed stripes for the various segments – mandibular (Mn), maxillary (Mx), labial (La) and the first thoracic segment (T1)). By 22%, a broad abdominal stripe appears (bracket in E). This is posterior to the A1 engrailed stripe, and is followed at 24% (F, in situ hybridization for hb in L. migratoria) with the appearance of a second abdominal domain (arrow indicates A4/A5 domain, arrowhead indicates the newly appearing A7-A9 domain). This second abdominal domain is also evident at the protein level at 26% (bracket in G, closer view in H), but the A4/A5 domain has faded away. The appearance of the engrailed A7 stripe (black, I) at 27%, allows placement of the anterior boundary of the second Hb domain at posterior A7. Immunostaining in S. americana (A-E,G-I) and in situ hybridization in L. migratoria (F), reveal similar expression domains.

View larger version (95K): [in a new window]   Fig. 8. Neural patterns of hb expression. Immunostaining for Hb (black) alone (in F, I, and K) or in combination with engrailed (brown in A-E, G, and H), or even-skipped (red in J, L, and M), or fasciclin II (brown in N). Grasshopper Hb protein is detected in all cells of the neuroepithelium before neuroblast delamination (A), but is then restricted to the newly delaminated neuroblasts (B). At 27%, NBs 6-1 and 6-2 (arrowhead in C) express both engrailed (brown) and Hb (black). At 29%, these same neuroblasts express only engrailed, but the initial GMCs that they have produced do express hb (arrow in D). Mesodermal expression in every segment is found as the mesoderm compacts and takes on a wedge shape (E). As the mesoderm moves out laterally, hb is expressed in the more distal cells in the abdomen (arrowhead in F), and in the more proximal cells at the limb bases of appendage-bearing segments (arrowhead in G). Expression is also seen in peripheral neurons of the leg (arrows in G,H). At 35% of development (I, closer view in K), expression can be seen in mesoderm at the base of the limbs (diamond), and in peripheral neurons of the gnathal segments (arrows in K indicate a few of these in the maxillary appendage) and several within each thoracic leg (arrowhead in K indicates the most distal Hb positive neuron in the T1 leg). By 42% (J, closer view in L, Hb in black, even-skipped in red), the pattern of Hb expression in the CNS resembles the pattern seen in Drosophila (see Fig. 1E,F). (M) A closer view of the lateral T1-T2 region from (J). Grasshopper Hb is expressed by several muscle fibers in the body wall (fibers 3 and 4, arrowhead) and at the base of the legs (arrow). The red staining in M shows eve expression in some pericardial tissue as well as muscle fibers 1 and 2. (N) At 50%, hb expression is still retained in some neurons, including aCC (arrow) and pCC (arrowhead).