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
Developmental Biology of the Sea Urchin XXIV, The Marine Biological Laboratory, Woods Hole, MA April 5-9, 2017
Wednesday, April 5
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 post- gastrula 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 one- sided 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 pro- nucleus. 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 non- bilaterian 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 two- dimensional 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 Talk A shift from normal to regeneration-specific proliferation in the sea star Patiria miniata 04/06/17 05:00 PM - 05:20 PM Wolff, Andrew (Carnegie Mellon University, Pittsburgh, PA, USA); Hinman, Veronica (Carnegie Mellon University, USA)
The production of new cells through proliferation has been observed during regeneration in many organisms. Cellular proliferation produces the cells necessary for the replacement of tissues lost due to injury or amputation. Regeneration requiring proliferation is often associated with the formation of a blastema. However, there is a distinction in the source of proliferation during regeneration in various model organisms, and the relative contribution of proliferative cells remains unresolved. In this study, we examine the role of cellular proliferation during whole-body regeneration of a larval sea star Patiria miniata. Labeling of proliferating cells using EdU identified distinct patterns of proliferation at various time points during regeneration. Early proliferation patterns resemble those in normal, uncut larvae, whereas at later time points there is a concentration of proliferating cells adjacent to the cut site, indicative of blastema formation. Pulse-chase labeling of cells suggests that blastemal proliferation provides the cells necessary for the re-formation of lost tissues, as labeled cells are found throughout regenerated structures. Labeling proliferative cells before and during blastema formation results in very little overlap in these two populations, pointing to the presence of proliferation that is specific to the regeneration context. Lastly, perturbation of Wnt signaling results in a reduction of proliferation, implicating this pathway in regeneration. These results show that during sea star regeneration, proliferation is distinct from normal growth and that this proliferation drives tissue reformation during this process.
Characterization of ciliary band neurons expressing pancreatic transcriptional factors 04/06/17 05:00 PM - 05:20 PM Perillo, Margherita (Boston College, chestnut hill, MA, USA); Arnone, Maria Ina (Stazione Zoologica Anton Dohrn, USA)
Neurons and insulin-producing B-cells share common features, including developmental programs and signature genes. The evolutionary origin of these two cell types, which do not have an obvious common developmental origin, is still unknown. Pancreatic-like hormones are mostly produced by neurons in protostomes. We have previously described that gut cells in the sea urchin Strongylocentrotus purpuratus produce an insulin-like peptide. We hypothesize that neurons and pancreatic endocrine cells share a common gene toolkit. The fact that the sea urchin represents the earliest branched deuterostome where a pancreatic hormone is produced by endodermal cells, and not by neurons, makes it an interesting model organism to investigate the features shared by these two cell-types. Our lab identified ventro-lateral clusters of cells below the ciliated band that co-express two transcription factors with known roles in both nervous system and pancreatic endocrine cell development: SpBrn1/2/4 and SpLox (Pdx1). We found that the SpLox/SpBrn1/2/4+ cells are lateral ganglion cells that express neuroendocrine markers. Moreover, these cells produce the sea urchin-specific neuropeptide SpPnp5, which is expressed also in other neurons of the apical organ and ciliated band. To further characterize these cells, we looked at the expression of other pancreatic markers and found that only Islet1 is co-expressed in one of the SpLox/SpBrn1/2/4+. Moreover, we found that, when SpLox is knocked-down, the neuropeptide disappears from one or two apical organ cells. In agreement with previously reported results, we hypothesize that the larval nervous system is a complex network, where perturbation of one cell affects the others. In conclusion, we defined new ectodermal cells expressing pancreatic transcriptional factors, which function should be further investigate.
The emerging Wnt signaling network that governs anterior-posterior neuroectoderm patterning in the sea urchin embryo 04/06/17 07:30 PM - 08:00 PM Range, Ryan C. (Mississippi State University, Mississippi State, MS, USA)
Studies in several deuterostome developmental models, including the sea urchin, suggest that an early, broad regulatory state initiates specification of the anterior neuroectoderm (ANE). During early development, a posterior-to-anterior wave of inductive signaling progressively positions this broad ANE potential along the anterior-posterior (AP) axis to a territory around the anterior pole. However, the molecular mechanisms used during this ANE restriction process are incompletely understood in any deuterostome embryo. Our recent results indicate that the ANE restriction mechanism in the sea urchin embryo involves integration of information from the Wnt/β-catenin, Wnt/JNK, and Wnt/PKC pathways. We have also found that secreted Wnt signaling modulators at the anterior pole act as a signaling center that is integrated into this Wnt network and establishes the ANE boundary and subsequently patterns the ANE territory. These studies provide the framework for our current focus on functional characterization of several transcription factors identified in whole-transcriptome differential screens whose expression is driven by the Fzl5/8 (Wnt/JNK) and Fzl1/2/7 (Wnt/PKC) signaling pathways, looking for functional interactions at the transcriptional level during ANE positioning. In addition, we are performing functional analyses on several potential extracellular and intracellular modulators of Wnt signaling in an effort to determine potential roles for these factors in the ANE restriction mechanism. Importantly, evidence from functional and expression studies in other species, including chordate embryos, strongly suggests that aspects of this fundamental developmental mechanism are conserved in deuterostome embryos. These efforts are the first steps in an our strategy to use a combination of high throughput genome-wide assays, molecular manipulations, and gene regulatory network analysis to produce a systems- level model of how a Wnt network governs a fundamental deuterostome developmental process.
9 Talk
Patterning Neurogenesis 04/06/17 08:00 PM - 08:30 PM Burke, Robert D. (University of Victoria, Victoria, BC, CAN)
At gastrulation neural progenitors enter S-phase coincident with expression of SoxC. We used a BAC (bacterial artificial chromosome) containing GFP knocked into the Sp-SoxC locus to label neural progenitors. Live imaging, and immunolocalizations indicate that Sp-SoxC expressing cells divide producing pairs of adjacent cells expressing GFP. Over an interval of about 6 h one cell fragments and undergoes apoptosis. Fragmenting cells contain high levels of activated Caspase3. A Notch reporter indicates that cells expressing Sp-SoxC activate Notch signaling in adjacent cells. Inhibition of g -secretase, injection of Sp-Delta morpholinos, or CRISPR/Cas9 mutation of Sp-Delta, results in supernumerary neural progenitors and neurons. Interfering with Notch signaling increases neural progenitor recruitment and pairs of neural progenitors. Thus, Notch signaling patterns neurogenesis by restricts the number of neural progenitors recruited and regulating the fate of progeny of the asymmetric division. We propose a model in which localized signaling converts ectodermal and ciliary band cells to neural progenitors that divide asymmetrically to produce a neural precursor and an apoptotic cell.
Eyes without the master: photoreceptor specification in the sea urchin larvae is independent of the retinal determination network. 04/06/17 08:30 PM - 09:00 PM Feuda, Roberto (California Institute of Technology, California, CA, USA); Valencia, Jon (California Institute of Technology, USA); Mollett, Dan (University of Victoria, USA); Burke, Robert (University of Victoria, USA); Peter, Isabelle (California Institute of Technology, USA)
The ability to respond to visual stimuli is crucial for every aspect of animal life. This process in the majority of metazoan is mediated by photoreceptors cells (PRCs) a specialized type neurons that expressing opsins. There are two main types of PRCs, the ciliary and the rhabdomeric the first are usually involved in the visual process in deuterostomes while the latter perform the same function in protostomes. It was shown recently that Strongylocentrotus purpuratus possess rhabdomeric PRCs in the tube feet.
In this work, we identified in a sea urchin larvae two clusters of bilaterally arranged cells expressing the transcription factor rx that is associated with PRCs. We show that rx+ cells express an RGR/Go opsin (opsin3.2), that opsins3.2 is expressed in neuronal cells and that these cells have the molecular signature of ciliary photoreceptors. Furthermore, PRCs are nearby pigments cells suggesting that probably they can discriminate light directionality. Next, analyzed the PRCs regulatory state, and we identified nine transcription factors that are expressed in a spatial arrangement similar to rx-opsin3.2+ cells. We performed a spatial analysis of the expression pattern of the nine transcription factors during PRCs development to identify the origin of the rx+ cells. The results suggest that rx+ cells most likely originate from the apical plate domain. Finally, we analyzed the spatial expression the retinal determination gene network (RDN, i.e. Pax6-Six1/2-Eya-Dach) which is widely conserved in the specification of PRCs in Bilateria. Surprisingly we identify that RDN is expressed neither in differentiated PRCs nor during their development. However, the RDN is expressed in hydropore canal an echinoderm specific morphological structure with osmotic function.
The result of this work indicates that the S. purpuratus genome encodes the regulatory program for specifying both ciliary and rhabdomeric PRCs, and more generally that the RDN is a dispensable component of a larger gene regulatory network (GRN) for PRCs developmental specification. Finally, the implication for the evolution of PRCs GRN will be discussed.
10 Talk Morphogenesis along the animal-vegetal axis: fates of primary quartet micromere daughters in the gastropod Crepidula fornicata 04/07/17 08:30 AM - 09:00 AM Lyons, Deirdre (Scripps Institution of Oceanography, USA); Henry, Jonathan (University of Illinois Urbana-Champaign, USA); Perry, Kimberly (University of Illinois Urbana-Champaign, USA)
The Spiralia are a large and morphologically diverse group of protostomes (e.g. molluscs, annelids, nemerteans) that share a homologous mode of early development called spiral cleavage. One of the most highly conserved features of spiralian development is the contribution of the primary quartet micromere cells, 1a-1d, to the anterior region of the embryo including the brain, eyes, and anterior ciliary band, called the prototroch. Yet, very few studies have followed primary quartet sub- lineages or examined the morphogenetic events that take place in the anterior region of the embryo. This study focuses on the caenogastropod slipper snail Crepidula fornicata, a model for molluscan developmental biology. Through direct lineage tracing of primary quartet daughter cells and examination of these cell during gastrulation and organogenesis stages, we uncovered behaviors never described before. For the first time we show that the 1a2-1d2 cells do not contribute to the prototroch, as they do in other species, and are lost before hatching. Instead, these cells appear to act as provisional epidermal cells on the dorsal side of the embryo during gastrulation and elongation stages. In the process of studying these cells and their sister cells, 1a1-1d1, we also observed an extensive re-arrangement of the pretrochal region during gastrulation stages. We speculate that the function of 1a2-1d2 cells is to serve as a provisional epithelium that allows for anterior displacement of the other progeny of the primary quartet towards the anterior-ventral side of the embryo. These data challenge earlier models suggesting that axial bending may be driven by differential proliferation of posterior dorsal cells, and suggest that examining sublineages in other spiralians might reveal greater variation than previously assumed.
Echinoderm GRNs are going omics: insight into the evolution of gut patterning. 04/07/17 09:00 AM - 09:30 AM Arnone, M. Ina (Stazione Zoologica Anton Dohrn, Naples, ITA)
Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN. As technology progresses, so do the methods used to address different biological questions. Next generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein- DNA interactions and DNA accessibility. We are using an approach integrating multiple NGS applications, including ATAC- Seq, for the prediction and validation of gene interactions within the GRNs governing gut patterning in two echinoderms, the sea urchin Strongylocentrotus purpuratus and the sea star Patiria miniata. An important novel node of the sea urchin gut GRN featuring the interaction between the Sp-Lox protein and the homeobox gene Sp-Meis has been revealed. The genome wide comparison of the differentially expressed genes after either Xlox or Cdx perturbation in sea urchin and sea star embryos has revealed that, although the absence of these two proteins affects similar digestive functions and developmental processes related to gut specification in each species, many of the regulatory genes involved in these mechanisms are rewired in different networks.
Establishment of body axes in the annelid Capitella teleta 04/07/17 10:30 AM - 11:00 AM Seaver, Elaine (University of Florida Whitney Marine Lab, Saint Augustine, FL, USA)
The establishment of the body axes during embryogenesis is often achieved as the result of signals from organizing centers. Localized to either an individual cell or group of cells, embryonic organizing activity induces the specification of other cells in the embryo as well as influencing the formation of the body axes. In the spiralian Capitella teleta, cell deletion studies show that organizing activity is localized to a single cell, 2d, and this cell induces the formation of the dorsal-ventral and left-right axes. Using various small chemical inhibitors, we attempt to identify the signaling pathway responsible for the organizing activity of 2d. Embryos at stages when organizing activity is occurring were exposed to inhibitors, raised to larval stages, and scored for axial anomalies analogous to previously described phenotypes. Our results suggest that the MAPK, Notch, BMP, and Wnt/β-catenin signaling pathways do not play a role in specification of the dorsal-ventral axis or bilateral symmetry through 2d. However, interference with the TGF beta signaling pathway, through a short 3 hour exposure to the inhibitor SB431542, results in larvae that lack bilateral symmetry and a detectable dorsal-ventral axis. These investigations shed light on the identity of the 2d signaling pathway involved with Capitella axes formation, and contribute to our understanding of how changes in developmental programs lead to the evolution of body plans.
