Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology Danielle M. Liubicicha, Julia M. Seranob, Anastasios Pavlopoulosc, Zacharias Kontarakisd, Meredith E. Protasb, Elaine Kwanb, Sandip Chatterjeeb,1, Khoa D. Tranb,2, Michalis Averofd, and Nipam H. Patela,b,3 aDepartment of Integrative Biology, University of California, Berkeley, CA 94720-3140; bDepartment of Molecular Cell Biology, Center for Integrative Genomics, University of California, Berkeley, CA 94720-3200; cLaboratory for Development and Evolution, Department of Zoology, University Museum of Zoology, Downing Street, Cambridge CB2 3EJ, United Kingdom; and dInstitute of Molecular Biology and Biotechnology, Nikolaou Plastira 100, 70013 Iraklio, Crete, Greece Edited by Sean B. Carroll, University of Wisconsin, Madison, WI, and approved May 29, 2009 (received for review March 24, 2009) Crustaceans possess remarkably diverse appendages, both be- transcription factors that specify regional identity in diverse tween segments of a single individual as well as between species. animal body plans (5). Experimentally altering boundaries of Previous studies in a wide range of crustaceans have demonstrated Hox gene expression has produced dramatic phenotypes in a correlation between the anterior expression boundary of the organisms such as flies and mice. Therefore, it is possible that homeotic (Hox) gene Ultrabithorax (Ubx) and the location and shifting Hox expression may have similar morphological conse- number of specialized thoracic feeding appendages, called maxil- quences in crustaceans and could provide one potential mech- lipeds. Given that Hox genes regulate regional identity in organ- anism contributing to the evolution of crustacean appendage isms as diverse as mice and flies, these observations in crustaceans diversity. led to the hypothesis that Ubx expression regulates the number of To test these hypotheses regarding the role of Ubx in crusta- maxillipeds and that evolutionary changes in Ubx expression have cean appendage specification and evolution, we characterized generated various aspects of crustacean appendage diversity. Ubx in the malacostracan amphipod crustacean Parhyale ha- GENETICS Specifically, evolutionary changes in the expression boundary of waiensis, an emerging model system. We examined both mRNA Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs and protein expression, and found Ubx expression throughout of thoracic maxillipeds. Here we test this hypothesis by altering the the walking and grasping appendages of the second through expression of Ubx in Parhyale hawaiensis, a crustacean that nor- eighth thoracic appendages, but no expression in the maxilliped mally possesses a single pair of maxillipeds. By reducing Ubx appendage of the first thoracic segment. We then developed an expression, we can generate Parhyale with additional maxillipeds siRNA-based approach to knock down gene function in Parhyale, in a pattern reminiscent of that seen in other crustacean species, and used this technique to functionally test the developmental and these morphological alterations are maintained as the animals role of PhUbx directly in this crustacean. By reducing Ubx molt and mature. These results provide critical evidence supporting expression in Parhyale, we were able to transform multiple the proposition that changes in Ubx expression have played a role walking legs to a maxilliped-like identity. The extent of trans- in generating crustacean appendage diversity and lend general formation varied among thoracic segments in a pattern that insights into the mechanisms of morphological evolution. replicates the morphological variation naturally occurring among wild crustacean species that possess multiple pairs of appendages ͉ arthropods ͉ development ͉ Hox maxillipeds. he morphology and structure of crustacean appendages are Results Tas diverse as their assorted functions, and these appendages Ubx Expression Correlates with Appendage Identity in Parhyale. not only vary between species, but between different segments Molecular characterization of the Parhyale ortholog of Ultra- of the same individual as well. Appendages of the posterior head bithorax (PhUbx) revealed that alternative splicing generates 2 segments are part of the jaw apparatus that crushes food and different mRNAs (PhUbx-I and PhUbx-II) that vary only in the moves it to the mouth during feeding. The more posterior first few amino acids they encode (see Fig. S1). We initially appendages of the crustacean trunk serve numerous roles in- analyzed the pattern of PhUbx expression by in situ hybridization cluding mating, defense, and locomotion. The pattern of these with a probe that recognizes both mRNAs and immunostaining segmental specializations varies between species, and is often with polyclonal antisera that recognizes both protein products used as a criterion for subdividing crustaceans into various groups. For example, brine shrimp possess appendages through- out the entire trunk that are used in locomotion. These append- Author contributions: D.M.L., J.M.S., M.E.P., E.K., and