A great deal is known about the molecular mechanismsresponsible for patterning and segmentation during Drosophilamelanogaster embryogenesis, and this knowledge provides anexcellent framework for examining both the conservation andthe evolution of these processes in the arthropod phylum. Suchcomparative data not only provide a more accurate assessmentof how these processes vary between insects, but also of howearly developmental mechanisms evolve in all animals.
Genetic and molecular analyses have demonstrated thatsegmentation in D. melanogaster depends on progressivesubdivision by a hierarchy of regulatory factors (Rivera-Pomarand Jckle, 1996; St Johnston and Nsslein-Volhard, 1992).Initially, maternal gradients of bicoid and nanos provideinformation that defines position along the length of thesyncytial embryo. Translational repression by bicoid and nanos
result, respectively, in gradients of caudal and hunchback (Hb)protein. The bicoid, Hb and caudal gradients lead to activationof zygotic gap gene transcription at distinct positions along theanteroposterior (AP) axis. Regulation by maternal and gapgene products then leads to the expression of pair-rule genesin stripes of a two-segment periodicity. The pair-rule genesthus define the initial reiterated pattern within the embryo.Segmental pattern is later maintained and refined by theexpression, under pair-rule regulation, of the segment polaritygenes.
Both the data summarized above, and the results of variousembryonic manipulations, show that in Drosophila the entirebody plan is established simultaneously across the length ofthe blastoderm embryo. This type of development (in whichbody regions are present at blastoderm in the same proportionsas are found in the hatching larva) is known as long germembryogenesis (reviewed by Sander, 1976). By contrast, short
3459Development 128, 3459-3472 (2001)Printed in Great Britain The Company of Biologists Limited 2001DEV5933
While the expression patterns of segment polarity genessuch as engrailed have been shown to be similar inDrosophila melanogaster and Schistocerca americana(grasshopper), the expression patterns of pair-rule genessuch as even-skipped are not conserved between thesespecies. This might suggest that the factors upstream ofpair-rule gene expression are not conserved across insectspecies. We find that, despite this, many aspects of theexpression of the Drosophila gap gene hunchback areshared with its orthologs in the grasshoppers S. americanaand L. migratoria.
We have analyzed both mRNA and protein expressionduring development, and find that the grasshopperhunchback orthologs appear to have a conserved role inearly axial patterning of the germ anlagen and in thespecification of gnathal and thoracic primordia. Inaddition, distinct stepped expression levels of hunchback inthe gnathal/thoracic domains suggest that grasshopperhunchback may act in a concentration-dependent fashion(as in Drosophila), although morphogenetic activity is notset up by diffusion to form a smooth gradient.
Axial patterning functions appear to be performed
entirely by zygotic hunchback, a fundamental differencefrom Drosophila in which maternal and zygotic hunchbackplay redundant roles. In grasshoppers, maternalhunchback activity is provided uniformly to the embryo asprotein and, we suggest, serves a distinct role indistinguishing embryonic from extra-embryonic cells alongthe anteroposterior axis from the outset of development a distinction made in Drosophila along the dorsoventralaxis later in development.
Later hunchback expression in the abdominal segmentsis conserved, as are patterns in the nervous system, and inboth Drosophila and grasshopper, hunchback is expressedin a subset of extra-embryonic cells. Thus, while theexpected domains of hunchback expression are conservedin Schistocerca, we have found surprising and fundamentaldifferences in axial patterning, and have identified apreviously unreported domain of expression in Drosophilathat suggests conservation of a function in extra-embryonicpatterning.
Key words: Hunchback, Drosophila, Schistocerca, Locusta,Segmentation, Axis formation, Grasshopper
Grasshopper hunchback expression reveals conserved and novel aspects of
axis formation and segmentation
Nipam H. Patel1,*, David C. Hayward2, Sabbi Lall1, Nicole R. Pirkl1, Daniel DiPietro1 and Eldon E. Ball2
1Department of Organismal Biology and Anatomy and Howard Hughes Medical Institute, University of Chicago, 5841 S. MarylandAve., MC1028, Chicago, IL 60637, USA2Molecular Genetics and Evolution Group, Research School of Biological Sciences, PO Box 475, Canberra, A.C.T. 2601, Australia*Author for correspondence (e-mail: email@example.com)
Accepted 25 June 2001
germ embryos, such as those of the grasshoppers Locusta andSchistocerca, appear to pattern only the head region of the bodybefore gastrulation, more posterior regions being patternedduring a subsequent growth phase. Intermediate germ insectsfall somewhere between these extremes. The long germ modeof development is seen only in the most phylogeneticallyderived insect orders, and thus ancestral insect developmentwas probably closer to short or intermediate germembryogenesis.