11 Talk Assessing the relationship between structure and function in developmental circuits 04/07/17 11:00 AM - 11:30 AM Peter, Isabelle (Caltech, Pasadena, CA, USA)
The gene regulatory network for sea urchin endomesoderm specification is experimentally well resolved, and a recent Boolean model demonstrated that this network contains sufficient information to causally explain the observed gene expression patterns during early development. The relative completeness of this model now permits the evaluation of specific regulatory features that determine the developmental function of this network. At least six different types of subcircuit occur in the endomesoderm network, including positive feedback, coherent feedforward, and mutual repression circuits. Using Boolean modeling I analyze the relationship between the specific architecture of the subcircuit and its developmental outcome. Computational perturbation experiments show that circuit function is not only determined by the structure of regulatory interactions but also by the regulatory logic by which regulatory inputs are interpreted at individual nodes. Thus in the presence of multiple activating inputs, the effect of exchanging AND for OR logic at an individual node can be as prominent as changing the structure of the entire circuit. Furthermore, as indicated by recent cis-regulatory analyses of key regulatory genes in the endomesoderm GRN, Wnt and Delta/Notch signaling inputs can have complex regulatory functions, and a Boolean model of signaling circuits supports the idea that signaling interactions operate by de- repression rather than by activation of gene expression. An analysis of the organization of different types of subcircuits within the endomesoderm GRN shows that subcircuits and signaling interactions frequently overlap, such that individual nodes and interactions may contribute to multiple network circuits. This analysis reveals the function of individual regulatory interactions not only on the expression of immediate target genes but also on the operation of subcircuits or even entire developmental networks they pertain.
Troponin system in the sea urchin embryos 04/07/17 11:30 AM - 12:00 PM Yaguchi, Shunsuke (University of Tsukuba, USA); Yaguchi, Junko (University of Tsukuba, USA); Tanaka, Hiroyuki (Hokkaido University, USA)
The troponin complex, composed of Troponin-I, Troponin-T and Troponin-C, is an essential mediator of the contraction of striated muscle downstream of calcium signaling in almost all bilaterians. However, in echinoderms and hemichordates, collectively termed Ambulacraria, the components of the troponin complex have never been isolated, thus suggesting that these organisms lost the troponin system during evolution. Here, by analyzing genomic information from sea urchins, we identify the troponin-I gene and isolate its complete mRNA sequence. Using this information, we reveal that the larval muscles express this gene and its translated product and that the protein is definitely a functional molecule expressed in sea urchin larvae by showing that troponin-I morphants are unable to swallow algae. We conclude that muscular contraction in all bilaterians universally depends on a regulatory system mediated by troponin-I, which emerged in the common ancestor of bilaterians. In addition, we will explain the function of troponin-I present at the non-muscular region of the embryos/larvae.
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 04/07/17 01:30 PM - 02:00 PM Quirin, Magali (Insitut Biologie Valrose, USA); Molina, Maria Dolores (Insitut Biologie Valrose, USA); Hernandez, Felipe (Institut Biologie Valrose, USA); Range, Ryan (University of Miami, USA); Lepage, Thierry (CNRS, Nice, FRA)
Specification of the dorsal-ventral axis of the sea urchin embryo critically relies on zygotic expression of nodal in the presumptive ventral ectoderm. nodal expression is initiated around the 16-32 cell-stage and is initially rather broad but it is rapidly restricted to the ventral ectoderm by mechanisms that are not completely understood. Dissecting how the spatial expression domain of nodal is established is therefore central to understand how the D/V is specified. Previous studies have shown that the nodal expression domain is shaped by short-range Nodal autoregulation and long-range inhibition by Lefty. Recently, the activity of the maternally expressed TGF beta ligand Panda was also shown to be required for the spatial restriction of nodal.
We previously reported that the maternal function of the ETS domain repressor Yan/Tel is critically required for the spatial restriction of nodal expression and that inhibition of translation of maternal yan/tel mRNA caused a massive ectopic expression of nodal starting from cleavage stages up to late in gastrulation, mimicking inactivation of Lefty. Conversely a non-phosphorylatable form of Yan/Tel acted as a hyperstable and constitutively active repressor of nodal expression.
We found that Yan cooperates and acts redundantly with Lefty and FoxQ2 to repress nodal expression in the animal pole domain and with Lefty to repress nodal expression in the vegetal pole region. In contrast, both Panda, Lefty and Yan/Tel are required non-redundantly to repress nodal in the dorsal ectoderm. Furthermore, removing the function of Tel rescues nodal expression and D/V patterning in embryos animalized by suppression of Vegetal/Wnt-beta Catenin signalling. We will present our recent progress on the identification of the kinases that control the stability of Yan/Tel and that allow this factor to integrate information from multiple signaling pathways and to operate with Lefty and Panda to establish the sharp boundaries of nodal expression. 12 Talk New Cues for Skeletal Patterning in Sea Urchin Embryos 04/07/17 02:00 PM - 02:30 PM Bradham, Cynthia A. (Boston University, Boston, MA, USA)
The mechanisms that underlie tissue patterning and morphogenesis remain central and challenging questions in developmental biology. The sea urchin larval skeleton serves as a simple model of patterning in which the ectoderm instructively communicates information to the primary mesenchyme cells (PMCs), which secrete the skeletal biomineral. Using a high throughput sequencing, we performed a screen to identify novel skeletal patterning cues that are expressed by the ectoderm. We discovered that sulfated proteoglycans (SPGs), 5-HETEs, the TGFß signal Univin, and BMP5-8 each serve as skeletal patterning cues, and that each cue is required for a distinct aspect of the skeletal pattern. SPGs accumulate ventrally, and are required for ventral PMC migration and ventral skeletal patterning. 5-HETEs are generated from apical and ventrolateral domains, and are required for midline patterning, consistent with 5-HETE exerting a repulsive effect on PMC migration. Zygotic BMP5-8 is required for left side patterning, and for normal left-right specification. Activity of the TGFß receptor Alk4/5/7 is required specifically for anterior PMC migration and skeletal patterning, while VEGFR activity is required for posterior secondary migration and patterning. Collectively, we’ve discovered conserved cues required for patterning most of the major skeletal regions; these results support a new model for ectodermally-mediated regulation of PMC migration, skeletal patterning, and morphogenesis.
VEGF activates a complex gene network with angiogenetic motifs to control sea urchin spiculogenesis 04/07/17 02:30 PM - 03:00 PM Roopin, Modi (The university of Haifa, USA); Morgulis, Miri (The University of Haifa, USA); Gildor, Tsvia (The University of Haifa, USA); Khalialy, Lama (The University of Haifa, USA); Ben-Tabou de-Leon, Smadar (The University of Haifa, Haifa, ISR)
Sea urchin larval skeletogenesis, particularly, Vascular Endothelial Growth Factor (VEGF) control of this process is an excellent system for deciphering how developmental gene regulatory networks control organogenesis and how they evolve to generate diverged body plans. Here we show that human VEGF is capable of inducing ectopic spicule branching in the sea urchin embryo. We discover that VEGF signaling is not necessary for calcium accumulation but essential for calcium deposition and the formation of the tubular cord in which the sea urchin skeletal spicules are formed. Our RNA-seq analysis reveals a network of biomineralization, regulatory and angiogenesis specific genes activated by VEGF signaling just before the spicules are formed. VEGF target genes are activated specifically at the skeletogenic clusters positioned at the closest vicinity to the ectodermal VEGF secreting cells, indicating a dose dependent transcriptional activation. Perturbation of VEGF target, rhopag24L/2, induces ectopic spicule branching in the sea urchin larva. The human homolog of rhogap24L/2 is highly expressed in endothelial cells where it inactivates RAC1; an essential factor for blood vessel branching. This study elucidates the cellular and molecular programs VEGF activates during sea urchin skeletogenesis. We propose that VEGF regulation of tube formation through the control of cytoskeleton rearrangement could have been the common evolutionary origin of sea urchin spiculogenesis and vertebrates angiogenesis.
Evolution of the Delta-HesC feedback mechanism driving mesodermal subtype specification in echinoderms 04/07/17 03:30 PM - 04:00 PM Cary, Greg (Carnegie Mellon University, Pittsburgh, PA, USA); McCauley, Brenna (USA); Huang, Katherine (USA); Shirai, Miwa (USA); Hinman, Veronica (Carnegie Mellon University, USA)
A central question in evolutionary developmental biology is how morphological differences between species are precipitated by alterations in developmental gene regulatory networks (dGRNs). In this study we focus on the dGRN changes leading to the formation of prominent coelomic pouches in sea star embryos compared with the specification program in sea urchin. In both embryos the segregation of mesodermal subtypes is governed by a highly conserved Delta-Notch-HesC regulatory motif. Sea urchin mesodermal territories are segregated by successive inductive Notch signaling events that establish and maintain territorial boundaries. First Delta expressing large micromeres, fated to become primary mesenchyme cells (PMC), are distinguished from non-skeletogenic mesenchyme (NSM). Later this pathway is used again to maintain the boundary between the NSM (now Delta+) and the small micromere descendants. In each case, the Delta repressor HesC is downregulated by the activity of a specific repressor (i.e. Pmar1 in PMCs and Blimp1 in NSM), which enables Delta expression in each territory. In sea star embryos we find evidence that the Delta-Notch-HesC motif is used to segregate mesoderm via a conserved lateral inhibition mechanism. We observe an early co-expression of Delta and HesC that is not resolved until mid-gastrula stage, at which point Delta+ cells are intercalated throughout the mesodermal bulb. Delta+ cells are fated to become mesenchyme and the intervening HesC+ cells contribute to the coeloms. We explain GRN alterations that permit the switch from inductive to lateral inhibition modes of Delta/Notch signaling. Finally, we examine the regulatory environment of the early coelom-fated mesoderm and find another conserved GRN motif in operation in both species. Thus, differences in the control of a conserved Delta-HesC regulatory motif in early development propagate changes that yield dramatic morphological differences while ultimately arriving at a highly similar regulatory environment in the early coelomic epithelium.
13 Talk Three populations of neurons in the sea urchin develop using gene regulatory networks highly conserved with vertebrates 04/07/17 04:00 PM - 04:20 PM Slota, Leslie (Duke University, Durham, NC, USA); McClay, David (Duke University, USA)
Conserved classes of transcription factors are used throughout metazoa to build diverse nervous systems. However, there are functional differences of some neural transcription factor orthologs between protostomes and deuterostomes, leaving their ancestral functions unknown. Here, we use a basally-branching deuterostome, the sea urchin Lytechinus variegatus, to determine how neurogenic transcription factors contribute to neurogenesis. We show that neurogenic gene regulatory network (GRN) components are conserved between echinoderms and vertebrates and distinct from known protostome GRNs involving those same genes. Like their role in vertebrates, Lv-achaete-scute and Lv-neurogenin are proneural, both being required for specification of different neuronal subtypes. Furthermore, we find that Lv-orthopedia is required for differentiation of the dopaminergic/cholinergic postoral neurons, a role strikingly similar to that in vertebrates. We propose that these GRN subcircuits are ancient components of the deuterostome lineage, possibly of all bilaterians, and were used by the common ancestor of chordates and ambulacrarians during neurogenesis.
SpMeis: a “novel” key factor shaping the gut of the sea urchin post-gastrula embryo 04/07/17 04:00 PM - 04:20 PM Cuomo, Claudia (Stazione Zoologica Anton Dohrn, napoli, ITA); Lowe, Elijah K. (Stazione Zoologica Anton Dohrn, USA); Arnone, Maria I. (Stazione Zoologica Anton Dohrn, USA)
SpMeis is a TALE homeodomain protein, which forms heterodimers and heterotrimers with different Hox protein to stabilize the homeoprotein-DNA complex. It is associated with several processes in Metazoa, such as the patterning and cell differentiation in the developing nervous system of vertebrates, Drosophila and C. elegans, the morphogenesis and specification of the mouse developing eye, and the developing of chicken limb and inner ear. In our differential transcriptomic data for sea urchin gut GRN reconstruction, SpMeis strongly decreases its expression after SpLox perturbation experiments, suggesting it as putative target of SpLox protein. Through the use of ATAC-seq we were able to identify a region of accessible chromatin in the first intron of SpMeis which contains the Xlox binding site, and the direct interaction between Xlox protein and SpMeis gene at this site has been demonstrated by ChIP-qPCR using an antibody against SpLox protein. Functional analysis experiments with SpMeis morpholino show a strong decrease of SpCdx transcripts and SpMeis itself, suggesting an autoregulatory mechanism of this gene and its role in SpCdx transcriptional regulation. Our results lead us to reveal a key module of the sea urchin gut specification GRN, involving a SpLox positive input on SpMeis gene that in turn activates SpCdx. The key role of SpMeis in the sea urchin gut patterning, compared to nervous system development in other deuterostomes and protostomes, suggests an evolutionary mechanism of co-option from the nervous system to endoderm formation occurred in the sea urchin lineage.
