bioRxiv preprint doi: https://doi.org/10.1101/712216; this version posted July 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Full Title: Evolution of abbreviated development in Heliocidaris erythrogramma 2 dramatically re-wired the highly conserved sea urchin developmental gene regulatory 3 network to decouple signaling center function from ultimate fate 4 5 Short Title: Heliocidaris life history switch rewired early gene regulatory network 6 7 Authors and affiliations: 8 Allison Edgar1,2, Maria Byrne 2,3, David R. McClay1, Gregory A. Wray1,4 9 1Department of Biology, Duke University, Durham, NC, USA 10 2School of Medical Science and Bosch Institute, Department of Anatomy and Histology, 11 The University of Sydney, Sydney, NSW, Australia 12 3School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 13 Australia 14 4Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, 15 USA. 16 17 Email addresses of corresponding authors: Allison Edgar: [email protected]; Gregory 18 A. Wray: [email protected]) 19 20 Email addresses of co-authors: 21 Maria Byrne: [email protected]; David R. McClay: [email protected] 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/712216; this version posted July 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Abstract 25 Developmental gene regulatory networks (GRNs) describe the interactions among gene 26 products that drive the differential transcriptional and cell regulatory states that pattern 27 the embryo and specify distinct cell fates. GRNs are often deeply conserved, but 28 whether this is the product of constraint inherent to the network structure or stabilizing 29 selection remains unclear. We have constructed the first formal GRN for early 30 development in Heliocidaris erythrogramma, a species with dramatically accelerated, 31 direct development. This life history switch has important ecological consequences, 32 arose rapidly, and has evolved independently many times in echinoderms, suggesting it 33 is a product of selection. We find that H. erythrogramma exhibits dramatic differences in 34 GRN topology compared with ancestral, indirect-developing sea urchins. In particular, 35 the GRN sub-circuit that directs the early and autonomous commitment of skeletogenic 36 cell precursors in indirect developers appears to be absent in H. erythrogramma, a 37 particularly striking change in relation to both the prior conservation of this sub-circuit 38 and the key role that these cells play ancestrally in early development as the embryonic 39 signaling center. These results show that even highly conserved molecular mechanisms 40 of early development can be substantially reconfigured in a relatively short evolutionary 41 time span, suggesting that selection rather than constraint is responsible for the striking 42 conservation of the GRN among other sea urchins. 43 44 Introduction 45 Instructions encoded in the genome are executed during development to specify distinct 46 cell types in specific spatial patterns. Developmental gene regulatory networks (GRNs) 2 bioRxiv preprint doi: https://doi.org/10.1101/712216; this version posted July 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 are formal models of the transcription factor cascades and cell signaling interactions 48 that specify these diverse cell fates and distinct embryonic territories. Evolutionary 49 changes in these processes are thought to underlie many interesting and novel 50 phenotypes. However, two fundamental challenges to understanding how GRNs evolve 51 are distinguishing stabilizing selection from inherent network features that promote 52 stability and discriminating directional selection from phenotypically neutral 53 developmental systems drift [1,2]. The sea urchin Heliocidaris erythrogramma is an 54 ideal model system to explore these questions because its development has changed 55 dramatically from the ancestral state in a relatively short evolutionary time, 56 approximately 4 million years ago (mya). Developmental GRNs from sea urchins 57 diverged ~40-270 mya, and from several echinoderm outgroups diverged up to 550 58 mya, are particularly well-studied (reviewed in [3-6]). Thus evolution of echinoderm 59 GRNs may be compared across orders of magnitude of divergence time. 60 The euechinoid genus Heliocidaris encompasses a dramatic shift in developmental life 61 history [7,8]. H. tuberculata exhibits the ancestral condition for sea urchins: small eggs 62 with indirect development via a feeding larva (planktotrophy). The developmental GRN 63 underlying this ancestral life history (Figure 1A) has been characterized in considerable 64 detail and is highly conserved across euechinoid sea urchins [6,9]. H. erythrogramma’s 65 ancestors diverged from the ancestral condition, acquiring much larger eggs and greatly 66 accelerated development via a nonfeeding larva (lecithotrophy) with highly derived 67 morphology [8,10,11] (Figure 1B). Despite these substantive differences, the post- 68 metamorphic phase of its life cycle is nearly indistinguishable from that of its congener 69 H. tuberculata. The Heliocidaris lineages with ancestral and accelerated development 3 bioRxiv preprint doi: https://doi.org/10.1101/712216; this version posted July 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 70 diverged only ~4 million years ago (mya) [7]. While this life history switch has arisen 71 multiple times in echinoderms [8,12,13], the Heliocidaris genus remains the best studied 72 example [14-33]. Loss of the feeding larval stage entails tradeoffs among maternal 73 investment, offspring survival, and dispersal [12,34-36], although the ecological 74 consequences of the transition to lecithotrophy are complex and incompletely 75 understood [37]. Lecithotrophic development in H. erythrogramma is accompanied by 76 dramatic changes to embryogenesis including changed timing of key developmental 77 events, altered cleavage pattern, axial patterning, and early cell fate specification. The 78 rapidity with which this developmental mode has arisen in H. erythrogramma and its 79 implications for ecology, suggest that accelerated development is a product of strong 80 selection for accelerated development rather than of evolutionary drift or selection on an 81 adult trait. 82 83 Figure 1. A Euechinoid mesodermal cell lineages are specified cell-autonomously and non- 84 autonomously. The skeletogenic mesenchyme (SM) cells arise by an unequal cleavage (smaller 85 pink cells) and are autonomously specified as the signaling center and prospective skeletogenic 86 cells. A signaling relay (arrows) initiated by SM cells specifies adjacent cells as non-skeletogenic 87 mesoderm (NSM) (green). The gene regulatory network activated in these cells was constructed 88 by individual gene perturbation experiments to identify transcription factors and ligands that must 89 be expressed in the SM cells for their cell-autonomous specification as prospective skeletogenic 90 cells (pink), their identity and/or function as a signaling center (green), or both (yellow). B Top: 91 Development in euechinoid planktotrophs drawn from literature. In the ancestral state, the SM 92 cells ultimately become larval skeleton (pink), but also function as the signaling center that induces 4 bioRxiv preprint doi: https://doi.org/10.1101/712216; this version posted July 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 specification of other cell lineages, such as coelomic pouch mesoderm, pigment cell mesoderm 94 and other non-skeletogenic mesenchyme (green). Coelomic pouch mesoderm is the source of 95 juvenile skeletogenic cells (not pictured). Bottom: Model of development in the lecithotroph H. 96 erythrogramma drawn from literature. The timing, lineage, and fate of the embryonic signaling 97 center is not yet known in detail. No mesodermal sub-types are segregated before the 64-cell stage 98 and descendants of the yellow shaded area will contribute to skeletogenic mesoderm, coelomic 99 pouch, pigment cell mesoderm and other NSM, and endoderm. Whether and when larval (pink) 100 and juvenile (not pictured) skeletogenic cells are segregated is unknown. 101 102 To understand how the ancestral GRN may have changed to accommodate the 103 shift to lecithotrophy, we chose to focus on a specialized cell lineage shared by all 104 euechinoid sea urchins [38] that is well studied for its unique developmental role, cell 105 behaviors, and specification process: the skeletogenic mesenchyme (SM). In the 106 planktotroph four cells specified early in development, the large