Cover Photo by Adi Kehn

Cover Photo by Adi Kehn

DEVELOPMENTAL BIOLOGY OF THE SEA URCHIN XXIV April 5-9, 2017 Marine Biological Laboratory Woods Hole, MA Organizers: Athula Wikramanayake Billie Swalla Thierry Lepage Amro Hamdoun The organizers would like to acknowledge the generous support of these sponsors: The organizers would like to thank MRD for providing the T-Shirts

Wednesday, April 5 Developmental Biology of the Sea Urchin XXIV, The Marine Biological Laboratory, Woods Hole, MA April 5-9, 2017 Arrival and check in Swope Building: 12:00 PM onwards Dinner 6:00-7:30 PM Opening Plenary Session: From egg to embryo: Developmental and evolutionary insights 7:30-9:10 PM Lillie Auditorium Chair: Athula Wikramanayake (University of Miami) 7:30-7:40 PM-Introductory comments 7:40-8:10 PM-Athula Wikramanayake (University of Miami) Evolution of the primary egg axis and the emergence of complex metazoans 8:10-8:40 PM-Uli Technau (University of Vienna) Cnidarians and the evolution of bilaterality 8:40-9:10 PM-Yi-Hsien Su (Academia Sinica, Taiwan) The roles of the maternal and zygotic hypoxia-inducible factor a in sea urchin development Mixer and posters 9:15-11:00 PM Meigs Room Thursday, April 6 Breakfast 7:00-8:30 AM Plenary Session II: Cell biology of the early embryo 8:30-12:00 AM Lillie Auditorium Chair: Zak Swartz (Whitehead-MIT) 8:30-9:00 AM- Zak Swartz (Whitehead-MIT) Ensuring transmission of the centromere through meiosis and development 9:00-9:30 AM- John Henson (Dickinson College) New insights into the organization of the contractile ring in dividing sea urchin embryos 9:30-10:00 AM- David Burgess (Boston College) Role of the Sperm Aster in Nuclear Centering and Determinant Localization 10:00-10:30 AM-Coffee break

10:30-11:00 AM-Mamiko Yajima (Brown University) Molecular modification of the G-protein regulator, AGS, contributes to asymmetric cell division in sea urchins 11:00-11:30 AM- Silvia Sepulveda-Ramirez (New Mexico State University) Role of cell polarity in morphogenesis 11:30-12:00 PM-Tony De Tomaso (UC Santa Barbara) Dissecting mechanotransduction in real time: How do cells respond to changes in their physical environment Lunch 12:00-1:30 PM Plenary Session III: Specifying primordial germ cells and stem cells 1:30-3:00 PM Lillie Auditorium Chair: Nathalie Ouhlen (Brown University) 1:30-2:00 PM-Nathalie Ouhlen (Brown University) Nanos is super important and very cool 2:00-2:30 PM-William Browne (University of Miami) Ancient functions in new contexts: the role of Krüppel-like factor genes in the ctenophore Mnemiopsis 2:30-3:00 PM-Paola Oliveri (University College London) Development and regeneration in the brittle star A. filiformis Coffee Break 3:00-3:30 PM, Lillie Auditorium Concurrent Session 1: Cell Biology and Development 3:30-5:20 PM Lillie Auditorium Session Chair: Charles Shuster (New Mexico State University) 3:30-4:00 PM-Charles Shuster (New Mexico State University) Evolving roles of Arp2/3 actin networks over the course of early development 4:00-4:20 PM- Wei Wu (University of Miami) Casein Kinase 1 delta/epsilon mediates anterior-posterior axis formation in the sea urchin embryo, potentially through localized activation of Disheveled 4:20-4:40 PM- Daphne Schatzberg (Boston University) V-type H+ ATPase activity is required for dorsal-ventral symmetry breaking in sea urchin embryos 4:40-5:00 PM- Maria Dolores Molina Jimenez (Institut Biologie Valrose, Nice) p38 MAPK as an essential regulator of D/V axis specification and skeletogenesis during sea urchin development: a re-evaluation 5:00-5:20 PM- Margherita Perillo (Boston College) Characterization of ciliary band neurons expressing pancreatic transcriptional factors

Concurrent Session 2: Primordial germ cells and regeneration 3:30-5:20 PM Speck Auditorium Session Chair: Mariko Kondo (MIBS, University of Tokyo) 3:30-4:00 PM- Mariko Kondo (MIBS, University of Tokyo) Regeneration studies in two echinoderms, feather star and sea cucumber 4:00-4:20 PM- Raymond Allen (Duke University) Cell reprogramming in the urchin embryo after PMC removal and embryo bisection 4:20-4:40 PM- Tara Fresques (Brown University) Identifying embryonic mechanisms that induce a germ cell fate in sea stars 4:40-5:00 PM- Minyan Zheng (Carnegie Mellon University) Characterization of apical pole domain associated gene regulatory networks during sea star larval regeneration 5:00-5:20 PM- Andrew Wolf (Carnegie Mellon University) A shift from normal to regeneration-specific proliferation in the sea star Patiria miniata Dinner 5:45-7:30 PM Swope Plenary Session IV: Making neurons and sensory organs 7:30-9:00 PM Lillie Auditorium Session Chair: Ryan Range (Mississippi State University) 7:30-8:00 PM-Ryan Range (Mississippi State University) The emerging Wnt signaling network that governs anterior-posterior neuroectoderm patterning in the sea urchin embryo 8:00-8:30 PM-Robert Burke (University of Victoria) Patterning Neurogenesis 8:30-9:00 PM-Roberto Feuda (California Institute of Technology) Eyes without the master: photoreceptor specification in the sea urchin larva is independent of the retinal determination network Posters and mixer 9:00-11:00 PM Meigs Room Friday, April 7 Breakfast 7:00-8:30 AM