The Regulatory States in the Sea Urchin Larva 04/07/17 04:20 PM - 04:40 PM Valencia, Jon E. (Caltech, Pasadena, CA, USA); Peter, Isabelle (Caltech, USA)
The gene regulatory networks that direct the development of the sea urchin larva remain for the most part unknown. At 72 hours post fertilization, the sea urchin early pluteus larva is a fully functional organism with quite a variety of distinct cell types and organs. To uncover the number of larval cell types and to better understand how the different parts of the sea urchin larva form, we analyzed the spatial expression of 332 genes encoding transcription factors during the time-window of larva-genesis at five consecutive developmental time-points from the onset of gastrulation to 72 hours post fertilization. Initially, we bookmarked all transcription factor spatial gene expression into five major embryonic territories in order to keep track of and create a data-matrix of expression patterns. By comparing the spatial expression patterns, we identified at least 78 regulatory state domains, i.e., regions that express unique combinations of transcription factors, in the larva. Importantly, we have gleaned an incredible amount of insight into the biology of the developing larva from our data. We’ve identified novel cell types such as photoreceptor cells and other cell types expressing unique regulatory states in the apical plate. Additionally, we used the identified regulatory state domains as markers to create process diagrams in all the major territories of the larva that detail the partitioning and/or progression of these domains throughout developmental time. Lastly, the results have provided us an image repository as well as a searchable gene expression database that will soon become publicly available.
14 Talk Small micromere migration and a possible role for lysophosphatidic acid signaling in left/right asymmetry 04/07/17 04:20 PM - 04:40 PM Espinoza, Jose A. (University of California San Diego, San Diego, CA, USA); Morton-Curl, Sarah (University of California San Diego, USA); Nicklisch, Sascha (University of California San Diego, USA); Hamdoun, Amro (Scripps Institution of Oceanography, La Jolla, CA, USA)
The Small Micromeres (Smics) are the primordial germ cells of the sea urchin. Previously, our lab demonstrated that these cells migrate into the left and right coelomic pouches and defined the major events of Smic migration. To better understand this process we used high resolution, in vivo imaging of Smics labeled with fluorescent protein fusions of Sp-Vasa and Pleckstrin Homology Domain of PLC-δ between the mesenchyme blastulae and early prism stages. The results extend our understanding of the three key events in the migratory process, which include de-epithelialization of Smics, filopodial extension toward and contact with the oral animal ectoderm, and extension of filopodia towards the lateral ectoderm in the direction of Smic movement. Based on these observations and analogous processes in other animal embryos, we hypothesize that Smics use filopodia to respond to extracellular signals, most likely lipids, expressed during the active phase of migration. Extracellular lipid signals can act over multiple cell distances, but are also subject to oxidation and degradation by extracellular enzymes, which allows for control of these signals on very short time scales and precise spatial scales. Here we investigated a possible role for lipid signals by measuring Smic migration asymmetry after exposure to; Palmityl trifluoroketone (PTK), a Phospholipase A2 inhibitor that blocks global phospholipid synthesis; SC-236 and EI-211 which block the formation of prostaglandin and leukotriene like eicosanoids, respectively; and HA-130 and PF-8380 which block production of lysophosphatidic acid (LPA). Of these, PTK, HA-130, and PF-8380 significantly increased Smic migration asymmetry, while Sc-236 and EI-211 did not. This suggests that LPA, but not eicosanoids, are involved in Smic left/right migration asymmetry. To study transient LPA signaling, we identified four homologs of the lipid phosphatase/phosphotransferase (LPT) superfamily of ectoenzymes, which have conserved roles regulating extracellular LPA levels and primordial germ cell migration. Current and future directions are to determine the expression of the sea urchin LPT homologs and define their potential roles in Smic migration.
Characterizing Epithelial to Mesenchymal Transition of Pigment Cells in the Sea Urchin Lytechinus variegatus. 04/07/17 04:40 PM - 05:00 PM George, Andrew N. (Duke University, Durham, NC, USA); McClay, David (Duke University, USA)
Epithelial-to-mesenchymal transition (EMT) is a dynamic cellular process during which cells must change polarity, de-adhere from neighboring cells, breach through the basement membrane and become motile. This occurs during early development and is similarly re-deployed during cancer metastasis. Much of our understanding of the mechanisms that control this process have been determined through cell culture models using artificial basement membranes. The sea urchin embryo however provides a tractable system to observe this process in vivo in multiple cell types during development. In the sea urchin there are a number of discrete cell types that undergo an EMT at different developmental stages. At the onset of gastrulation, pigment cell precursors undergo an EMT at the tip of the archenteron. This begins one to two hours after the EMT of PMCs. Previous work in the McClay lab identified 13 transcription factors (TFs) that govern the dynamic cellular changes that occur during EMT in the primary mesenchymal cells. Here we ask whether the EMT in pigment cells occurs by a similar mechanism under the same regulatory control as the PMCs. We show that most of the TFs deployed in PMCs are not expressed in pigment cells during EMT, which suggests that there are different mechanisms controlling EMT in the pigment cells. We also show that morpholino knock down of twist disrupts EMT in pigment cells indicating that there is some overlapping control. By understanding the molecular mechanisms that govern EMT in multiple cell types within the sea urchin, we will gain valuable insights into the genetic control of this process during development allowing for a better understanding of this process in many disease contexts.
15 Talk 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 04/07/17 04:40 PM - 05:00 PM Martinez-Bartolome, Marina (Mississippi State University, USA); Range, Ryan (Mississippi State University, USA)
Anterior neuroectoderm (ANE) specification, positioning, and patterning is a crucial event in body plan formation in all deuterostomes. Studies from diverse metazoan embryos indicate that Wnt/β-catenin signaling is essential for the specification and patterning of the neuroectoderm along the primary axis. In early development of the sea urchin embryo, ANE positioning depends on integrated information from the Wnt/β-catenin, Wnt/JNK, and Wnt/PKC pathways, forming an interconnected Wnt signaling network. We have previously shown that Fzl1/2/7-PKC pathway antagonizes the down- regulation of the ANE GRN by Wnt1/Wnt8-Fzl5/8-JNK signaling in the anterior ectodermal half of early cleavage and blastula staged embryos, allowing for the proper positioning of the ANE territory around the anterior pole. Yet, the exact mechanism by which Fzl1/2/7-PKC signaling antagonizes Fzl5/8-JNK signaling during this process is still unclear. Hence, our research aims to better characterize the Fzl1/2/7 pathway and the gene regulatory network (GRN) it activates to identify possible interactions between these different Wnt signaling branches during this fundamental developmental process. Using a candidate gene approach in combination with whole-transcriptome differential screens, we identified a candidate Wnt ligand, Wnt16, necessary to activate the Fzl1/2/7 signaling during ANE positioning, a potential transcriptional effector, NFAT, of the Fzl1/2/7-PKC signaling, an intracellular signal transduction modulator, Siah1, and several transcription factors in the GRN activated by Fzl1/2/7 signaling. We use morpholino knockdown assays to demonstrate that many of these regulatory factors are necessary to antagonize the ANE restriction mechanism mediated by Wnt1/Wnt8-Fzl5/8-JNK signaling. Our results indicate that Wnt16 and NFAT are necessary for the specification of the ANE and activation of the putative GRN activated by Fzl1/2/7 signaling. Together, our data are consistent with the idea that Wnt16 activates the Fzl1/2/7 pathway and that NFAT acts downstream of Fzl1/2/7 signaling as its transcriptional effector necessary to antagonize Fzl5/8-JNK signaling mediated down regulation of ANE GRN.
Testing the utility of sea urchin embryos to discover human embryonic cis-regulatory modules 04/07/17 05:00 PM - 05:20 PM Guay, Catherine (Rutgers, USA); Nam, Jongmin (Rutgers, USA)
A large number of embryonic cis-regulatory modules (CRMs) can be a useful resource to elucidate and validate gene regulatory programs for early embryonic development including germ layer specification. Despite their importance, only a minute fraction of human embryonic CRMs has been discovered due to limited accessibility to embryos and/or lack of efficient tools. Sea urchin embryos have the potential to be an ideal model system to uncover human embryonic CRMs thanks to the virtually unlimited number of synchronized embryos for high-throughput CRM discovery. Considering the vast evolutionary distance between human and sea urchin, an obvious concern for this idea is that there may not be many human CRMs that are active in sea urchin embryo. Therefore, we first estimated the number of potential human CRMs that are active in the mesenchyme blastula stage of sea urchin embryos by using our high-throughput reporter assay method. When a randomly selected set of ~1,500 800bp-long human DNA fragments were tested in sea urchin embryos, 26 (~1.7%) drove significant reporter expressions. At this rate, we estimate ~68,000 fragments in the entire human genome to be active in sea urchin embryos at the mesenchyme blastula stage. This number should increase, if we include other developmental stages. We next asked if these potential CRMs exhibit diverse spatial patterns. To address this question, we employed our recently developed method for high-throughput spatial cis-regulatory analysis, MMOSAIC. In brief, the method computationally clusters CRMs based on their quantitative profiles of single-embryo resolution reporter expressions; CRMs in the same cluster are expected to have similar spatial patterns. In the analysis, the 26 human fragments displayed diverse spatial patterns in sea urchin embryos. Together, these results foretell the utility of sea urchin embryos to discover and examine an unprecedentedly large number of human embryonic CRMs.
16 Talk Upstream regulation of sea urchin skeletal patterning and VEGF signaling 04/07/17 05:00 PM - 05:20 PM Ben Tabou de Leon, Smadar (University of Haifa, USA); Gildor, Tsvia (University of Haifa, USA); Khalaily, Lama (University of Haifa, Haifa, ISR)
Understanding how signaling pathways regulate cell fate specification and organogenesis is an essential step toward deciphering the molecular control of embryo development. The vascular endothelial growth factor (VEGF) pathway is an example of a highly conserved signaling pathway that participates in the regulation of organogenesis through inter-tissue interactions. In vertebrates, VEGF has a major role in promoting cancer by inducing blood vessel growth into tumors that provide them with nutrients and allow them to grow and metastases. In the sea urchin embryo that does not have a vascular system, VEGF signaling is critical for larval skeletogenesis. Interestingly, hypoxic conditions strongly affect skeletal patterning in the sea urchin embryo but the molecular mechanisms that underlie this phenotype are not known. In my study, I aimed to understand the upstream regulation of sea urchin skeletal patterning and VEGF signaling and how it compares with its activation in vertebrates. The transcription factor, hypoxia inducible factor 1α (Hif1α) is a key activator of VEGF in vertebrates and therefore a key candidate to drive the hypoxia skeletal phenotype in sea urchin. Yet, I found that perturbation of Hif1α does not affect either VEGF expression or the skeletogenesis process in normal development. On the other hand, I discovered that the Bone Morphogenetic Protein, BMP2/4, represses VEGF, VEGFR and skeletogenic gene expression and by that restricts them into a localized expression domain that required for normal skeletal patterning. My studies suggest that the BMP pathway that in vertebrates drives bone and cartilage formation, controls sea urchin skeletal patterning directly and through the restriction of VEGF, VEGFR, and skeletogenic genes. These findings illuminate the conserved role of common signaling pathways in driving comparable morphogenetic processes.
Regeneration of Larval P. miniata 04/07/17 07:30 PM - 08:00 PM Hinman, Veronica (Carnegie Mellon U, Pittsburgh, PA, USA); Cary, Greg (Carnegie Mellon University, Pittsburgh, PA, USA); Wolff, Andrew (Carnegie Mellon University, Pittsburgh, PA, USA); Zheng, Minyan (Carnegie Mellon University, Pittsburgh, PA, USA)
Echinoderm larva exhibit an extraordinary capacity for regeneration following bisection and dramatic loss of tissue. We are using Patiria miniata to characterize this process and to ask how, and whether, developmental GRNs are reactivated during regeneration. Data from an RNA-Seq screen suggests that regeneration is initiated by conserved wound forming processes, a restoration of embryonic AP axis and then an upregulation of regeneration-specific proliferation. Edu labeling further shows how proliferating cells establish a blastema and how these are in turn form the regenerating tissues of the larva.
