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First published online August 2, 2005
doi: 10.1242/10.1242/dev.01939

Development 132, 3705-3715 (2005)
Published by The Company of Biologists 2005
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A major role for zygotic hunchback in patterning the Nasonia embryo

Mary Anne Pultz1,¶, Lori Westendorf1,*, Samuel D. Gale1,{dagger}, Kyle Hawkins1, Jeremy Lynch2, Jason N. Pitt1,{ddagger}, Nick L. Reeves1,§, Jennifer C. Y. Yao1, Stephen Small2, Claude Desplan2 and David S. Leaf1

1 Department of Biology, Western Washington University, Bellingham, WA 98225, USA
2 Department of Biology, New York University, New York, NY 10003, USA



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  		Fig. 1. Comparison of mutant phenotypes and embryonic timing. (A) Comparison of Nasonia zygotic headless (hl) and Drosophila zygotic hunchback (hb) mutant phenotypes. The black bars indicate regions with pattern deletions. (B) Comparative timing of embryogenesis in Nasonia and Drosophila. At 25°C, development from gastrulation to hatching is completed in about 20 hours in both insects, but approximately threefold more time is allocated to early development, prior to gastrulation, in Nasonia.

 


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  		Fig. 2. Linkage analysis of the headless mutation. (A) Primers used for mapping N. vitripennis (Nv) and N. giraulti (Ng) hunchback. Lowercase letters on the Ng-specific primer indicate sites of mismatch: a T/C SNP at the 3' end and a destabilizing mismatch four bases from the 3' end. (B) PCR controls with Nv and Ng genomic DNA demonstrating the efficacy of the Ng-specific primer. (C) Strategy for inter-specific cross to test linkage of Nv hunchback to headless. If hunchback is linked to headless then surviving hemizygous sons of the experimental F1 mothers should all have Ng hunchback.

 


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  		Fig. 3. Nasonia hunchback gene structure and the headless (hbhl) deletion. (A) Nasonia hunchback gene structure [GenBank accession numbers: DQ116756 (cDNA), DQ116757 (cDNA), DQ116758 (genomic)]. Exons are boxed. Arrows indicate putative transcription start sites. ATG indicates putative initiating methionines. NF1 Zf, MF 1-4 Zf, and CF1-2 Zf refer to C2H2 zinc fingers, and are indicated as bars. The NF1 Zf is interrupted by an intron. The HB-GST region, against which the anti-Nv-Hunchback antibody was raised, is indicated as a stippled box in exon 4. TAA indicates the stop codon. The shaded box indicates a 3' UTR. (B) A deletion in Nasonia hunchback in the DNA from headless (hbhl) mutant embryos. The open reading frames from headless and wild-type genomic DNA show that the breakpoints of the 1.497 kb hbhl deletion generate a frameshift mutation in Nasonia hunchback (GenBank accession number: DQ116759). The dotted lines on the wild-type Nv hb indicate contiguous sequence. (C) The alignment of candidate NREs from the 3' UTR of Nasonia hunchback (Nvhb.1-4) with NREs of D. melanogaster (Dm), and with candidate NREs from hunchback genes of other insects: D. virilis (Dv), Tribolium (Tc), Locusta (Lm) and Schistocerca (Sa), as well as with Dm.cycB1.1.

 


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  		Fig. 4. Maternal expression of Nasonia hunchback. (A) Nasonia hunchback mRNA is loaded from the nurse cells (Nc) – of which there are 15, as in Drosophila – into the maturing oocyte (Oc). The non-staining cells surrounding the oocyte are the follicle cells (Fc). (B) Negative-control staining using a sense Nasonia hunchback probe.

 


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  		Fig. 5. Nasonia hunchback expression during blastoderm development. (A) Timeline of Nasonia embryogenesis at 28°C. The embryos in B and C are from the same two-hour egg collection as the embryos in D and E. (B) mRNA expression soon after the nuclei begin dividing at the surface of the embryo. (C) Anterior nuclear gradient of protein expression after the nuclei begin dividing at the surface of the embryo. (D) The next phase of mRNA expression after that shown in B, localized in a posterior and central domain. (E) Anterior Hunchback domain with sharper boundary several cell cycles later than is shown in C. (F,G) Subsequent mRNA and protein during early cellularization. (H,I) mRNA and protein expression shortly before gastrulation.

 


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  		Fig. 6. Nasonia Hunchback in serosa and nervous system of wild-type embryos. (A) A dorsal stripe of mRNA expression initiates shortly before gastrulation. (B) Protein expression in the nuclei of the developing serosa, soon after germ-band extension. (C,D) Continued protein expression in the serosa as it begins to expand to envelop the entire embryo. (E) Protein expression in the nervous system, seen here during head involution. (F) Ventral view of embryo shown in E.

 


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  		Fig. 7. How late is maternal Hunchback expressed in Nasonia and Drosophila? (A) Zygotic expression of Nasonia Hunchback during the onset of cellularization and the beginning of posterior cap expression. (B) Lack of residual maternal Hunchback expression in similarly aged hunchbackhl mutant embryo. The embryos in A and B were from a very tightly staged collection, and, therefore, are very similar in age (see Materials and methods). (C) Zygotic expression of Drosophila Hunchback, during the onset of cellularization and the beginning of posterior cap expression. (D) Residual maternal expression in nuclei at the surface of a Drosophila embryo lacking zygotic hunchback, very similar in age to the embryo in C. The A,B and C,D embryo pairs were photographed together in the same frames.

 


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  		Fig. 8. Comparison of hunchback mutant phenotypes. All embryos are stained with the FP6.87 antibody (Kelsh et al., 1994Go), which recognizes both Ultrabithorax (Ubx) and Abdominal-A (Abd-A) proteins. The embryos in A,B,D and E are segmented. The embryos in C and F are younger, at the age of onset of Ubx-Abd-A expression. (A) Wild-type Drosophila embryo. (B) Drosophila embryo lacking zygotic hunchback function. (C) Drosophila embryo lacking both maternal and zygotic hunchback function. (D) Wild-type Nasonia. (E) Nasonia hunchbackhl, segmented embryo. (F) Nasonia hunchbackhl as Hox gene expression is initiating, to ensure that no head rearrangements have yet taken place.

 

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© The Company of Biologists Ltd 2005