Plenary Session V: Cellular and molecular mechanisms underlying specification and morphogenesis 8:30-12:00 PM Lillie Auditorium Chair: Ina Arnone (Stazione Zoologica) 8:30-9:00 AM- Dede Lyons (Scripps Institute of Oceanography) Morphogenesis along the animal-vegetal axis:fates of primary quartet micromere daughters in the gastropod Crepidula fornicata 9:00-9:30 AM-Ina Arnone (Stazione Zoologica) Echinoderm GRNs are going omics: insight into the evolution of gut patterning 9:30-10:00 AM- David McClay (Duke University) GRNs regulating morphogenesis 10:00-10:30 AM Coffee Break 10:30-11:00 AM-Elaine Seaver (UF-Whitney Marine Laboratory) Establishment of body axes in the annelid Capitella teleta 11:00-11:30 AM-Isabel Peter (California Institute of Technology) Assessing the relationship between structure and function in developmental circuits (California Institute of Technology) 11:30-12:00 PM-Shunsuke Yaguchi (University of Tsukuba) Troponin system in the sea urchin embryos Lunch 12:00-1:30 PM Plenary Session VI: Patterning the ectoderm 1:30-3:00 PM Lillie Auditorium Chair: Cyndi Bradham (Boston University) 1:30-2:00 PM-Thierry Lepage (Institut Biologie Valrose, Nice) The ETS domain YanTel as a key maternal regulator of axis formation at the crossroads of Wnt and Nodal signaling in the sea urchin embryo 2:00-2:30 PM-Cyndi Bradham (Boston University) New Cues for Skeletal Patterning in Sea Urchin Embryos 2:30-3:00 PM-Smadar ben Tabou de Leon (University of Haifa) VEGF activates a complex gene network with angiogenetic motifs to control sea urchin spiculogenesis Coffee Break 3:00-3:30 PM, Lillie Auditorium Concurrent Session 3: Morphogenesis 3:30-5:20 PM Lillie Auditorium Session Chair: Jose Espinoza (Scripps Institute of Oceanography) 3:30-4:00 PM Derk Joester (Northwestern University) Title TBA

4:00-4:20 PM- Claudia Cuomo (Statione Zoologica) SpMeis: a novel key factor shaping the gut of the sea urchin postgastrula embryo 4:20-4:40 PM- Jose Espinosa (Scripps Institute of Oceanography) Small micromere migration and a possible role for lysophosphatidic acid signaling in left/right asymmetry 4:40-5:00 PM- Andrew George (Duke University) Characterizing Epithelial to mesenchymal transition of pigment cells in the sea urchin Lytechinus variegatus 5:00-5:20 PM- Lama Khalaily (University of Haifa) Upstream regulation of sea urchin skeletal patterning and VEGF signaling Concurrent Session 4: GRNs in development and evolution 3:30-5:40 PM Speck Auditorium Session Chair: Greg Cary (Carnegie Mellon University) 3:30-4:00 PM- Greg Cary (Carnegie Mellon University) Evolution of the Delta-HesC feedback mechanism driving mesodermal subtype specification in echinoderms 4:00-4:20 PM- Leslie Slota (Duke University) Three populations of neurons in the sea urchin develop using gene regulatory networks highly conserved with vertebrates 4:20-4:40 PM- Jon Valencia (California Institute of Technology) The regulatory states in the sea urchin larva 4:40-5:00 PM- Marina Martinez-Bartolome (Mississippi State University) Wnt16-Fzl1/2/7-NFAT signaling antagonizes Wnt1/Wnt8-Fzl5/8-JNK signaling mediated restriction of the anterior neuroectoderm along the anterior-posterior axis in the sea urchin embryo 5:00-5:20 PM-Catherine Guay (Rutgers University) Testing the utility of sea urchin embryos to discover human embryonic cis-regulatory modules Dinner 5:45-7:30 PM Plenary Session VII: Genomic and developmental evolution 7:30-9:00 PM Lillie Auditorium Chair: Veronica Hinman (Carnegie Mellon University) 7:30-8:00 PM-Veronica Hinman (Carnegie Mellon University) Regeneration of larval P. miniata 8:00-8:30 PM-Billie Swalla (University of Washington) Evolution, development and regeneration in hemichordates 8:30-9:00 PM-Greg Wray (Duke University) Rapid evolution of key GRN interactions within the sea urchin genus Heliocidaris

Posters and mixer 9:00-11:00 PM Meigs Room Saturday, April 8 Breakfast 7:00-8:30 AM Plenary Session VIII: Environmental and epigenetic influences on development 8:30-12:00 PM Lillie Auditorium Chair: Meike Stumpp (Christian-Albrechts-University Kiel) 8:30-9:00 AM- Meike Stumpp (Christian-Albrechts-University Kiel) The alkaline larval gut a key trait under environmental change? 9:00-9:30 AM-Jonathan Rast (University of Toronto) The gene regulatory underpinnings of deuterostome immunity. 9:30-10:00 AM- Jia Song (University of Delaware) Function of microrna-31 in the sea urchin embryo Coffee Break 10:00-10:30 AM, Lillie Auditorium 10:30-11:00 AM-Donal Manahan (University of Southern California) Animal evolution and development: An environmental and physiological perspective 11:00-11:30 AM-Catherine Schrankel (University of Toronto) Developmental and immune-regulated expression of the perforin-like SpMacpf gene family in the purple sea urchin 11:30-12:00 PM-Avery Andrus (California State University-Long Beach) Cellular and proteomic characterization of the innate immune response in wasting bat stars (Patiria miniata) Lunch 12:00-1:30 PM Plenary Session IX: Functional genomics and development 1:30-3:00 PM Lillie Auditorium Chair: Jongmin Nam (Rutgers University-Camden) 1:30-2:00 PM-Hector Escriva (Observatoire Oceanologique de Banyuls sur Mer) A genomic view of chordates' evolution 2:00-2:30 PM- Cesar Arenas-Mena (College of Staten Island-CUNY) The origins of developmental gene regulation and the genome-wide identification of active blastula enhancers 2:30-3:00 PM- Jongmin Nam (Rutgers University-Camden) Ins and outs of transgenes in the sea urchin Coffee Break 3:00-3:30 PM