Evolution, Development and Regeneration in Hemichordates 04/07/17 08:00 PM - 08:30 PM Swalla, Billie J. (University of Washington, Seattle, WA, USA)
Vertebrates share several distinct morphological characters with three invertebrate groups, lancelets, tunicates and hemichordates. Tunicates, lancelets and vertebrates are a monophyletic group, the chordates, which share five morphological characters: a notochord, a dorsal neural tube, an endostyle, a muscular, post-anal tail and pharyngeal gill slits. Hemichordates share some of these chordate features, the pharyngeal gill slits, an endostyle and a post-anal tail, and were thought to contain a notochord homolog, the stomochord, and a dorsal neural tube in the neck region. Developmental genetics and genomics have allowed re-examination of the question of chordate origins, comparing developmental gene expression in embryos of different phyla has allowed insights into the molecular mechanisms underlying morphological changes. Genomics has allowed insight into the phylogenetic relationships of the chordates and their invertebrate relatives, and comparison of the shared genetic pathways in related embryos. Recent evidence from my lab and others has shown that the chordate ancestor was likely a benthic worm, with a mouth and pharyngeal gill slits, supported by cartilaginous gill bars. It is possible that the deuterostome ancestor was actually a chordate and that notochord was lost in the ambulacrarian ancestor, with echinoderms losing all of the chordate features. We are using phylogenies, genomes, and developmental characters to test these contrasting hypotheses.
17 Talk Rapid evolution of key GRN interactions within the sea urchin genus Heliocidaris 04/07/17 08:30 PM - 09:00 PM Wray, Gregory (Duke University, USA)
Many aspects of the core developmental GRN in echinoderms are conserved among species that last shared an ancestor hundreds of millions of years ago. This high degree of conservation is consistent with two distinct interpretations that are not mutually exclusive: one is that key regulatory interactions within the GRN are so critical for later events that substantial evolutionary changes are no longer possible, the other that these interactions are required for producing particular traits but can still evolve if there is selection to alter those traits. The sea urchin genus Heliocidaris, which contains two closely related species with highly distinct larval morphology and life history traits, provides a powerful system for distinguishing between these possibilities. Based on our prior evolutionary analyses of developmental transcriptomic time-series (Israel et al. 2016), we identified several candidates for altered interactions within the GRN. We are now following up with morpholino knockdowns, over-expression, and drug treatments to test the role of specific GRN components. As expected, we find the roles of some GRN components are likely conserved in H. erythrogramma, the species with the highly derived larval morphology and life history. An example is Alx1, which appears to retain its ancestral function, although at a different relative time during development. More surprisingly, we also find that some key gene interactions are qualitatively altered. A striking example is the double-negative gate that specifies the fate of the skeletogenic cell precursors, where HesC no longer plays a role. Our findings support the hypothesis that some critical gene interactions within the otherwise highly conserved GRN can evolve rapidly in response to natural selection to alter morphological and life history traits.
18 Talk The alkaline larval gut - a key trait under environmental change? 04/08/17 08:30 AM - 09:00 AM Stumpp, Meike (Zoological Institute, Taipei, TWN); Tseng, Yung-Che (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan, USA); Su, Yi-Hsien (ICOB, Academia Sinica, Taiwan, USA); Lein, Etienne (Helmholtz Centre for Ocean Research Kiel (GEOMAR), Kiel, Germany, USA); Lee, Jay-Ron (ICOB, Academia Sinica, Taiwan, USA); Dupont, Sam (SLC, University of Gothenburg, Sweden, USA); Hu, Marian Y. (Institute of Physiology, Christian-Albrechts-University of Kiel, Germany, USA)
Digestion is a central physiological process common to all metazoans and the digestive system evolved early during metazoan evolution. Most Echinoderm larvae are planktotrophic and have a simple tripartite digestive system. Depending on the species, the Echinoderm larva generates highly alkaline (pH 8.5-9.7) conditions through evolutionary conserved ATP- consuming ion regulatory processes (Na+K+-ATPase) in its digestive system. Gastric pH plays an important role in food breakdown, by optimizing digestive conditions for digestive enzymes e.g. proteases and has, therefore, a double sided role as an energy source (provision of nutrients) and energy sink (maintenance of alkaline pH). Additionally, the functionality of the larval gut is highly regulated under environmental fluctuations (e.g. pH and food). Consequently, the alkaline gastric pH can be regarded as a key trait with outstanding significance for animal-environment interactions, affecting larval energetics and development, and can also be used to understand observed patterns of sensitivity/resilience of echinoderm larval species to seawater acidification and climate change.
The gene regulatory underpinnings of deuterostome immunity. 04/08/17 09:00 AM - 09:30 AM Rast, Jonathan (University of Toronto, Toronto, ON, CAN); Buckley, Katherine M. (University of Toronto, Sunnybrook Research Institute, Toronto, ON, CAN); Schrankel, Catherine S. (University of Toronto, Toronto, ON, CAN); Schuh, Nicholas (University of Toronto, Toronto, ON, CAN); Ho, Eric (University of Toronto, Toronto, ON, CAN)
Many aspects of immune function are ancient among bilaterians, although it is not yet clear how they relate among animal phyla. The sea urchin larva has a relatively simple morphology, yet displays a complex immune response to microbial disturbance that involves changes in the activity of hundreds of genes expressed throughout the organism as well as complex interactions among several types of differentiated immune cells and the cells of the gut epithelium. We have developed a model to characterize gene regulatory control of this response using the marine bacterium Vibrio diazotrophicus. When exposed to Vibrio in the surrounding seawater, bacteria accumulate in the gut and later invade the body cavity. Bacteria that enter the blastocoel are cleared by a coordinated response of phagocytic and granular immune cells. Expression of immune factors over the course of response is distributed in many tissues and involves homologs of factors that are also important in vertebrate immunity (e.g., IL-17), response genes that are specific to sea urchins (e.g., 185/333) and an interesting set of novel genes with homologs that are widely distributed in bilaterians, but absent in vertebrates and ecdysozoan model organisms. The morphological simplicity of this system provides a model to investigate system-wide molecular interactions at single-cell resolution and to characterize the distributed gene regulatory network that underpins immune response. More generally, what we find in the sea urchin larva can be related to understanding the evolutionary origins of molecularly unusual aspects of vertebrate immunity.
Function of microRNA-31 in the sea urchin embryo 04/08/17 09:30 AM - 10:00 AM Song, Jia L. (University of Delaware, Newark, DE, USA); Stepicheva, Nadezda (University of Delaware, USA)
The microRNAs are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. microRNA-31 (miR-31) has been found to play a role in cancer, bone formation, and lymphatic development. However, the function of miR-31 in embryogenesis is not well described. We examined the role of miR-31 in early development, using the sea urchin embryo as a model. We found that miR-31 is expressed in all stages of development and its knockdown (KD) resulted in defects in the patterning and function of the primary mesenchyme cells (PMCs). Using bioinformatics and luciferase reporter constructs, we identified miR-31 to repress genes within the PMC gene regulatory network (GRN). The majority of the mislocalized PMCs in miR-31 KD embryos did not express VegfR10, indicating that miR-31 regulates the ability of PMCs to respond to positioning cues. We also found that miR-31 directly suppresses expression of Eve and indirectly regulates Vegf3 in the ectoderm. These results indicate that miR-31 coordinately suppresses genes within the GRN of PMCs and in the ectoderm to impact PMC patterning and skeletogenesis. To understand the function of miR-31 at a systems level, we used miR-31 pull down assays to identify its direct targets. This study will reveal how miR-31 cross- regulates GRNs and signaling pathways to ensure proper development. Since miR-31, GRNs, and signaling pathways are highly conserved in animals, this study will enhance the understanding of fundamental mechanisms used by a developing embryo to build its precise organization.
19 Talk Animal Evolution and Development: An Environmental and Physiological Perspective 04/08/17 10:30 AM - 11:00 AM Manahan, Donal T. (University of Southern California, Los Angeles, CA, USA)
"How Animals Work" is a theme of mutual interest to developmental biologists and physiologists. The approaches to study function taken by each sub-discipline are, however, often markedly different. Studies of gene function and regulatory networks have yielded important insights into the evolution of animal shape and form. In contrast, physiological processes are less well understood that may regulate the energetics of genome complexity and developmental responses to environmental change. A fuller understanding of how animals function requires a merging of information on genetic, environmental, and physiological processes. Recent studies are now providing new insights into the bases of genotype-by- environment interactions in the survival and growth (fitness) of early developmental stages of marine invertebrates. Experimental breeding of pedigreed lines to produce contrasting phenotypes has allowed for analysis of biochemical, metabolic, and genetic bases of adaptation. Such experimental approaches are providing a mechanistic understanding of the physiological capacities of developmental stages and their responses to a changing Earth system.
Developmental and immune-regulated expression of the perforin-like SpMacpf gene family in the purple sea urchin 04/08/17 11:00 AM - 11:30 AM Schrankel, Catherine S. (University of Toronto, Toronto, ON, CAN); Buckley, Katherine (Department of Immunology, University of Toronto, Toronto, ON , USA); Rast, Jonathan (Department of Immunology, University of Toronto, Toronto, ON , USA)
The genome sequence of the purple sea urchin (S. purpuratus) encodes a complex repertoire of innate immune recognition and effector proteins. Transcriptome screens of feeding larvae indicate that many genes are tightly regulated in response to immune challenge with Vibrio diazotrophicus. A candidate gene family involved in sea urchin immunity is the MACPF family of perforin-like molecules. Developmental transcriptomes, RNA-Seq analysis of immune challenge, WMISH, and fluorescent reporters were used to characterize MACPF genes. There are 22 SpMacpf genes organized into eight subfamilies (A-H). SpMacpf genes are encoded in multi-exon loci located across 17 scaffolds (v4.2). All encoded proteins share a single membrane-attack complex/perforin like domain (MACPF) that is implicated in lytic pore-formation and immune processes across several phyla. The SpMacpf subfamilies cluster based on MACPF domain size and sequence features. Most genes also encode a C-terminal apextrin-like “ApeC” domain. MACPF and ApeC domains are present in the Apextrin protein, first characterized in the sea urchin Heliocidaris erythrogamma (Haag 1999). The domains are also present in a diverse family of amphioxus (Branchiostoma) genes involved in gut immunity (Huang 2014). SpMacpf subfamilies exhibit differential expression patterns in embryonic, larval and adult tissues, as well as in adult coelomocytes. Several SpMacpf genes are tightly regulated during larval immune responses. SpMacpfA2 was found in a subset of larval blastocoelar cell immunocytes. The number of MacpfA2+ cells decreases upon immune challenge and may be used up during the acute immune response. SpMacpfE2 was found in a ring of cells encircling the anterior portion of the stomach of post-feeding larvae, where it may function in gut immunity. The SpMacpfB subfamily was present in adult coelomocytes and expressed at high levels in the ectoderm of early blastulae, a pattern reminiscent of both embryonic and adult HeApextrin expression. The apextrin-like ApeC and MACPF domains date back to the common ancestor of eumetazoa and are present in diversified gene families of several invertebrates. However, vertebrates lack ApeC-related proteins entirely. Thus the MACPF and ApeC domains may have been linked early in metazoan immunity, but this association has been lost in Gnathostomata. The purple sea urchin represents a unique system to explore the evolution and function of ApeC and MACPF-domain related genes in deuterostome development and immunity.
Cellular and Proteomic characterization of the innate immune response in wasting bat stars (Patiria miniata) 04/08/17 11:30 AM - 12:00 PM Andrus, Avery K. (California State University Long Beach, Long Beach, CA, USA); Livingston, Brian (California State University Long Beach, USA)
Sea stars of multiple species in western North America have been degrading and dying from an outbreak of the epidemic Sea Star Wasting Syndrome. Infected individuals develop white skin lesions which spread until the whole body decays. The Sea Star-associated Densovirus (SSaDV) discovered in symptomatic individuals was implicated as a potential initiator of the disease, although the mechanisms of defense and tissue disintegration are unknown. Echinoderm innate immune reactions include increasing numbers of coelomocytes (immune cells), increasing phagocytic activity, clumping cells, and the expression of defensive proteins. We examined the coelomic fluid of the bat star (Patiria miniata) before and throughout disease progression to determine differences in the coelomocytes of healthy and wasting bat stars, and after exposure to isolated virus. We identified several immune cell types and increases in the concentration, clumping, and phagocytic activity of coelomocytes throughout disease progression. The increase in phagocytosis and cell death before the symptoms appear suggests an aggressive immune response to the virus. Our analysis of the coelomic fluid proteome shows an increase in signaling, adhesion, and immune response proteins before and after symptoms appear. Many of these peptides and domains participate in pathways of defense and protein disintegration brought about by infection and cell death. The increase in cell signaling, phagocytosis, and death is consistent with evidence suggesting an autoimmune response may be involved in the death of the sea stars’ tissue. 20 Talk A genomic view of chordates' evolution 04/08/17 01:30 PM - 02:00 PM Escriva, Hector (Observatoire Oceanologique de Banyuls sur Mer, Banyuls sur Mer, FRA); Irimia, Manu (Observatoire Oceanologique de Banyuls sur Mer, USA); Maeso, Nacho (Observatoire Oceanologique de Banyuls sur Mer, USA); Marletaz, Ferdinand (Observatoire Oceanologique de Banyuls sur Mer, USA); Gomez Skarmeta, Jose Luis (Observatoire Oceanologique de Banyuls sur Mer, USA)
The evolution of vertebrates is a major issue in evodevo research. An important animal model used for comparative studies in this field is the cephalochordate amphioxus due to its close phylogenetic relationship with vertebrates but also because of its conserved bodyplan with vertebrates. Thus, amphioxus shares with vertebrates all the chordate characteristics such as the notochord, the dorsal hollow neural tube, the ventral gut, the pharynx perforated with gill slits, segmented muscles and gonads, post anal tail, pronephric kidney and homologues of the thyroid gland and adenohypophysis. However, they lack vertebrate-specific characters, such as migratory neural crest cells and placodes, an endoskeleton, and a morphologically segmented hindbrain. Comparative gene expression or gene function studies during embryonic development have shown that amphioxus embryonic development is highly conserved with vertebrates. Moreover, complete genome sequencing of two amphioxus species also showed a high degree of conservation both in terms of gene content and synteny. However, despite this high degree of conservation, amphioxus is not a vertebrate and here we used an epigenome wide approach in order to try to find structural and functional differences between amphioxus and vertebrates capable to explain the appearance of vertebrate specific traits. Our preliminary results show an increase of the complexity of genome structure and gene regulation mechanisms in vertebrates. These results suggest that structural changes in the genome played a major role in the appearance of vertebrates.