N.H.P. designed research; D.M.L., ages are similar to one another, yet they are quite distinct from J.M.S., M.E.P., E.K., S.C., K.D.T., and N.H.P. performed research; D.M.L., J.M.S., A.P., Z.K., M.E.P., E.K., S.C., K.D.T., and M.A. contributed new reagents/analytic tools; D.M.L., J.M.S., the head appendages used in feeding. Other crustaceans, how- M.E.P., S.C., and N.H.P. analyzed data; and D.M.L., J.M.S., M.E.P., and N.H.P. wrote the ever, possess a variety of specialized appendages within the paper. trunk. Lobsters, for example, have certain anterior, thoracic The authors declare no conflict of interest. appendages that are morphologically similar to the mouthparts This article is a PNAS Direct Submission. and serve as additional feeding appendages. These modified Freely available online through the PNAS open access option. thoracic appendages are called maxillipeds (‘‘jaw-feet’’), and 1Present address: Department of Biopharmaceutical Sciences, University of California, San crustaceans may possess up to 3 pairs of these specialized Francisco, CA 94143-2811. appendages. 2Present address: Institute of Neuroscience and Molecular Biology, University of Oregon, A striking correlation has been found between the anterior Eugene, OR 97403. expression boundary of the Hox gene Ultrabithorax (Ubx) and the 3To whom correspondence should be addressed. E-mail: [email protected]. position and number of maxillipeds that develop in crustaceans This article contains supporting information online at www.pnas.org/cgi/content/full/ (1–4). Hox genes are members of a highly conserved family of 0903105106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903105106 PNAS Early Edition ͉ 1of5 Downloaded by guest on October 2, 2021 from individual rows of cells (called parasegment precursor AB C D rows, or PSPRs) that become organized during early germband stages (6). We found that PhUbx transcripts are first detected at stage 12 in the most medial cells of PSPR 5 and 6 (Fig. 1D and E)—the progeny of these PSPRs will produce ectodermal de- rivatives stretching from posterior T2 to anterior T4. At stage 14 (Fig. 1F and FЈ), expression expands laterally in PSPR 5 and 6, anteriorly into progeny of PSPR 4, and posteriorly into PSPRs that form the remaining thoracic segments. PhUbx mRNA E expression in PSPR 4 is detected only after this row has divided into a/b and c/d progeny. At stages 15–16, PhUbx expression in the 4 a/b row (and subsequently formed 4a and 4b rows) is confined to the medial (ventral) region and does not extend F GH laterally to the cells that contribute to the posterior portion of the developing T1 appendage. This leads to an expression pattern at stage 18 (Fig. 1H) where Ubx mRNA expression has a paraseg- mental anterior boundary in the medially located neuroecto- derm, but a segmental boundary more laterally so that expres- sion is seen throughout the T2 limb primordia, but not in the T1 ‘ limb primordia. This same pattern can also be seen for PhUbx F protein at stage 18 (Fig. 1G). At these stages, expression is also seen throughout the abdomen. By stage 25, however, abdominal expression is restricted to a small number of segmentally re- peated cells. It should be noted that there is a significant delay between the appearance of PhUbx mRNA and PhUbx protein. PhUbx protein is not detected before stage 16–17. Even for Fig. 1. Parhyale Ubx expression during embryonic development. (A) and (F) mRNA, PhUbx transcripts are restricted to the nucleus until are brightfield images of PhUbx expression (purple) and (C–E and FЈ–H) are stage 15. Probes specific to each splice variant revealed an DAPI images (highlighting nuclei) with PhUbx expression overlaid in red. All identical expression pattern with respect to segmental bound- images display a ventral view of the embryo with anterior to the top except for aries, although expression of PhUbx-I appears weaker than (B), which shows ventral views of hatchling appendages. (A) PhUbx mRNA in PhUbx-II, but this difference in signal intensity may be due to the a stage 23 Parhyale embryo is expressed at lower levels in the second and third necessarily short length of the PhUbx-I specific probe. thoracic segments (T2 and T3) and appendages, at higher levels throughout the remaining thoracic segments and appendages (T4–T8), and is absent from Knockdown of PhUbx in Parhyale. the T1 segment and maxillipeds. (B) Hatchling thoracic (T1–T4) and head (MxII) To test the hypothesis that Ubx appendages corresponding to the embryonic appendages shown in (A). Colors defines the distinction between the maxillipeds and the remain- of each arrow reflect the level of PhUbx expression in that appendage. (C) ing walking and grasping legs of the thorax in crustaceans we Ventral view of a stage 24 Parhyale embryo stained with an antibody recog- developed a strategy to knock down gene function during nizing PhUbx protein. The PhUbx protein expression boundaries and levels are Parhayle development using injection of siRNAs, a technique consistent with the mRNA expression in (A).