Concurrent Session 5: Using marine invertebrate embryos in undergraduate research and teaching 3:30-4:45 PM Lillie Auditorium Session Chair: Christine Byrum (College of Charleston) 3:30-3:45 PM- Christine Byrum (College of Charleston) Discovery in the classroom: Using the sea urchin to teach fundamental molecular skills and examine gene expression 3:45-4:00 PM- Pei Yun Lee (UCLA) The sea urchin embryo as a model system in an undergraduate research immersion laboratory course 4:00-4:15 PM-Laura Romano (Denison University) Using art to help students visualize processes in a developmental biology course 4:15-4:30 PM- Thomas Onorato (LaGuardia Community College) PRIMO experiences strengthen the URGE to democratize undergraduate research at a diverse large public urban community college 4:30-4:45 PM-Cyndi Bradham (Boston University) Teaching gene regulatory network logic to undergraduates 5:00-6:00 PM- Business Meeting Lillie Auditorium Mixer 6:30-7:30 PM Meigs Room Lobster Banquet 7:30 9:00 PM Sunday, April 9 Breakfast 7:00-8:30 AM Departure

Contents Talk... 1 Poster... 24

Talk Developmental Biology of the Sea Urchin 2017 Talk Evolution of the primary egg axis and the emergence of complex metazoans 04/05/17 07:40 PM - 08:10 PM Wikramanayake, Athula H. (University of Miami, Miami, FL, USA) The animal-vegetal (AV) axis is a cytoplasmic polarity present in most animal eggs that strongly influences the formation of the anterior-posterior (AP) axis during embryogenesis. A critical early step during AP axis formation is the segregation of the germ layers; a process usually mediated by asymmetrically distributed developmental information in the ovum. In many bilaterians and their closest relatives, the Cnidaria, endoderm segregation is mediated by localized activation of canonical Wnt (cwnt) signaling. These observations suggest that a cwnt signaling-dependent gene regulatory network (GRN) specified endoderm in the last common ancestor (LCA) to bilaterians and cnidarians over 600 million years ago. Strikingly, however, endoderm specification occurs at the vegetal pole in most bilaterians and at the animal pole in cnidarians. We have proposed that a switch in the localization of critical upstream activators of cwnt signaling along the AV axis led to the redeployment of endoderm specification from the animal pole of the cnidarian-bilaterian LCA to the vegetal pole of the bilaterian LCA. This change may have been critical for the evolution of cephalization, a key trait underlying the success of the Bilateria. Our long-term objective is to reconstruct the cell biology of this key evolutionary change in egg polarity. To begin to address this problem we are taking advantage of our discoveries that Disheveled (Dvl), a key upstream regulator of cwnt signaling is enriched at the animal pole in cnidarian eggs and at a novel vegetal cortical domain (VCD) of sea urchin eggs, and that cwnt signaling and endoderm specification are induced by localized Dvl "activation" in sea urchins. We have hypothesized that the VCD serves as a cortical scaffold to locally activate Dvl during cwnt signaling. Hence, identification of proteins that target Dvl to the VCD and selectively activate it in the cwnt pathway may provide key insight into how deployment of this pathway may have been shifted from one pole of the egg to the other during evolution. To identify candidate factors we conducted Dvl Co-Immunoprecipitation of isolated egg cortex and micromere lysates coupled with mass spectrometry. One protein identified from this screen, Casein Kinase 1δ (CK1δ), is highly enriched and co-localized with Dvl in the VCD. I will present data that supports the hypothesis that CK1δ is one factor that locally regulates Dvl activity in the cwnt pathway during endoderm specification in the sea urchin embryo. I will also present preliminary analysis of CK1δ function in Nematostella. Cnidarians and the evolution of bilaterality 04/05/17 08:10 PM - 08:40 PM Technau, Ulrich (University of Vienna, Vienna, AUT) Most cnidarians display a single body axis, the oral-aboral axis and hence are radially symmetric. However, anthozoans (corals, sea anemones) have an internal asymmetry, the directive axis and hence are bilaterally symmetric. The directive axis is characterized by the asymmetric distribution of retractor muscles in the mesenteries and - in some species - the onesided syphonoglyph in the pharynx. This has fueled questions as to whether bilaterality in cnidarians and bilaterians is homologous, or whether it has evolved independently in both lineages. We used the anthozoan model organism Nematostella vectensis to address this question by elucidating the molecular basis of the formation of both axes. We show that the oral-aboral axis is specified by Wnt signaling. The Wnt-expressing blastopore lip has axis inducing properties in transplantation experiments, similar to the vertebrate Mangold-Spemann organizer. Functional analysis attributed this tissue property to two distinct Wnt ligands expressed in the blastopore lip. Wnt signaling initiates BMP and chordin expression at the oral pole. Inherent double negative feedback loops of BMPs and Chordin lead to a symmetry break at mid-gastrula stage, which requires BMP signaling. Subsequently, a gradient of psmad is established along the directive axis, hence perpendicular to the main oral-aboral axis. I will discuss the evolutionary implications on the basis of functional studies as well as mathematical simulations, which analysed the constraints of the components of the network. 1