The origins of developmental gene regulation and the genome-wide identification of active blastula enhancers 04/08/17 02:00 PM - 02:30 PM Arenas-Mena, Cesar (CSI-CUNY, STATEN ISLAND, NY, USA)
It is proposed that developmental enhancers evolved from unicellular inducible promoters that diversified the expression regulatory genes during metazoan evolution. Constitutive-type promoters of regulatory genes would have acquired novel receptivity to distal regulatory inputs from promoters of inducible genes that eventually specialized as enhancers during metazoan evolution. Promoters and enhancers are functionally similar; both can regulate the transcription of distal promoters and both direct local transcription. Additionally, enhancers have structural features and motifs, such as the TATA box, that reveal their origin from inducible-type promoters. The distal cooperative regulation among promoters identified in unicellular opisthokonts possibly represents the precursor of distal regulation of promoters by enhancers. We capitalize on these features to identify active enhancers in blastula stage embryos. Our genome-wide ATAC-seq, RNA Polymerase II Chip-seq and PRO-seq characterizations reveal that active enhancers are also transcribed in sea urchins. The activity of these enhancers is validated by previous and our own functional cis-regulatory analyses. In addition, the distribution of Pol II pausing is associated with developmental genes in sea urchins, in agreement with similar findings in other organisms. We have also proposed that metazoan evolution involved the developmental suppression of cell proliferation and transcriptional potency. My original hypothesis of a transcriptional multipotency role of H2A.Z has been experimentally confirmed. Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and it is developmentally regulated in indirectly developing sea urchins and polychaetes. Photomorpholino-controlled depletion of H2A.Z reveals that it is essential during development. We are currently characterizing the transcriptional and post-transcriptional regulation of H2A.Z and Cyclin D in Strongylocentrotus purpuratus. Our results reveal that embryonic expression of H2A.Z is primarily driven by a single cis-regulatory module, and that undetermined Myb-family and GATA transcription factors contribute to control the transcriptional regulation of H2A.Z during the maternal to zygotic transition.
Ins and outs of transgenes in the sea urchin 04/08/17 02:30 PM - 03:00 PM Nam, Jongmin (Rutgers University-Camden, USA)
DNA transgenesis is a key component of studying the function of cis-regulatory modules (CRMs) in sea urchin embryos. Since the establishment of the method in the 80s, sea urchin embryos have been the major model system for cis-regulatory analysis by employing green fluorescent reporter proteins. More recently, high-throughput reporter assays by using barcoded DNA reporters have been pioneered in the sea urchin. Despite these technological advances, there are still several basic questions that need to be addressed. In this work, by using the purple urchin, Strongylocentrotus purpuratus, we will tackle the following two questions on the fate of transgenes: i) when is the earliest time for transgene expression?; ii) can multiplex reporter assays be applied to the late larval stage and the juvenile stage urchins? For the first question, our detailed QPCR analysis showed that the earliest time point for detectable reporter expression is 5h - 6h post fertilization. For the second question, our QPCR and sequencing analysis showed that, despite the gradual loss of transgenes during larval stages, the integrity and the diversity of transgenes are still maintained. Building upon these observations, we demonstrate the feasibility of multiplex reporter assays in larval stage urchins. We anticipate that application of the multiplex reporter assays in larval stage urchins will accelerate the discovery of gene regulatory programs that are responsible for dramatic changes during metamorphosis. 21 Talk Discovery in the classroom: Using the sea urchin to teach fundamental molecular skills and examine gene expression 04/08/17 03:30 PM - 03:45 PM Byrum, Christine (College of Charleston, Charleston, SC, USA)
Teaching an upper level course to both undergraduates and graduate students can be challenging. Often the students come from varying backgrounds and they may have different expectations and future goals. To help bridge these gaps, I designed a lab for my genomics course in which students collaborate in a group project, exploring a previously uncharacterized gene family of the sea urchin Lytechinus variegatus. Students experience the excitement of addressing real scientific questions, while becoming more proficient in the laboratory, learning not only how to use important bioinformatics resources, but also developing fundamental skills in the wet lab. In this course, all students study the selected gene family while each student investigates a particular gene family member in greater detail. This presentation will discuss approaches used to teach students about: 1) identification and characterization of new genes, 2) gene expression analysis (standard PCR and qPCR techniques), and 3) basic phylogenetic analysis. I have found that students in the course gain confidence in their abilities as the semester progresses and that they leave with valuable skills that help them succeed in future endeavors.
The Sea Urchin Embryo as a Model System in an Undergraduate Research Immersion Laboratory Course 04/08/17 03:45 PM - 04:00 PM Lee, Pei Yun (University of California Los Angeles, Los Angeles, CA, USA)
One of the greatest challenges in undergraduate laboratory education is to create a curriculum that provides hands on technical experience while being intellectually stimulating. Traditional laboratory courses tend to be “cookbook” in structure where all students perform a series of tried and tested experiments with predetermined outcomes. Here I describe a discovery based undergraduate laboratory course that uses the sea urchin as its model organism. Characteristics of the sea urchin embryo, such as its transparency, fast development, and ease of culture all make it ideal for experimentation in the undergraduate laboratory setting. In my course, students are given DNA sequences that they identify and characterize using bioinformatic tools. The majority of the course involves wet lab experiments whereby students attempt to clone their assigned genes and determine gene expression patterns through whole mount in situ hybridization. In a second companion course, students write a research paper based on the experimental data that they generated, and create a poster to be presented at UCLA Science Poster Day. This immersive approach to undergraduate laboratory education is superior to traditional methods because it mirrors the research process, allowing students to conduct an entire project from the identification of a research question, followed by practical experimentation in the laboratory, culminating in the analysis and presentation of results. Such activities involve higher order cognitive skills on the part of the students, and also have the added of benefit of increasing their understanding of biological concepts.
Using art to help students visualize processes in a developmental biology course 04/08/17 04:00 PM - 04:15 PM Romano, Laura (Denison University, Granville, OH, USA)
The discipline of developmental biology requires four-dimensional thinking that can be a challenge for undergraduate students. In particular, it requires them to understand a suite of changes in cell shape, division, adhesion, and motility that are occurring over time as the fertilized egg transforms itself into an embryo (which often becomes a larva that then transform itself into an adult, as in the case of most species of sea urchin). The availability of time-lapse videos is very useful in teaching a course in developmental biology, but often insufficient. For example, they do not often reveal the behavior of a particular cell or group of cells enclosed by the ectoderm unless the embryo is fairly transparent. Also, there have been relatively few efforts to create animations that provide a view of all of the cells as they rearrange themselves and undergo the aforementioned changes that are necessary to create an embryo. Therefore, one approach that I have been using in my course for several years is to incorporate art projects that allow students to develop their four-dimensional thinking so that they can better appreciate the complexity of developmental biology. The creation of sculptures, paintings, animations, poetry, interpretive dances, and other works of "art" seems to be effective in the context of my students at Denison, with a quantitative assessment currently being established.
22 Talk PRIMO experiences strengthen the URGE to democratize undergraduate research at a diverse large public urban community college 04/08/17 04:15 PM - 04:30 PM Onorato, Thomas M. (LaGuardia Community College, Long Island City, NY, USA); Veras, Ingrid D. (LaGuardia Community College, USA); Oulhen, Nathalie (Brown University, USA); Wessel, Gary M. (Brown University, USA)
The bat star, Patiria miniata, is not only an emerging model for studying gene regulatory networks, development and regeneration but is also emerging as a model for bridging scientific and educational silos via enhancing faculty scholarship, democratizing undergraduate research experiences and strengthening academic transitions. Sea stars are a key component of the NIH-funded experiences with the Providence Rhode Island Institute of Molecular Oogenesis (PRIMO) at Brown University that have led to the establishment of the Undergraduate Research Group on Echinoderms (URGE) at LaGuardia Community College of the City University of New York. Here we illustrate how echinoderm-based PRIMO experiences have strengthened the URGE to democratize authentic undergraduate research experiences, pay mentorship forward, and bridge academic transitions to broaden impacts for underrepresented students. We will discuss: 1) how research in the curriculum is being utilized to overcome the limitations of the classical apprenticeship model to provide more students with the opportunity to perform authentic undergraduate research at community colleges (i.e. democratize), where these opportunities are scarce due to limited resources and socioeconomic factors; 2) how PRIMO and the URGE are collaborating to pay mentorship forward to positively impact undergraduate, graduate, postdoctoral and faculty development; 3) how the URGE plans to bridge academic transitions for community college students studying STEM through echinoderm- based research. Here we present a paradigm-shifting model that bridges scientific and educational silos to create sustainable authentic undergraduate research experiences for underrepresented students through a genuine collaboration between a diverse large public urban community college and a research intensive institution.
Teaching Gene Regulatory Network Logic to Undergraduates 04/08/17 04:30 PM - 04:45 PM Bradham, Cynthia A. (Boston University, Boston, MA, USA)
Gene regulatory network (GRN) models for developmental specification are intimidating and overwhelming for undergraduate students to learn, yet these network models form the core of our understanding of the specification process in the developing embryo. Because GRN models were pioneered in sea urchin embryos, this model organism is an ideal system for teaching and illustrating GRNs. To appropriately teach specification in the classroom requires approaches that make the networks accessible to students intellectually and emotionally. In this talk, I will share the strategy I’ve developed to teach GRN models and logic in the classroom. The strategy rests on first teaching the student a series of network motifs via their exploration of foundational publications from the sea urchin community, including classic papers on Notch-mediated SMC specification (Sherwood and McClay, 1999), pmar and the double-negative gate for PMC specification (Oliveri et al, Developmental Biology 2003), and the evolutionary conservation of GRN architecture and the endomesoderm lock-down loop (Hinman et al, PNAS 2003). These papers are taught during discussion sections in conjunction with lectures that cover specification in sea urchins, in Drosophila, and in Xenopus and other vertebrates. Thus, when we arrive at the lecture on GRNs after 5 weeks, the students have already learned quite a lot about GRNs without realizing it, and are positioned to integrate the information in a coherent network model. Aspects of the GRN lecture are drawn from Eric Davidson’s books, and, as a whole, the lecture is organized around teaching networks from the perspective of five rules or principles, as first defined by Eric Davidson: 1. Double negative gates as a means to initiate a network; 2. Lock-down loops as a means to install an irreversible specification state; 3. The community effect as means to spread a specification state; 4. Mutual exclusion of alternative cell specification states; and 5. Differentiation gene batteries as a check point for specification and mechanism for coordination of differentiation gene expression. We explore these principles using the endomesoderm GRN from sea urchin embryos, and also compare it to GRNs for Drosophila and Xenopus DV specification. Because they have learned the examples first via the publications, the students are highly receptive to the lecture that ties it all together, and report a deep intellectual engagement and satisfaction with the integrated perspective that the GRN models provide. Based on examination metrics, this approach is quite successful in conveying the core principles of GRN logic to undergraduate students in the classroom.