Significantly, features of short germ development suggestthat the mechanisms of pattern formation in these embryos maybe fundamentally different from those seen in Drosophila. Forexample, during formation of the grasshopper abdomen,segments are defined sequentially along the AP axis,suggesting temporal disparity in the response to positionalinformation along this axis. In addition, cellularization of thegrasshopper blastoderm occurs relatively early (Ho et al.,1997), so that positional information must be interpreted in acellular environment: this differs from the fly segmentationparadigm, where a syncytial environment seems crucial topatterning.
Despite the very different embryogenesis of short germinsects, there is evidence that some molecular aspects ofDrosophila segmentation are conserved. In particular thesegment polarity genes engrailed and wingless are expressedin a conserved pattern at parasegment boundaries in manyinsects including the grasshopper, Schistocerca, and beetle,Tribolium castaneum (Brown et al., 1994b; Dearden andAkam, 2001; Nagy and Carroll, 1994; Patel et al., 1989a; Patelet al., 1989b). However, earlier events in Drosophilasegmentation appear less conserved in short germ insects.Although genetic and molecular data suggest that pair-rulepatterning is conserved in Tribolium (Brown et al., 1994a;Maderspacher et al., 1998; Patel et al., 1992; Patel et al., 1994;Schrder et al., 1999; Sommer and Tautz, 1993), such evidencehas proven more elusive in the grasshopper. Recently, ahomolog of Drosophila paired (prd) has been found to beexpressed with two-segment periodicity during grasshoppersegmentation (Davis et al., 2001). Given, however, that thegrasshopper eve and ftz orthologs are not expressed in a pair-rule pattern, significant differences in the mechanisms ofsegmentation between Drosophila and the grasshopper mayexist (Dawes et al., 1994; Patel et al., 1992). Reasoning thatthe source of these differences might lie upstream, wewondered whether the expression of regulators of Drosophilapair-rule genes might also differ significantly in thegrasshopper.
In order to better understand the similarities and differencesbetween long and short germ embryogenesis, we have tried toidentify orthologs of the earliest factors involved in theDrosophila segmentation hierarchy, and we focus here on hb.The gap gene hb encodes a zinc-finger transcription factor thatfunctions early in development, both as a maternal morphogenand a gap gene. During Drosophila oogenesis, the egg is loadedubiquitously with maternal hb transcript (Tautz et al., 1987).Upon fertilization, maternal hb transcript is translated onlyanteriorly, owing to translational repression by a gradient ofmaternal nanos protein emanating from the posterior end of theembryo (Hlskamp et al., 1989; Irish et al., 1989; Wang andLehmann, 1991). This translational repression results in theformation of a Hb protein gradient derived from the maternally
supplied mRNA (Tautz, 1988). Previous studies in Drosophilareveal that this translational repression depends on the 3untranslated region (3UTR) of the hb transcript. Here, abipartite sequence (the nanos response element, NRE) isrecognized by pumilio protein, which in turn recruits nanos(Murata and Wharton, 1995; Sonoda and Wharton, 1999;Wharton and Struhl, 1991). Together nanos and pumilio act toprevent hb translation in the posterior part of the embryo.The resulting Hb protein gradient is known to behavemorphogenetically, regulating its own promoter, as well asother gap genes such as Krppel, knirps and giant in aconcentration-dependent manner (Hlskamp et al., 1990;Simpson-Brose et al., 1994; Struhl et al., 1992).
Zygotic expression of Drosophila hb begins at theblastoderm stage (Fig. 1A; Tautz et al., 1987). Drosophila hbmRNA is transcribed from two promoters, P1 and P2, resultingin transcripts of 3.2 and 2.9 kb, respectively (Schrder et al.,1988). The P2 promoter is activated by the anterior morphogenbicoid, while the 3.2 kb transcript is activated acrossparasegment 4 (PS4) by, among other factors, Hb itself(Schrder et al., 1988; Tautz, 1988). The zygotic Hb proteingradient (Fig. 1A) is similar to its maternal counterpart. Indeedthe two gradients are almost redundant: maternal hb isdispensable for AP patterning, and zygotic hb activation bybicoid almost is (Hlskamp et al., 1989; Irish et al., 1989;Wimmer et al., 2000).
Anterior hb expression is crucial for the development ofthoracic segments. In addition, a posterior domain of zygotichb, under independent control, emerges and refines to a domainspanning P