Talk The roles of the maternal and zygotic hypoxia-inducible factor a in sea urchin development 04/05/17 08:40 PM - 09:10 PM Chang, Wei-Lun (1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan 2Graduate Institute of Life Sciences, National Defense Medical Ce, USA); Chang, Yi-Cheng (Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, USA); Lin, Kuan-Ting (Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, USA); Li, Han-Ru (Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, USA); Pai, Chih-Yu (Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, USA); Chen, Jen-Hao (Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, USA); Su, Yi- Hsien (Institute of Cellular and Organismic Biology Academia Sinica, Taipei, TWN) Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs the hypoxic acclimation and results in various diseases, including cancers. The role of the hypoxia pathway in early embryogenesis remains unclear. In the sea urchin Strongylocentrotus purpuratus blastula, we discovered that the maternal hypoxia-inducible factor α (HIFα), the downstream transcription factor of this pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricted the expression of nodal to the ventral side and activated several genes encoding transcription factors on the dorsal side. Later during gastrulation, the zygotic hifα gene is expressed in the mesodermal cells. Knock-down of the zygotic HIFα by a splicing morpholino resulted in skeletogenesis defect. Knock-out of the hifα gene by CRISPR/Cas9 showed similar phenotype. Our results present unprecedented roles of the hypoxia pathway in animal development. 2

Talk Ensuring transmission of the centromere through meiosis and development 04/06/17 08:30 AM - 09:00 AM Swartz, Zak (Whitehead Institute for Biomedical Research, Cambridge, MA, USA); Cheeseman, Iain (Whitehead Institute for Biomedical Research, USA) The oocyte-to-embryo transition is a defining time in preparation for animal development. Oocytes are arrested in the ovary for great lengths of time decades in humans. Upon hormonal stimulation, the oocyte must then re-enter the cell cycle and undergo meiosis, a unique program that reduces the female genome content by half, through highly asymmetric divisions that produce the polar bodies. The site of polar body extrusion then classically defines the animal pole of the oocyte. After fertilization, the division program must then convert for embryonic mitosis. Female meiosis is thus a pivotal time in animal development, which sets the stage for proper genome inheritance and embryonic polarity. The sea star oocyte provides a powerful system to understand these transitions from a cell biological perspective. Accurate segregation of the genome through this changing environment requires the centromere, the locus at which chromosomes interact with spindle microtubules. The centromere is defined epigenetically by a histone 3 variant called CENP-A, and a suite of interacting factors. The mechanisms that maintain these factors during the extended oocyte arrest, through meiosis, and in early development are very poorly defined. We find that CENP-A is not deposited during meiosis, but rapidly incorporates at centromeres in G1 phase in the egg pronucleus. This observation is consistent with the deposition dynamics of CENP-A in human mitotic cells. Furthermore, it implies that transmission of the centromere through the germ line relies upon the inherent stability of these epigenetic factors. Surprisingly, however, we observe slow incorporation of CENP-A at centromeres in prophase-arrested oocytes during extended culture. By proteomics, we have identified candidate factors for mediating this novel CENP-A incorporation. These results indicate a novel pathway for maintenance of the centromere during oocyte arrest, and provide new mechanistic insight into its inheritance through the animal germ line. New insights into the organization of the contractile ring in dividing sea urchin embryos 04/06/17 09:00 AM - 09:30 AM Irons, Zoe (Dickinson College, USA); Gamache, Courtney (Dickinson College, USA); Garno, Chelsea (New Mexico State University, USA); Williams, Erik (Dickinson College, USA); Shuster, C. Bradley (New Mexico State University, USA); Henson, John (Dickinson College, USA) The process of cell division, or cytokinesis, has long been known to be driven by the interaction of actin filaments with the motor protein myosin II within a structure termed the contractile ring (CR). Recently we have established for the first time the precise structural organization of CR actin and myosin II filaments in cortices isolated from first division sea urchin embryos. In the present study we use 3D structured illumination super-resolution microscopy (SIM) to extend this work to examine the x,y and z axial arrangement of the CR components actin, myosin II, septin2 and anillin as well as the impact of drug-based actin disruption. The localization results suggest that early in cytokinesis actin, myosin II (labeled in a way that allows for the recognition of head and tail regions), septin2, and anillin are organized into discrete clusters arranged in a broad stripe in the region of the early cleavage furrow. As division progresses, this arrangement transforms into an aligned linear array of actin, myosin II, septin2, and anillin oriented parallel to the CR long axis. The septin2 staining patterns suggests the presence of a gauze-like network of filaments in the CR in close association with myosin II. Anillin staining indicates that this protein is also codistributed with myosin II filaments in the CR. Z axial imaging indicates that myosin II head groups are oriented towards the invaginating membrane of the cleavage furrow relative to the tail regions. We have also begun to use SIM imaging of whole embryos to examine the distribution of CR components relative to the astral microtubules in the vicinity of the cleavage furrow. Our results emphasize the utility of employing 3D SIM imaging in order to further elucidate the high resolution structural organization of the CR apparatus that drives cytokinesis. 3