23 Poster Poster
1. Larvae of the echinoid Dendraster excentricus avoid acidified seawater in stratified columns with different pH Batzel, Grant (Scripps Institution of Oceanography, San Francisco, CA, USA); Maboloc, Elizaldy (Hong Kong University of Science, USA); Grunbaum, Daniel (University of Washington, USA)
By the end of the century, the world’s oceans are expected to drop in pH due to the diffusion of greenhouse gasses, in a process called Ocean Acidification (OA). The effects of OA on the abundance and distribution of marine larvae, a swimming life-history stage of many benthic invertebrate animals, is currently unknown. Different bodies of water are expected to fluctuate in pH, which could act as a barrier between once connected marine populations. In order to understand the effects of OA on the behavior and vertical distribution of larvae, we used plutei of the sand dollar Dendraster excentricus to study their vertical distributions when confronted with a choice between acidified and ambient seawater. Stratified layers were achieved by varying the salinity of seawater; however, salinity controls showed no differences in the vertical distribution. Larvae were introduced into columns with ambient seawater overlaying acidified seawater, as well as the reciprocal treatment of ambient seawater underlying acidified seawater. As larvae approached the gradient from ambient to acidified water, they performed a twirling swimming response followed by downward movement. Larvae transitioning from acidified to ambient seawater maintained upward swimming to the top of the column. For both treatments, larvae changed their swimming behavior to avoid acidified seawater. We show preliminary data of larval tracking, which can be used as a tool to assess swimming speeds. This study provides evidence for avoidance behavior of acidified seawater by larvae of Dendraster excentricus.
2. Major Yolk Protein (MYP) is expressed in bipinnaria larvae of the sea star, Patiria miniata. Bonilla, Alejandro (LaGuardia Community College, USA); Tadros, Albert (LaGuardia Community College, USA); Veras, Ingrid D. (LaGuardia Community College, USA); Oulhen, Nathalie (Brown University, USA); Vargas, Henry (LaGuardia Community College, USA); Fresques, Tara (Brown Univeristy, USA); Onorato, Thomas M. (LaGuardia Community College, Long Island City, NY, USA); Wessel, Gary M. (Brown University, USA)
Yolk proteins are important for both providing the components necessary for gametogenesis and a nutritional source for larvae during early embryogenesis. Vitellogenin (Vtg) and the major yolk protein (MYP) are two of the most important yolk proteins among diverse species of invertebrates. Sea stars, unlike sea urchins, express two Vtgs (Vtg-1 and Vtg-2) in addition to MYP. Vtg-1, Vtg-2, and MYP mRNAs are differentially expressed in the ovary and during early embryogenesis in the sea star, Patiria miniata. Interestingly, MYP mRNA is expressed in larvae, including larvae that are already feeding, and is enriched in the posterior enterocoel (PE), which is thought to give rise to the germline in sea stars, coelomic pouches and intestine. We performed Western Blot analysis and immunofluorescence microscopy to determine MYP protein expression during larval development. Embryos and bipinnaria larvae express MYP. Confocal microscopy reveals MYP is found in the right and left coeloms, PE, esophagus, stomach and anus. Future experiments will investigate the function of MYP in bipinnaria larvae. We will use CRISPR genome editing to mutate MYP and analyze its function and role in the PE and germline formation. Additionally, regulation of MYP expression and function by signaling pathways such as Wnt and TGF-b will be investigated.
3. DISTRIBUTION OF KARYOPHERIN BETA IMPORTINS AND TRANSPORTINS IN EARLY EMBRYONIC DEVELOPMENT OF THE SEA URCHIN Hathaway, Devon (College of Charleston, USA); Siegwald, Paul (College of Charleston, USA); McFadden, Greg (College of Charleston, USA); Overcash, Melanie (College of Charleston, USA); Byrum, Christine (College of Charleston, USA)
Karyopherins are a group of nuclear transport proteins that transfer important cargo, including transcription factors (TFs), into and out of the nucleus, potentially affecting cell fates. Recent studies indicate that these proteins may also be useful predictors of human health. For example, the karyopherin KPNA2 is a biomarker misexpressed in many late stage cancers (Christiansen and Dyrskjøt, 2013) and is also mislocalized in hippocampal neurons of patients with Alzheimer’s disease (Lee et al., 2006). Our investigation is an important step in developing hypotheses about how these proteins could influence early development and in exploring use of the sea urchin embryo as a model for studying nuclear transport. Traditionally, studies examining roles of karyopherins in nucleocytoplasmic localization of cargo have utilized yeast or isolated cell lines, but the sea urchin provides investigators with a valuable opportunity to explore roles of nuclear transport proteins in an intact developing organism.
Karyopherins in sea urchins and other organisms are subdivided into two groups: the karyopherin alpha (KAP-α) forms and the karyopherin beta (KAP-β) forms. In a previous investigation, we used wholemount in situ hybridization (WMISH) to examine distribution of the 3 KAP-α importins in developing Lytechinus variegatus embryos. Each was found to be spatiotemporally restricted. The current study focuses on the KAP-β importins and transportins. Using reverse transcriptase PCR we confirmed that L. variegatus has at least 7 KAP-β importins and 2 KAP-β transportins. The spatiotemporal distribution of KAP-β importins and transportins has been mapped between the 4-cell and pluteus stages and is reported here. In many cases (KPNB1, IPO5, IPO9), we have found that the KAP-β forms, when present, are more likely to be generally distributed. 24 Poster 4. The role of hypoxia-inducible factor a in sea urchin embryonic development Chang, WeiLun (Institute of Cellular and Organismic Biology, Taipei, TWN)
The hypoxia-inducible factor alpha (HIF-α) is crucial for cells to adapt to fluctuating oxygen levels by activating downstream genes in responding to a hypoxia environment. However, HIF-α has been proved to play an important role in regulating embryogenesis in zebrafish, chicks, and mouse embryos that grow in normoxic conditions. Previous studies in sea urchin embryos have shown that maternal HIF-α is identified as a player in the aboral gene regulatory network (GRN), although it is unknown whether physiological hypoxia is present to activate HIF-α in normoxic embryos. Moreover, the function of the zygotically expressed HIF-α is still unclear. According to our preliminary data, the canonical HIF pathway exists in sea urchin embryos and is regulated by physiological hypoxia during early development. Moreover, based on the expression pattern of HIF-α and the phenotype of zygotic HIF-α knockdown/knockout sea urchin embryos, zygotic HIF-α may be functional in skeletogenic and coelomic pouch cells. In order to thoroughly elucidate the roles of HIF-α in sea urchin embryonic development, it is necessary to examine the expression of the early and late developmental genes associated with primary mesenchyme cells (PMCs), secondary mesenchyme cells (SMCs), and small micromeres (SMMs) in the zygotic HIF-α knockdown/knockout sea urchin embryos. Furthermore, because HIF-α is an ancient molecule, its roles in sea urchins may be applicable to other animals. This study may also provide molecular mechanisms to explain the role of hypoxia during normal embryogenesis, such as the hypoxic environment in the uteri of mice.
5. Specification of larval and adult skeletogenic lineages in the lecithotrophic urchin Heliocidaris erythrogramma Edgar, Allison (Duke University, Durham, NC, USA); Wray, Greg (Duke University, USA)
In the lecithotrophic urchin Heliocidaris erythrogramma, which has lost the feeding larval stage, the pentameral juvenile spicules and (vestigial) larval spicules appear nearly simultaneously instead of weeks apart as in planktotrophs. It is not known whether H. erythrogramma’s larval and adult skeletogenic cells are derived from a common cell lineage or from distinct lineages as in the planktotroph. Since the planktotroph’s skeletogenic gene regulatory network (GRN) is well characterized, we have chosen this system to ask how GRN deployment changes when development is temporally compressed. We first characterized spatial and temporal expression patterns of skeletogenic markers in H. erythrogramma. We ask whether the gene regulatory network (GRN) that specifies each type of skeletogenic cell in H. erythrogramma resembles the planktotrophic larval GRN, the planktotrophic adult GRN, or an alternative GRN by manipulating expression of key GRN nodes with knockdowns, mis-expression, and small molecule inhibitors.
6. Parallel embryonic transcriptional programs evolve under distinct constraints that enable morphological conservation amidst adaptation Gildor, Tsvia (University of Haifa, USA); Ben-Tabou de-Leon, Smadar (University of Haifa, USA); Malik, Assaf (USA); Sher, Noa (USA)
Embryonic development evolves by balancing stringent morphological constraints with genetic and environmental variation. The design principle that allows developmental transcriptional programs to conserve embryonic morphology while adapting to environmental changes is still not fully understood. To address this fundamental challenge in development and evolution, we compare developmental transcriptomes of two sea urchin species that are morphologically similar, yet genetically and geographically distant. We find that both developmental and housekeeping genes show highly dynamic and strongly conserved expression patterns. However, of these, only the interspecies correlation of developmental gene expression is tightly linked to morphological similarity. Other gene sets, such as homeostasis, response and signaling genes, show divergent expression which could reflect either adaptation to dissimilar environmental conditions or evolutionary drift due to lower developmental constraints. The interspecies correlation of each of these sets positions the phylotypic stage at a different developmental time: developmental gene expression shows highest conservation at mid-developmental stage, in agreement with the hourglass model while the conservation of housekeeping genes keeps increasing with developmental time. When all gene sets are combined, the association to morphological similarity is partially masked compared to that of only the developmental gene subset. Our study illustrates the various transcriptional programs that coexist in the developing embryo, evolve under different constraints and enable the conservation of similar body plans while allowing adaption to local conditions.
25 Poster 7. The role of Wnt Inhibitory Factor-1 in the Wnt signaling network governing anterior-posterior patterning of sea urchin embryos Grant, Daisy (Mississippi State University, USA); Stocks, Christian D. (Mississippi State University, USA); Range, Ryan (Mississippi State University, USA)
The specification and patterning of the anterior-posterior (AP) axis in many metazoan embryos is dependent on a posterior- to-anterior gradient of Wnt signaling. In the sea urchin embryo, patterning of the anterior neuroectoderm (ANE) along the AP axis is dependent on a network involving three interconnected Wnt signal transduction pathways: Wnt/β-catenin, Wnt-JNK, and Wnt/Ca2+. While much has been learned about the roles of each of these individual Wnt signaling branches in development and disease, our understanding is still limited and little is known about how they interact with one another in any context. Wnt inhibitory factor-1 (Wif-1), one of the least understood secreted Wnt signaling modulators, has been shown to bind Wnt ligands resulting in Wnt/β-catenin signaling inhibition. In this study, we report that wif-1 is zygotically expressed in two different germ layers during early AP and dorsal-ventral (DV) patterning in the sea urchin embryo: the endomesoderm and the dorsal ectoderm. Pharmaceutical manipulations suggest that wif-1 expression is activated by Wnt/β-catenin signaling in the endomesoderm and a mechanism dependent on Nodal signaling in the dorsal ectoderm. We show that perturbing Wif-1 function disrupts gastrulation and specifically perturbs the correct positioning of the ANE along the AP axis, possibly though the inhibition of the Wnt/Ca2+ pathway. Together, these data suggest Wif-1 may represent an important and direct link between the gene regulatory networks that control AP and DV patterning in the early sea urchin embryo.
8. Bicarbonate transport regulates intracellular pH critical for biomineralization in the sea urchin larva Hu, Marian Y. (Institute of Physiology, Christian-Albrechts University of Kiel, Germany, USA); Yan, Jia-Jiun (ICOB, Academia Sinica, Taipei, Taiwan, USA); Himmerkus, Nina (Physiological Institute, Christian-Albrechts-University, Kiel, Germany, USA); Bleich, Markus (Physiological Institute, Christian-Albrechts-University, Kiel, Germany, USA); Stumpp, Meike (Zoological Institute, Taipei, TWN)
Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants. Protons generated during biomineralization need to be buffered and excreted to promote further precipitation of CaCO3. The calcite endoskeleton of the sea urchin larva is formed through intracelllar precipitation of amorphous calcium carbonate within vesicles of the primary mesenchyme cells (PMCs). To date, little is known about pH regulatory mechanisms of PMCs and their role in calcification.
- Here, we demonstrate the importance of a solute carrier 4 (SLC4) family transporter in the aquisition of HCO3 , and - intracellular pH (pHi) regulation of PMCs. Among the four SLC4 family HCO3 transporter genes found in the genome of Strongylocentrotus purpuratus, SLC4a10 is predominantly expressed in PMCs and highest mRNA levels were detected + - during de novo formation of the larval skeleton. Morphants lacking this Na /HCO3 cotransporter are characterized by decreased PMC pHi and impaired ability to compensate an intracellular acidosis. These acid-base regulatory defects are accompanied by decreased calcification and spicule deformations. Pharmacological studies underline this observation and demonstrate DIDS sensitive pHi regulation in PMCs. Increased expression of Slc4a10 under acidified conditions relevant for ocean acidification scenarios suggests a critical role of this transporter in maintaining calcification rates under reductions in seawater pH.
- This work identified and characterized a HCO3 transport mechanism critical for pHi homeostasis of the calcifying PMCs in the sea urchin embryo. Since pH regulation is fundamentally linked to the biological precipitation of CaCO3, a better understanding regarding acid-base regulatory mechanisms in calcifying systems will be required to broaden our understanding regarding biomineralization strategies in animals.
