The beetle amnion and serosa functionally interact as apposed epithelia

ACCEPTED MANUSCRIPT The beetle amnion and serosa functionally interact as apposed epithelia Maarten Hilbrant, Thorsten Horn, Stefan Koelzer, Kristen A Panfilio DOI: http://dx.doi.org/10.7554/eLife.13834 Cite as: eLife 2016;10.7554/eLife.13834 Received: 16 December 2015 Accepted: 28 January 2016 Published: 29 January 2016 This PDF is the version of the article that was accepted for publication after peer review. Fully formatted HTML, PDF, and XML versions will be made available after technical processing, editing, and proofing. Stay current on the latest in life science and biomedical research from eLife. Sign up for alerts at elife.elifesciences.org 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 The beetle amnion and serosa functionally interact as apposed epithelia Maarten Hilbrant, Thorsten Horn, Stefan Koelzer, Kristen A. Panfilio * Institute for Developmental Biology, University of Cologne Cologne Biocenter, Zülpicher Straße 47b, 50674 Cologne, Germany * Author for correspondence: E-mail: Telephone: +49 (0)221 470 8563 Fax: +49 (0)221 470 5164 Running Title: Coordinated tissue morphogenesis in Tribolium Keywords: epithelial morphogenesis; extraembryonic development; insects; RNA interference (RNAi); fluorescent live imaging; light sheet microscopy 20 21 1 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ABSTRACT Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues – the amnion and serosa – actively rupture and withdraw in late embryogenesis. Withdrawal is essential for development and has been a morphogenetic puzzle. Here, we use new fluorescent transgenic lines in the beetle Tribolium castaneum to show that the EE tissues dynamically form a basal-basal epithelial bilayer, contradicting the previous hypothesis of EE intercalation. We find that the EE tissues repeatedly detach and reattach throughout development and have distinct roles. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap. RNAi phenotypes demonstrate that the serosa contracts autonomously. Thus, apposition in a bilayer enables the amnion as “initiator” to coordinate with the serosa as “driver” to achieve withdrawal. This EE strategy may reflect evolutionary changes within the holometabolous insects and serves as a model to study interactions between developing epithelia. 2 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 INTRODUCTION Embryogenesis requires dynamic interaction between tissues to create changing threedimensional configurations, culminating in the completion of the body. In parallel to the amniote vertebrates (Calvin and Oyen, 2007), the insects have evolved extraembryonic (EE) tissues that arise in early embryogenesis to envelop the embryo (Panfilio, 2008). These are the amnion and the serosa, which are both simple, squamous epithelia. Like its vertebrate namesake, in most insect species the amnion encloses a fluid-filled cavity around the embryo. As the outermost cellular layer, the serosa provides mechanical and physiological protection (Farnesi et al., 2015; Jacobs et al., 2013; Jacobs et al., 2014; Rezende et al., 2008). This protective configuration is not permanent, though, and a major reorganization of the EE tissues is essential for embryos to correctly close their backs in late development. Reorganization involves whole tissue eversion, contraction, and final apoptosis of both EE tissues (Panfilio et al., 2013). For these events, perhaps the nearest morphogenetic equivalent in the model system Drosophila is eversion of the wing imaginal disc during metamorphosis, where the squamous peripodial epithelium also exhibits these behaviors (Aldaz et al., 2010). However, research on Drosophila cannot address the morphogenesis of the two EE epithelia directly, due to the secondarily derived nature of its single EE tissue, the amnioserosa, which does not surround the embryo (Rafiqi et al., 2012; Schmidt-Ott, 2000). Insect EE withdrawal – the active process whereby the EE tissues withdraw from the embryo and leave it uncovered – has been addressed at the level of gross morphology in classical descriptions for many species (reviewed in Panfilio, 2008). However, a major open question has been the organization and role of the amnion. This is primarily because it is difficult to visualize in its native topography with respect to other tissues. A lack of amnion-specific molecular markers (discussed in Koelzer et al., 2014) and the histological similarity and proximity of the serosa (Panfilio and Roth, 2010; van der Zee et al., 2005) have been particular challenges. Here, we present the first clear determination of the relative topography and role of the amnion in late development in a holometabolous insect, the red flour beetle, Tribolium castaneum. We characterize an enhancer trap line that labels the amnion and use this in conjunction with recently characterized serosal lines (Koelzer et al., 2014) to morphogenetically dissect which tissue is responsible for which aspects of EE tissue withdrawal. The topographical arrangement of the tissues differs strikingly from what was previously known in hemimetabolous insects and what had previously been hypothesized for Tribolium. To better appreciate the implications of this arrangement for morphogenesis, we situate these observations in the larger context of EE development at preceding and following stages. This global, mesoscopic approach to evaluating tissue interactions significantly improves our understanding the entire withdrawal process, including the first detailed examination of EE rupture in any insect. Furthermore, we provide evidence that while the serosa strongly drives the contraction and folding of the tissues, the amnion initiates EE rupture. 3 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 RESULTS AND DISCUSSION The amnion and serosa form a bilayer during late development To augment the toolkit for tissue-specific visualization in Tribolium, we identified and characterized an enhancer trap line with amniotic EGFP expression (Figure 1, see also Figure 1-figure supplement 1, Video 1). Prior to withdrawal morphogenesis, the EGFP-labeled tissue fully envelops the embryo but does not cover the yolk (Figure 1A-B). To confirm that this tissue is indeed the amnion, and not a specialized region of the serosa, we examined EGFP expression after RNAi for Tc-zen1, thereby eliminating serosal tissue identity (van der Zee et al., 2005). In the absence of the serosa, the amnion occupies a dorsal position over the yolk, and indeed this tissue expresses EGFP (Figure 1C-D). We then used the tissue-specific EE imaging lines to address the arrangement of the amnion and serosa during late deve (...truncated)