Talk Role of the Sperm Aster in Nuclear Centering and Determinant Localization 04/06/17 09:30 AM - 10:00 AM Burgess, David (Boston College, Chestnut Hill, MA, USA); Meaders, Johanathan (Boston College, USA); McDougall, Alex (UPMC Villefranche sur mer, USA) The fertilizing sperm brings with it centrioles from which the sperm aster and mitotic spindle are formed. The role of the sperm aster is to capture the female pro-nucleus for pronuclear fusion, to position the nucleus for the first cleavage division, and in many organisms to localize determinants for specific elements of the embryo. Nuclear centering was recently explained by dynein-dependent MT cytoplasmic pulling forces (Tanimoto, Kimura and Minc, 2016). We have re-analyzed the role of sperm astral microtubule (MT) growth and dynamics in these processes in two organisms: The primitive chordate ascidian Phallusia mammilata and the higher invertebrate echinoderm Lytechinus pictus. We find that astral MTs are longer on the cortical facing side than on the cytoplasmic side, which is inconsistent with the MT length-dependent cytoplasmic pulling model. Ethyl carbamate (Urethane) shortens MTs by increasing catastrophe followed by brief periods of MT growth, while hexylene glycol increases polymerization resulting in longer MTs (Strickland et al, 2005). Neither compound affects fertilization. We find that sperm astral MTs in urethane-treated eggs are shorter than in control zygotes and thus have less cortical contact. The result of suppressing sperm aster MT growth in L. Pictus is arrested sperm-egg pronuclear migration. The suppression of sperm astral MT growth in Phallusia also results in the failure of the sperm aster to capture the egg pronucleus. The result of suppressing MT growth in both zygotes is failure to center the nucleus and delayed cytokinesis, which results in heart shaped cleavage or unequal cell divisions. Conversely, increasing MT polymerization rates causes faster sperm aster migration, without affecting female pronuclear migration. Finally, we observe higher tyrosination of astral MTs on the leading side of the aster than on cortical-facing MTs. Recent studies have indicated a tyrosinated tubulin preference for dynein (McKenney et al, 2016) and a detyrosinated preference for different kinesins (Sirajuddin, et al, 2014). Taken together, we predict a model in which the tyrosination profile of the sperm aster allows more dynein transport on the cytoplasmic astral MTs and more kinesin transport on the cortical-facing astral MTs, leading to a MT motor asymmetry. We hypothesize that this motor asymmetry is essential for dynein to generate greater pulling forces at the front of the aster, while kinesins generate greater pushing forces from the cortical side to center the sperm aster during fertilization. In ascidians, the sperm aster also plays a role in the localization of determinants critical for proper development. Suppressing MT dynamics leads to failure to properly localize determinants and thus results in embryonic defects. Thus, we infer that sperm astral MTs must reach the cell cortex for proper pronuclear fusion, centration, normal cleavage, and localization of determinants. Molecular modification of the G-protein regulator, AGS, contributes to asymmetric cell division in sea urchins 04/06/17 10:30 AM - 11:00 AM Yajima, Mamiko (Brown University, Providence, RI, USA) The sea urchin micromeres, formed at the 4th cleavage, function as organizers to induce endomesodermal fates in adjacent cells. This classically defined inductive interaction has been defined experimentally and is engrained in textbooks, but the mechanism responsible for formation of this important lineage remains enigmatic. Here we report the discovery of a key functional mechanism in the asymmetric cell division of the embryo leading to the fate of micromeres. We found that an evolutionarily conserved cortical protein complex (Gαi-AGS-NuMA-Dynein, GAND) is stabilized at the vegetal cortex by reduction of PLK1 activity from the vegetal side of the spindle. This leads to asymmetric segregation of molecules. AGS appears to be dominant in this process: Its overexpression at the cortex resulted in ectopic recruitment and anchoring of spindle poles to the cortex of every blastomere and even induced asymmetric cell divisions during early embryogenesis in a sea star embryo, another echinoderm, which normally undergoes symmetric cell divisions. AGS mis-expression in the sea star also led to additional tissue invaginations. These results suggest that sea urchin AGS is essential for its asymmetric cell divisions and micromere formation. Taking these and other results together, we propose that the modifications of AGS and formation of a unique GAND complex was key in the evolutionary transition to an asymmetric cell division, which has led to the current developmental style of sea urchins with organizing centers and early cell fate specifications not seen in other echinoderms. We believe the findings in this study could explain a fundamental piece in how the asymmetric cell divisions are introduced into the developmental program, and which contributes to species diversification during animal evolution. 4

Talk Dissecting Mechanotransduction in Real Time: How do Cells Respond to Changes in their Physical Environment 04/06/17 11:30 AM - 12:00 PM De Tomaso, Tony (UC Santa Barbara, Santa Barbara, CA, USA) We have been developing a new model to study the interactions of cells and their physical environment: the extracorporeal vasculature of the colonial ascidian, Botryllus schlosseri. Botryllus is a powerful model for these studies as it has an extracorporeal vasculature consisting of large blood vessels (ca. 0.25 mm in diameter) ramifying over tens of square centimeters. The vessels are sessile, completely transparent and can be touched with a pipette tip. We have recently developed a vascular lineage tracing mechanism such that we can live image and isolate pure populations of vascular cells by FACS. During studies on vascular regeneration, we found that we could manipulate the stiffness of the ECM in vivo via inhibition of lysyl oxidase (LOX) activity. This induced anoikis in a subset of vascular cells; causing the vessels to regress as apoptotic cells were selectively removed from the vessel wall, which could be easily visualized and took ca. 20 hours. This occurred without loss of barrier function, and was reversible. We have found this is due to changes integrin binding pathways, and regression can also be induced by pharmacological inhibition of different signal transduction molecules, including FAK, ILK, and PI3K. These vessels are also large enough to directly physically manipulate, and we are in the process of doing so now. In summary, we are developing a new model that will allow us to study mechanotransduction pathways in vivo, characterize both genetic and proteomic changes from a purified population of cells, and have an overnite visual assay for anoikis and epithelial homeostasis. Current results will be discussed. Nanos is super important and very cool 04/06/17 01:30 PM - 02:00 PM Oulhen, Nathalie (Brown University, Providence, RI, USA); Swartz, S. Zachary (USA); Wang, Lingyu (USA); Wikramanayake, Athula (USA); Wessel, Gary M. (USA) Nanos is a translational regulator required for the survival and maintenance of primordial germ cells (PGCs) in all species where it has been tested. In the sea urchin Strongylocentrotus purpuratus (Sp), we found recently that Nanos2 is essential also to maintain the translational quiescence of the PGCs. To function specifically in the PGCs, we have learned that Nanos2 is tightly regulated post-transcriptionally. Mechanisms of RNA and protein stability, as well as the translation of its protein, are particularly evident in this specific PGC function. However, the mechanisms by which the Nanos gene is transcriptionally regulated remain elusive. Nanos2 is not maternally supplied but rather embryonically activated in the PGCs shortly after their formation. We report here that activation of Nanos2 requires the maternal Wnt pathway. Beta-catenin is highly nuclearized in the sea urchin PGCs and we found that the earliest expression of Nanos2 in the PGCs requires maternally activated beta-catenin. Using Chromatin ImmunoPrecipitation (ChIP), we identified a region in the promotor of Nanos2 that is recognized by the beta-catenin complex. Surprisingly, Nanos2 mrna accumulation expands into adjacent somatic mesodermal cells during gastrulation. This secondary Nanos expression requires Delta/Notch signaling, the Wnt pathway, as well as the forkhead transcription factor FoxY. Altogether, these data provide new insights into the multiples pathways and dynamics involved in the critical PGC determinant, Nanos2, for the germ line, and for their closely associated somatic cells. Ancient functions in new contexts: the role of Kr ppel-like factor genes in the ctenophore Mnemiopsis 04/06/17 02:00 PM - 02:30 PM Browne, William E. (University of Miami, Coral Gables, FL, USA) In bilaterians the Krüppel-like factor (Klf) transcription factor genes play critical roles in stem cell maintenance and balancing aspects of both cell proliferation and differentiation. Information regarding Klf expression and function are lacking in nonbilaterian lineages. We have examined the molecular function of Klf orthologs in the non-bilaterian lobate ctenophore Mnemiopsis leidyi using a combination of developmental expression analyses, immunohistochemistry, cell proliferation assays and targeted gene knockdown via two independent methods. The Mnemiopsis genome contains three Klf genes; MleKlf5a, MleKlf5b, and MleKlfX. MleKLF5a and MleKLF5b are closely related to bilaterian KLF5 and KLF4, which have been shown to play an important role in the maintenance of embryonic stem cell pluripotency as well as having competing roles in vertebrate gut epithelium proliferation and differentiation. In marked contrast MleKLFX has no clear orthology to any other metazoan KLF. Here we show that MleKlf5a and MleKlf5b are expressed in tissues and organs derived from endoderm whereas MleKlfX expression is restricted to late embryonic development in presumptive neural cells located in the ctenophore apical organ. Removing zygotic MleKLF5a and MleKLF5b function results in extensive endodermal defects associated with stem cell niches and gut patterning. Our results suggest a phylogenetically ancient role for Klf transcription factor genes in mediating aspects of transcriptional regulation associated with the maintenance of proliferation in endodermal stem cell niches and gut patterning in metazoans. 5