26 Poster 9. VEGF signaling during gastrulation is necessary for proper PMC positioning and gene expression during secondary skeletal patterning in Lytechinus variegatus Huth, James (Boston University, Boston, MA, USA); Zuch, Daniel T. (Boston University, Boston, MA, USA); Bradham, Cynthia A. (Boston University, Boston, MA, USA)
Vascular endothelial growth factor (VEGF) signaling has been implicated in regulating biomineralization, primary mesenchyme cell (PMC) migration, and skeletal patterning in sea urchin embryos. Inhibition of VEGF signaling by the VEGF Receptor antagonist axitinib in gastrulating Lytechinus variegatus embryos leads to the loss of the aboral rods, a secondary skeletal element, in mature larvae. Because PMCs are responsible for larval urchin skeletogenesis, and because their migration is directed by ectodermal signaling cues such as VEGF, we sought to characterize the effects of axitinib treatment on PMC positioning and gene expression in L. variegatus embryos. We observed that a plug of PMCs that goes on to deposit the aboral rods migrates laterally from the ventrolateral clusters in prism stage control embryos. In contrast, PMCs in axitinib-treated embryos failed to accumulate at the site of aboral rod branching. In order to characterize how axitinib treatment alters PMC migration, we performed fluorescence in situ hybridization (FISH) for genes that are spatially and temporally differentially expressed among PMCs. We found that Lv-Jun and Lv-Otop2L were dysregulated in axitinib-treated embryos at prism stage but not in gastrula or pluteus embryos. In control embryos, Lv-Otop2L was highly expressed in PMCs of control prism embryos at the site of aboral branching, but this expression was greatly diminished in axitinib-treated embryos. Similarly, Lv-Jun is not expressed in terminal PMCs of prism embryos at the site of aboral branching, while all PMCs at this site expressed Lv-Jun in treated embryos. No differential expression of these genes was observed between control and treated gastrula or pluteus embryos. These results suggest that VEGF signaling is specifically necessary for appropriate gene expression and migration of the PMCs that are involved in the formation of the aboral rods during secondary skeletal patterning.
10. The skeletal proteome of Eucidaris tribuloides test and spines Lopez, Lupita (California State University, Fountain Valley, CA, USA); Flores, Rachel L. (California State University, USA); Livingston, Brian T. (Cal State Long Beach, Long Beach, CA, USA)
The skeletal proteomes of Strongylocentrotus purpuratus test, spine, tooth and larval skeleton have been determined and revealed that the prominent proteins were a family spicule matrix proteins and MSP130. We have recently characterized the skeletal proteomes of two adult brittle stars and the sea star Patiria miniata. Surprisingly, we did not find either spicule matrix proteins or MSP130 occluded in the mineralized tissue. We have identified a number of other proteins that are conserved among all of these echinoderm groups. In order to understand the evolution of the proteins involved in echinoderm skeleton formation, we have characterized the test and spine proteome of the cidaroid urchin Eucidaris tribuloides. We have found C- type lectin proteins in the skeleton homologous to the spicule matrix proteins, but they lack the extensive repetitive domains. The cidaroid urchin also contains MSP130 homologues in the skeleton. The relationships between these proteins and those identified in other echinoderms has been determined. We also compare the overall proteomes of E. tribuloides to other echinoderms. Additionally, we have characterized the proteins present in the insoluble material that appears during skeletal protein isolations. We find most of the proteins present in the soluble fraction, as well as some additional proteins. E. tribuloides spines are pigmented, and the pigment segregates entirely to the insoluble fraction. We discuss what this analysis tells us about skeleton formation in echinoderms.
11. The crowns have eyes: An investigation of opsin diversity and function in Crown of Thorn Starfish Acanthaster planci Lowe, Elijah K. (Stazione Zoologica Anton Dohrn, Napoli, ITA); Garm, Anders (University of Copenhagen, USA); Ullrich- Lüter, Esther (Museum fuer Naturkunde Berlin, USA); Arnone, M. Ina (Stazione Zoologica Anton Dohrn, USA)
In the late nineteenth century, the examination of visual pigments led to the discovery of a protein—opsin—covalently bound to a chromophore. Opsins are G-couple proteins receptors (GPCR) used for both visual and non-visual photoreception, and these proteins date back to the base of the bilaterians. In the current sequencing age, phylogenomic analysis has proven to be a powerful tool, facilitating the increase in knowledge about diversity within the opsin subclass and so far, nine paralogs have been identified. While phylogeny may help infer function, direct functional studies of opsins in vertebrates, cephalopod mollusks, and fruit flies have shown that there are multiple pathways involving various opsins for visual photoreception along with several other processes. Within echinoderms, opsins have been studied in Echinoidea and Ophiuroidea, but these two groups do not possess proper image forming eyes, only dermal non-visual photoreception. However, most species of Asteroidea, the starfish, possess true eyes and studying them will shed light on the diversity of opsin usage within echinoderms and help resolve their evolutionary history. Using high-throughput RNA sequencing, we have sequenced and analyzed the transcriptomes of different Acanthaster planci tissue samples — eyes, radial nerve, tube feet and a mixture of other organelle tissue — allowing us to identify at least 7 potential opsin paralogs, and which of those opsins are differentially expressed in both eyes and radial nerve, providing new important insight into the involvement of opsins in visual and nonvisual photoreception in echinoderms.
27 Poster 12. Expression of stem cell factors in the adult sea cucumber digestive tube Mashanov, Vladimir (University of North Florida, Rio Piedras, PR, USA)
Homeostatic cell turnover has been extensively characterized in mammals. In their adult tissues, lost or aging differentiated cells are replenished by a self-renewing cohort of stem cells. The stem cells have been particularly well studied in the intestine and are clearly identified by the expression of marker genes including Lgr5 and Bmi1. It is, however, unknown if the established principles of tissue renewal learned from mammals would be operating in non-mammalian systems. Here, we study homeostatic cell turnover in the sea cucumber digestive tube, the organ with high tissue plasticity even in adult animals. Both the luminal epithelium and mesothelium express orthologs of mammalian Lgr5 and Bmi1. However, unlike in mammals, there is no segregation of these positively labeled cells to specific regions within the epithelia where most of the cell proliferation would take place. In the mesothelium, the cells expressing the stem cell markers are tentatively identified as peritoneocytes. There are significant differences among the five anatomical gut regions in cell renewal dynamics and stem factor expression. The cloaca differs from the rest of the digestive tube as the region with the highest expression of the Lgr5 ortholog, lowest level of Bmi1, and the longest retention of BrdU-labeled cells.
13. Sperm aster growth and dynamics during pronuclear migration Meaders, Johnathan (Boston College, USA); Burgess, David (Boston College, 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 and to centrally position the nucleus for the first cleavage division. Nuclear centering was recently explained by microtubule (MT) length-dependent cytoplasmic pulling forces in which aster asymmetry and dynein are essential for male pronuclear centration (Tanimoto et al, 2016). We have re- analyzed the role of sperm astral MT growth and dynamics during pronuclear migration in the higher invertebrate Lytechinus pictus. We find that astral MTs are longer on the cortical facing side than on the cytoplasmic side, which is inconsistent with a MT length-dependent cytoplasmic pulling model. We next manipulated aster asymmetry in 1-cell zygotes with ethyl carbamate (urethane) or hexylene glycol, which increases MT catastrophe or polymerization, respectively (Strickland et al, 2005). Neither compound affects fertilization. Sperm astral MTs in urethane-treated eggs are shorter than in control zygotes. The result of suppressing sperm aster MT growth in L. Pictus is arrested sperm-egg pronuclear migration, resulting in failure to center the nucleus and delayed cytokinesis. 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 motor asymmetry 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, allowing the force-balance required for male pronuclear centration.
14. The role of cAMP in gastrulation of the sea urchin, Strongylocentrotus purpuratus Nesbit, Katherine (Scripps Institution of Oceanography, La Jolla, CA, USA); Hamdoun, Amro (Scripps Institution of Oceanography, USA)
A central problem in developmental biology is to understand how spatially segregated groups of cells coordinate ontogenetic events. ABCC5/MRP5 is a member of the ATP-binding cassette C family of transporters most commonly known for their involvement in drug resistance of cancers. However, more recently, these transporters have been demonstrated to play important roles in efflux of signaling molecules. A previous study from our lab implicated MRP5 (aka ABCC5a) in secretion of a cAMP signal produced in pigment cells, and necessary for hindgut invagination of S. purpuratus. Here we aim to elaborate upon these findings by pursuing three specific goals, which include direct demonstration of cAMP transport by MRP5, determination of the timing of cAMP efflux, and identification of a cAMP receptor(s). To address the first goal, MRP5 will be heterologously overexpressed, and its transport function and ATP hydrolysis activity assessed in the presence of potential substrates. For the second goal, we will express a live-cell fluorescent cAMP sensor which, based on previous results, we hypothesize will reveal cAMP efflux during late blastula stages, and continuing through completion of gastrulation. Finally, in pursuit of the third goal, we have identified a putative receptor for cAMP, analogous to the well- known mammalian intracellular cAMP receptor ADORA2a (A2A). We will examine cellular and subcellular distribution of this gene and protein, focusing on a relevant hindgut cell population which we have found to have enhanced uptake of MRP5 substrates. In parallel, we will use these tools to test whether knockout of A2A, using CRISPR-Cas9 effectively blocks cAMP signal reception and recapitulates cell behaviors seen in the MRP5 KO. Collectively these approaches will illuminate the role of cAMP and its receptors in gastrulation, and more precisely define the role of transport in control of this crucial developmental signal.
28 Poster 15. Defining the Mechanism by which Sulfated Proteoglycans Direct Migration during Embryonic Development Nizhnik, Anastasia (Boston University, Boston, MA, USA); Zuch, Daniel (Boston University, Boston, MA, USA); Bradham, Cynthia A. (Boston University, Boston, MA, USA)
Sulfated proteoglycans (SPGs) are components of the extracellular matrix (ECM) that provide cues for migratory cells during embryogenesis. The Bradham Lab has shown that SPGs coordinate ventral migration of skeletogenic primary mesenchyme cells (PMCs) in the sea urchin embryo. We hypothesize that ventrally concentrated SPGs induce differential gene expression within a subset of the PMCs, thereby subdividing the ventral PMCs and driving their territory-specific migration. In this project, we aim to define the gene expression profile of PMCs that are responsive to SPGs as a migratory cue using an in vitro migration assay. To determine the specific role of SPGs in vitro, ECM is extracted from L. variegatus embryos in which SPG concentration has been experimentally manipulated, then applied to latex beads to induce directional migration of isolated PMCs. Using a novel technique developed for this project, we captured migratory cells in an agarose- formaldehyde matrix and subjected them to fluorescence in-situ hybridization analysis to assess PMC subset-specific gene expression. Later, by perturbing PMC gene expression and correlating the effects on SPG-mediated migration, we will begin to define the mechanism by which SPGs direct ventral PMCs. This understanding will provide insight into the complex, dynamic relationships that drive tissue patterning during early embryogenesis.
16. The spirochaetaceae family of bacteria is a major constituent of the pyloric caeca (gut) microbiome of the adult sea star, Patiria miniata. Veras, Ingrid D. (LaGuardia Community College, USA); Bonilla, Alejandro (LaGuardia Community College, USA); Vargas, Henry (LaGuardia Community College, USA); Onorato, Thomas M. (LaGuardia Community College, Long Island City, NY, USA)
Microbiomes have a significant role in animal health and disease. While current research primarily focuses on understanding human microbiomes and secondarily focuses on microbiomes of other terrestrial animals, there exists a dearth of information on echinoderm microbiomes, especially microbiomes of animals from the class Asteroidea. Characterizing sea star microbiomes is essential for understanding these animals’ normal physiology and pathophysiology associated with such diseases like Sea Star Wasting Disease (SSWD). Therefore, we utilized next-generation DNA sequencing to analyze the biodiversity of the coelomic fluid and pyloric caeca (gut) prokaryotic microbiomes of the adult sea star, Patiria miniata. Samples were collected from animals purchased from South Coast Bio-Marine, LLC (San Pedro, CA) during Fall 2014 and Fall 2015. We found that the spirochaetaceae family of bacteria is the main constituent of the gut microbiome but not the coleomic fluid microbiome of P. miniata. Interestingly, the spirochaetaceae family were not abundant in gut from four of the 14 sea stars sampled. The stark difference in the percentage of spirochaetacae in individual organisms suggests that these bacteria maybe essential constituents of the microbiome of adult P. miniata animals. Future experiments need to be conducted to identify the species of spirochetes present in the gut of P. miniata and their role in sea star physiology.
17. Early-specified vegetal domain may determine the plane of the first embryonic cleavage in starfish, independently of meiotic structures localisation Piñeiro López, Cristina (EMBL, HEIDELBERG, DEU); Somogyi, Kalman (USA); Lénárt, Peter (USA)
In echinoderms, and many other animal species, the first zygotic spindle positioned perpendicular to the animal-vegetal (A/V) polarity axis defines the plane of the first cleavage, directing the subsequent stereotypic cleavages and thus setting the initial pattern of the embryo. Historically, the animal pole has been defined by the meiotic structures found there, such as the oocyte nucleus (germinal vesicle), the meiotic spindles, and most prominently the polar bodies. However, recent studies by us and others showed that experimentally dislocating meiotic structures has no effect on the positioning of the zygotic spindle. Then, what determines A/V polarity positioning the zygotic spindle?