Talk Development and regeneration in the brittle star A. filiformis 04/06/17 02:30 PM - 03:00 PM Oliveri, Paola (University College London, London, GBR); Czarkwiani, Anna (USA); Dylus, David (USA); Piovani, Laura (USA); Sugni, Michela (University of Milano, USA) The ability to regenerate is represented throughout the animal kingdom, however the highly variable phylogenetic distribution supports the notion that regeneration might have arisen independently several times as a reactivation of developmental program. Ophiuroidea class of echinoderms provides an ideal system to address how much of the developmental program is in common between embryonic development ad adult regeneration. We use the species Amphiura filiformis, which develops in to a pluteus larva and also rapidly regenerates its arms, to compare at cellular and molecular level the process of skeleton formation. Combining classical embryological and histological data with transcriptome and large-scale expression data, we explored the conservation of regulatory states and molecular signatures of larval and regenerating arm skeletogenic cells. Furthermore, inhibiting signaling pathways during those two developmental stages revealed a conservation of a cohort of genes affected by SU5402. Taken together our result show a large conservation of the developmental programs of these two processes. Regeneration studies in two echinoderms, feather star and sea cucumber 04/06/17 03:30 PM - 04:00 PM Majic, Paco (MMBS, The Univ. of Tokyo, USA); Okada, Akari (MMBS, The Univ. of Tokyo, USA); Kensuke, Takatani (MMBS, The Univ. of Tokyo, USA); Omori, Akihito (MMBS, The Univ. of Tokyo, USA); Kondo, Mariko (The Univ. of Tokyo, Miura, Kanagawa, JPN) All five classes of echinoderms possess outstanding potentials for regeneration. Class Crinoidea (sea lilies and feather stars) often exhibits arm regeneration following self-amputation or damages caused by external factors. Stimulation causing evisceration leads Class Holothuroidea (sea cucumbers) to completely regenerate their intestine. Here, we studied the arm regeneration of a feather star Oxycomanthus japonicus and gut regeneration of a sea cucumber Apostichopus japonicus from a molecular perspective. For arm regeneration, we examined the expression patterns of genes associated to the maintenance of stem cells, dedifferentiation, positional identity and differentiation of cells during sequential stages, to obtain an overview of the relevance of the different cellular processes during regeneration. The results have shown that (1) vasa and members of the Piwi family, typically associated to the maintenance of stemness in the germ line, are expressed in the blastema and the tip of the regenerate, (2) two homologs to the vertebrate Yamanaka factors, soxb1 and klf1/2/4, are expressed in a manner suggesting they may be playing a role in the regenerative process, (3) some of the hox genes are differentially expressed in the regenerate and blastema, implying their involvement in the restitution of the positional identity, (4) the potentiality to become bone-forming cells is defined very early in regeneration, and (5) elav, generally considered to be a neural marker or related to multipotency in echinoderms, is expressed in the regenerating nerve cord and ectodermal tissues. As for the sea cucumber, since the intestines regenerate from both the anterior and posterior ends, we have cloned ParaHox genes and compared expression of these genes in the intact intestine and regenerating tissues, to see if these genes may reflect positional identities. Taken together, we consider these results to complement histological and microscopic observations and set a more fundamental base for studying regeneration. Evolving roles of Arp2/3 actin networks over the course of early development 04/06/17 03:30 PM - 04:00 PM Ellis, Andrea (New Mexico State University, USA); Sepulveda, Silvia (New Mexico State University, USA); Toledo, Leslie (New Mexico State University, USA); Salgado, Torey (New Mexico State University, USA); Henson, John (Dickinson College, USA); Shuster, Charles b. (New Mexico State University, Las Cruces, NM, USA) In nonmuscle cells, actin is organized into either branched or linear networks, whose polymerization is mediated by the Arp2/3 and formin families of actin nucleators, respectively. Arp2/3 acts downstream of Rho family GTPases to form branched, dendritic networks of actin that are crucial for lamellapodial cell protrusions. But while the role of Arp2/3 in twodimensional motility is well characterized, less is known about the role of Arp2/3 during early development. During the early cleavages, Arp2/3 is recruited to the cortical cytoskeleton, where it is cleared from the cleavage furrow during cytokinesis. This removal of branched actin networks appears to be critical for embryonic cleavage, as misregulation of Rac/Cdc42 signaling blocks cytokinesis in an Arp2/3-dependent manner. By the 16-cell stage, Arp2/3 localization becomes restricted to the apical membrane, and inhibition of Arp2/3 during the early cleavages results in epithelialization defects and formation of a blastula. During gastrulation, Arp2/3 depletion or inhibition had effects on both collective and individual cell migration. Primary Mesenchymal Cell (PMC) ingression is delayed in the absence of Arp2/3, and both filopodial extension and skeletogenesis are affected. If Arp2/3 is blocked after the tri-radiate spicule has formed, filopodia fail to form but the cytoplasmic sheath enveloping the spicule is maintained. During gut formation, loss of Arp2/3 resulted both in delays in primary invagination but also in convergent extension of the archenteron. Together, these results suggest that while Arp2/3 is dispensable for early cell shape changes such as cytokinesis, Arp2/3 is essential for the spherical-to-epithelial transition of early blastomeres as well as morphogenesis. 6

Talk Cell reprogramming in the urchin embryo after PMC removal and embryo bisection 04/06/17 04:00 PM - 04:20 PM Allen, Raymond L. (Duke University, Durham, NC, USA); Reardon, Riley (Duke University, USA); McClay, David (Duke University, USA) The embryos of sea urchins display a remarkable plasticity in the ability to replace missing cells or tissues. Removal of the skeleton-forming primary mesenchymal cells (PMCs) at the mesenchyme blastula stage leads to reprogramming a subpopulation of secondary mesenchymal cells (SMCs) to replace the missing skeletal cells. Transcription factors present in the PMC gene regulatory network (GRN) were shown to be ectopically expressed in reprogramming SMCs alongside SMC specific factors. This current work in Lytechinus variegatus further examined PMC GRN factors expressed in reprogramming SMCs during PMC removal. Concurrently, embryonic plasticity was investigated during multiple tissue type loss at the mesenchyme blastula stage by full horizontal and vertical bisections of the embryo. Using DIC and immunostaining, select embryo halves that were already under regulative development, are able to recover and develop into a swimming pluteus. Casein Kinase 1 delta/epsilon mediates anterior-posterior axis formation in the sea urchin embryo, potentially through localized activation of Disheveled 04/06/17 04:00 PM - 04:20 PM Wu, Wei (University of Miami, Coral Gables, FL, USA); Wang, Lingyu (Duke University, Durham, NC, USA); Smith, Lauren (Department of Biology, University of Miami, USA); Wikramanayake, Athula H. (University of Miami, Miami, FL, USA) Wnt signaling plays a central role in establishing anterior-posterior (AP) polarity in metazoan embryos. A key cytoplasmic component mediating Wnt signaling is the Disheveled (Dvl) protein, which is generally considered to be the central "hub" of the Wnt signaling pathway. In the sea urchin, Dvl is highly enriched and differentially post-translationally modified in a specialized vegetal cortical domain (VCD) of the egg, and the vegetal blastomeres that inherit the VCD during embryogenesis. Functional analysis has shown that localized Dvl activity mediates canonical Wnt signaling in vegetal blastomeres, but the molecular basis of Dvl asymmetric localization and activation remain unresolved. Therefore, identification and functional characterization of proteins interacting with Dvl (DIPs) in the VCD will help us better understand how Dvl partners regulate Dvl activity and Wnt signaling. By applying Dvl Co-immunoprecipitation coupled with mass spectrometry we have identified several potential Dvl-interacting-proteins (DIPs) from isolated egg cortices and 16-cell-stage micromeres. Casein Kinase 1 δ/ε (CK1δ/ε), one of our newly identified DIP candidates, is highly enriched and co-localized with Dvl at the vegetal pole of the sea urchin embryo. Downregulation of CK1δ/ε by overexpressing a dominant-negative form of CK1δ/ε resulted in severe downregulation of genes expressed in the endomesoderm and the anteriorization of embryos. Furthermore, overexpression of CK1δ/ε by injecting synthesized CK1δ/ε mrna into fertilized eggs induced upregulation of endomesoderm genes and posteriorization of embryos. Intriguingly, we found that co-overexpressing CK1δ/ε and Dvl induces a more severe posteriorized phenotype than when overexpressing CK1δ/ε alone, suggesting that CK1δ/ε synergizes with Dvl to positively regulate Wnt signaling. This hypothesis was further supported by the observation that the expression levels of endomesoderm genes were significantly higher in CK1δ/ε-Dvl co-overexpressed embryos compared to expression levels of endomesoderm genes in embryos overexpressing Dvl or CK1δ/ε only. This work establishes CK1δ/ε as a critical regulator of Dvl activation and AP axis specification in sea urchin embryos. Identifying embryonic mechanisms that induce a germ cell fate in sea stars 04/06/17 04:20 PM - 04:40 PM Fresques, Tara (Brown University, USA); Wessel, Gary (Brown University, USA) Germ cell specification is required for reproduction in all sexually reproducing animals. During animal development, animals can specify their germ cells through inheritance of maternal molecules or through induction by cell-cell signaling events. Although the ancestral mode of germ cell specification appears to occur by inductive mechanisms it is still not clear how signals selectively induce a germ cell fate. I use the sea star as a model to determine how signaling mechanisms direct germ cell formation. First I use RNA in situ hybridizations to identify when genes associated with germ cell formation are expressed during sea star embryogenesis. I find that the conserved germ cell factors Nanos, Vasa, and Piwi all localize in a germ cell pouch at the larva stage. In addition, Nanos and Vasa mrna appears to be serially restricted into smaller and smaller embryonic domains during early embryogenesis. One of these restriction events involves Left/Right asymmetry because the germ cell pouch forms on the left side of the embryo. Since Nodal is conserved and required for specification of the Left/Right axis I use a gene perturbation strategy, primarily by injecting a translation blocking morpholino into sea star oocytes, to determine the role that the Nodal signal has on germ cell specification. My results show that Nodal inhibits retention of Nanos and Vasa mrna in the ventral and right sides of the embryo by inhibiting transcription and by stimulating RNA degradation. In addition, my work suggests that Nodal also inhibits cell morphogenetic events in the ventral and right sides of the embryo required for germ cell pouch morphogenesis. Finally, I use RNA in situ hybridizations to determine when Nanos accumulates in diverse Echinoderm species to determine how germ cell specification mechanisms have changed during evolution. 7

Talk V-type H+ ATPase activity is required for dorsal-ventral symmetry breaking in sea urchin embryos 04/06/17 04:20 PM - 04:40 PM Schatzberg, Daphne (Boston University, Boston, MA, USA); Reidy, Patrick (Boston University, USA); Hadyniak, Sarah (Boston University, USA); Lawton, Matthew (Boston University, USA); Dojer, Brielle (Boston University, USA); Kitchloo, Shweta (Boston University, USA); Bradham, Cynthia (Boston University, USA) Bioelectricity in the form of differential membrane potential and intracellular ion concentrations encodes large-scale polarity in developing embryos. Sea urchin dorsal-ventral (DV) axis specification is initiated by Nodal signaling. We previously showed that the asymmetric initiation of Nodal expression occurs downstream from p38 MAPK, which itself is transiently asymmetrically active and is required for DV polarity. Here, we describe distinct voltage and ph gradients along the DV axis of developing sea urchin embryos. We show that the DV voltage and ph gradients are flattened by V-type H+ ATPase (VHA) inhibition, which results in ventralization of the ectoderm. This effect is rescued by experimentally enforcing Nodal signaling asymmetry. VHA-inhibited embryos maintain globally active p38 MAPK, and subsequently initiate Nodal expression globally. Together, these results suggest a model in which VHA-dependent bioelectrical gradients relay the maternal mitochondrial asymmetry to the transient asymmetry of active p38 MAPK, and indicate that VHA activity is required for DV symmetry breaking in sea urchin embryos. Characterization of Apical Pole Domain Associated Gene Regulatory Networks during Sea Star Larval Regeneration 04/06/17 04:40 PM - 05:00 PM Zheng, Minyan (Carnegie Mellon University, Pittsburgh, PA, USA); Cary, Greg (Carnegie Mellon University, USA); Wolff, Andrew (Carnegie Mellon University, USA); Hinman, Veronica (Carnegie Mellon University, USA) Sea star embryos are an ideal model system to study developmental gene regulatory networks (GRNs). We now seek to extend on the well-studied GRNs and establish the starfish Patiria miniata as a model to study the GRNs controlling regeneration. Starfish larvae have extraordinary regenerative capacity and can grow back their anterior structures, including the serotonergic ganglia within 1 week post decapitation. An important, open question is how and whether developmental GRNs are recapitulated for the reformation of structures during regeneration. To understand whether the neural regenerative processes recapitulate the developmental paradigm, here we examine the expression of transcription factors and signaling molecules that regulate neurogenesis at different time points over the course of regeneration. We also characterized cell proliferation profile of the regenerating larvae to better probe the regenerative mechanisms. p38 MAPK as an essential regulator of D/V axis specification and skeletogenesis during sea urchin development: a re-evaluation 04/06/17 04:40 PM - 05:00 PM Molina Jimenez, Maria Dolores (Institut Biologie Valrose, Nice, FRA); Quirin, Magali (USA); Haillot, Emmanuel (USA); Hernandez, Felipe (USA); Chessel, Aline (USA); Lepage, Thierry (USA) In the sea urchin, the dorsal-ventral axis is specified after fertilization by the asymmetrical expression of the TGF-beta Nodal. Nodal expression is initiated at the 16-32 cell stage and is rapidly restricted to an ectodermal domain of the early blastula. The regionalization of nodal expression is the first zygotic molecular asymmetry associated with specification of the D/V axis. Understanding how is regulated is therefore essential to understand how the D/V axis is specified. The p38 MAPK pathway has been proposed to be essential for the initiation of nodal expression (Bradham and McClay, 2006). Embryos treated with the pharmacological inhibitor of p38 SB203580 lacked expression of nodal and of its downstream target genes in the ventral ectoderm. However, SB203580 apparently did not prevent the induction of Nodal target genes in nodal overexpressing embryos suggesting that p38 functions upstream of nodal expression. Although no transcription factor acting downstream of p38 has been yet identified, an early expressed homeobox gene orthologous to the PMC lineage regulatory gene pmar has been recently proposed to act upstream of p38, as a negative spatial modulator of p38 activity and as a repressor of nodal expression (Cavalieri and Spinelli, 2014). We have revisited the role of p38 in D/V axis and in the regulation of nodal expression using a combination of functional and biochemical experiments. We discovered that p38 inhibitors strongly disrupted specification of all germ layers by blocking signalling from the Nodal receptor and by deregulating the ERK pathway. Therefore, our data invites to reconsider the role of p38 upstream of skeletogenesis and of nodal expression, and to reinterpret the results of recent studies linking the spatial regulation of p38 to transcriptional repressors of the Hbox12/pMar family and to nodal expression. 8