In a wide range of species, the canonical Wnt pathway (cWnt) has been identified as a key component in the establishment of the A/V axis, localizing to the vegetal pole, and at later stages involved in gastrulation and endomesoderm specification. Our preliminary data in starfish shows that Dishevelled (Dsh), a conserved activator of the cWnt pathway, localizes at the vegetal pole. By expressing Dsh-mEGFP protein we show that Dsh re-localizes to the vegetal domain concomitant with nuclear envelope breakdown at meiotic entry. Thereafter, a clearly visible vegetal patch of Dsh is maintained throughout meiosis and early embryonic development. Interestingly, displacing the oocyte nucleus by centrifugation and by other perturbations of meiotic structures did not affect Dsh localization, suggesting that the positioning of the vegetal domain marked by Dsh is independent of meiotic structures.
We are currently further characterizing this vegetal domain, which also appears to be associated with a specific network of microtubules that may be involved in transporting and anchoring determinants (protein and mRNA) defining vegetal identity. Our hypothesis is that the vegetal domain and potentially cWnt components are critically involved in the orientation of the zygotic spindle for the first cleavage that we are currently testing.
29 Poster 18. Compensation for the genetic knockout of the protective transporter P-glycoprotein Rosenblatt, Hannah (Scripps Institution of Oceanography, USA); Hamdoun, Amro (Scripps Institution of Oceanography, USA)
Embryos precisely execute a complex set of processes and rearrangements under variable environmental conditions. Responding to this variation requires extensive flexibility in regulation of developmental networks. Although there is thorough research on canonical pathways involved in normal development, the potential of embryos to alter their own biology and compensate for improper function or mutation is less understood.
My research explores the mechanism of compensation for the genetic knockout of P-glycoprotein (P-gp), an ABC transporter important for defense from toxicants during development. P-gp is rapidly depleted in knockout urchin embryos, as determined by western blotting, however the embryos maintain high efflux activity and show P-gp substrate accumulation levels on par with wild type embryos. To determine if this continued efflux activity in knockouts resulted from persistence of some P-gp or an alternative mechanism, embryos were exposed to PSC-833, a specific P-gp inhibitor. While PSC-833 dramatically increased the accumulation of substrate in wild type embryos, it barely raised accumulation in knockout embryos which accumulated on average 6.4 times less substrate than wild types (p < 0.00001). This suggests that the high efflux activity observed in knockout embryos is due to some compensatory mechanism.
I am currently investigating how this compensation occurs, focusing on identification of the checkpoint between P-gp transcription or trafficking to the membrane that activates the alternative pathway capable of carrying out P-gp activities. The possibility of close coordination between regulatory elements of gene expression and functionally redundant pathways could provide valuable insight into how embryos maintain robustness despite genetic variation or cellular malfunctions.
19. MicroRNA regulation of Dishevelled Sampilo, Nina Faye (University of Delaware, Newark, DE, USA); Stepicheva, Nadezda (University of Delaware, USA); Zaidi, Syed A. (University of Delaware, USA); Wang, Lingyu (University of Miami, USA); Wu, Wei (University of Miami, USA); Wikramanayake, Athula H. (University of Miami, USA); Song, Jia L. (University of Delaware, USA)
MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3’ untranslated region (UTR) of target mRNAs and silence translation. MiRNAs are key regulators of the Wnt signaling pathway that are known to affect cell proliferation, migration, polarity and other developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled (Dsh/Dvl), an important signaling protein located upstream of β- catenin. The Dvl mRNA isoforms have similar spatial localization in early development but later show distinct ciliary staining in select isoforms. Using luciferase assays and site-directed mutagenesis, we demonstrated that the different isoforms of Dvl are directly regulated by miRNAs. By blocking miRNA regulation of all Dvl isoforms using miRNA target protector morpholino oligonucleotides (miRNA TP MASO), we observed dose-dependent defects of the length of the dorsal ventral rods and patterning of the primary mesenchyme cells. We will identify the molecular mechanism of how miRNA regulation of Dvl impacts early development.
20. Evolution of the Wnt/ β -catenin signaling pathway in metazoans: Insights from sea anemones and sea urchins Sun, Hongyan (University of Miami, Coral Gables, FL, USA); Cabrera, Claudia (Department of Biology, University of Miami, USA); Wikramanayake, Athula (Department of Biology, University of Miami, USA)
The Wnt/β-catenin (cWnt) pathway is highly conserved from non-bilaterians to bilaterians, however whether all critical components of the pathway function similarly across taxa is not known. Here, we focus on Axin, a critical negative regulator of cWnt signaling to test if this protein is functionally conserved between sea urchins and the cnidarian Nematostella. In bilaterians, Axin interacts with β-catenin through a conserved β-catenin binding domain and targets it for degradation. Intriguingly, Axin in non-bilaterians lack the β-catenin binding domain, questioning a conserved role for this critical negative regulator in regulating β-catenin stability across taxa. To address this question, we first examined sea urchin Axin function in sea urchins by morpholino-mediated knockdown of sea urchin Axin in sea urchin embryos and determining the spatial and temporal expression of gene markers for different germ layers in the Axin knock-down embryos. Knock down of sea urchin Axin led to upregulation of endomesodermal markers indicating that cWnt signaling was ectopically activated in the absence of Axin. To directly test the idea that Axin was negatively regulating cWnt signaling in animal half blastomeres, we isolated animal halves from Axin morpholino (AxinMO) injected embryos and asked if downregulating Axin in mesomeres would activate endomesoderm. The results showed that downregulating Axin in the mesomeres activated endomesoderm indicating that cWnt signaling was ectopically activated in these cells. To determine if a non-bilaterian Axin is functionally active in bilaterians, we overexpressed sea urchin and Nematostella Axin in sea urchin embryos. Overexpression of sea urchin Axin in sea urchin embryos led to downregulation of cWnt signaling, but overexpression of Nematostella Axin in sea urchin embryos had no effect. However, when we deleted the β-catenin binding domain of sea urchin Axin (SpAxin △β-cat) to mimic NvAxin, SpAxin △β-cat was still able to inhibit endomesoderm formation. In sum, these results indicated that sea urchin Axin globally downregulated cWnt signaling in sea urchins, and that Nematostella Axin had no apparent effect on cWnt signaling in sea urchins. Moreover, our results indicated that SpAxin △β-cat may have additional domains that interact with β-catenin and regulate its stability in sea urchins. Elucidating how Axin regulates Wnt signaling in non-bilaterians and bilaterians will provide critical insight into the early evolution of the Axin/destruction complex in the Wnt signaling pathway. 30 Poster
21. Reconstructing Ancestral Development in Echinoderm Evolutionary Developmental Biology Thompson, Jeffrey R. (University of Southern California, Los Angeles, CA, USA); Erkenbrack, Eric (Yale University, USA)
Understanding how alterations to gene regulatory networks (GRNs) translate to the evolution of developmental processes is a central focus of evolutionary developmental biology. Research programs involved in illuminating this process prioritize representatives of a few taxa that either have plentiful molecular data or are amenable to laboratory research—or both. A consequence of this bounded research program is that we are frequently forced to make evolutionary statements about GRN evolution utilizing data from a few key outgroups, and by appealing to parsimonious explanations of character evolution. Furthermore, the evolutionary events that underlie GRN and developmental evolution occurred throughout the long course of evolutionary time, and are thus associated with marked uncertainty. We report here that these issues can be accounted for by employing ancestral state reconstruction, an under utilized phylogenetic comparative method used to estimate the evolutionary path of character evolution. Here, we use three different methods of ancestral state reconstruction to examine the evolution of alx1-expressing cells in echinoderm taxa. Our analyses support the counter intuitive hypothesis that the cidaroid state of mesenchyme ingression is a derived novelty unique to cidaroids and likely did not exist in the most recent common ancestor of euechinoids and cidaroids. Our study highlights the difficult nature of using modern species as a proxy to understanding ancestral conditions and the importance of applying a robust statistical framework in order to unravel these complex evolutionary problems.
22. Using CRISPR-Cas9 gene knockouts to investigate the role of neuropeptides in the early development of the sea urchin Strongylocentrotus purpuratus Wood, Natalie (University College London, Gower Street, GBR); Mattiello, Teresa (Department of Genetics, Evolution and Environment, University College London, Gower Street, London, WC1E 6BT, UK, USA); Rowe, Matt (School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK 3Stazione Zoologica Anton Dohrn, Villa, USA); Cocurullo, Maria (Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy, USA); Arnone, Maria Ina (Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy, USA); Elphick, Maurice (School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK, USA); Oliveri, Paola (Department of Genetics, Evolution and Environment, University College London, Gower Street, London, WC1E 6BT, UK, USA)
My project focuses on the role neuropeptides have in the early development of the sea urchin, Strongylocentrotus purpuratus, with the possibility to uncover new important function(s) for this exciting class of molecules.
Neuropeptides are ancient neuronal signalling molecules that bind to receptor proteins on target cells. A huge variety of neuropeptide precursors and their receptors exist across the bilaterian phyla, and some have been found in non-bilaterian metazoans, such as Cnidaria and Placozoa. Previous studies have shown that neuropeptides are present in animals that lack nerve cells, such as Trichoplax adherens, belonging to the Placozoa phylum (Nikitin 2015). This led to the hypothesis that neuropeptides may have a non-neuronal function. To investigate this hypothesis, we decided to study the pre-neuronal development of the purple sea urchin S.purpuratus.
Thirty-seven neuropeptide genes have so far been identified in the sea urchin genome and at least eleven are expressed in the early phase of embryogenesis. Further some of these are spatially localised. For example, a pedal peptide- like neuropeptide (PPLN1) shows oral/aboral asymmetric expression, suggesting a role in axis specification. Deducing their innovative function(s) will help to elucidate the evolutionary history of these molecules.
To identify the putative function of these neuropeptides we plan to undertake knockdown/out experiments using morpholino antisense oligos and the CRISPR/Cas9 system. We have already successful injected a gRNA targeting the pigment producing gene PKS1, as a positive control which produced greater than 90% albino embryos (Oulhen and Wessel 2016). PKS1 has been a good positive control and proven the efficiency of CRISPR/Cas9 as a genome editing tool in sea urchins.
31 Poster 23. Investigating the role of Wnt signaling and its cis-regulatory basis during the evolution of lecithotrophy Wang, Lingyu (Duke University, Durham, NC, USA); Koop, Demian (University of Sydney, USA); Byrne, Maria (University of Sydney, USA); Wray, Gregory (Duke University, USA)
The evolution from planktotrophy to lecithotrophy in Heliocidaris was accompanied by changes in gene expression and especially the gene regulatory network (GRN). Previous studies suggest evolutionary rewiring and/or acceleration of GRN interactions during the rapid evolution of lecithotrophy. To further study the rewired or accelerated GRN interactions in a finer scale, we have initiated experiments to perturb the early Wnt signaling in embryo development to identify such candidate genes and their interactions.
Wnt signaling is essential for cell fate specification throughout the embryo and it is an important component in the GRN. We have inhibited the Wnt signaling using C59 inhibitor and over-activated it using drug 1-azakenpaullone in the lecithotroph Heliocidaris erythrogramma, with two opposite and strong phenotypes. Next, we are comparing the gene expression changes in the planktotroph and lecithotroph to identify interactions that were rewired or accelerated in the lecithotroph.
Besides looking for what the changes are, we are also looking for how these changes evolved. We plan to investigate this question using the allelic imbalance approach.
We have obtained the transcriptomes of hybrid embryos from planktotrophic male and lecithotrophic female and the parent species. Preliminary data of a subset of genes have shown allelic imbalance is present in the planktotrophic or lecithotrophic alleles in hybrids. We are expanding the analysis to the whole transcriptome to identify all alleles that produce divergent expression levels in the hybrid embryos. This approach is allowing us to locate genes that are likely to carry cis-regulatory changes during lecithotrophy evolution.
Taking together, these studies will further our understanding of how life history shifts during evolution.
24. LvLipoxygenase activity regulates skeletal patterning and PMC migration throughout sea urchin larval skeletogenesis Zuch, Daniel (Boston University, Boston, MA, USA); Dionne, Kristen (Boston University, USA); Piacentino, Michael (Boston University, Boston, MA, USA); Rose, Sviatlana (Boston University, USA); Bradham, Cynthia A. (Boston University, Boston, MA, USA)
Patterning of the sea urchin larval endoskeleton offers a unique platform to gauge developmental plasticity in vivo. The 3D configuration of skeletal elements in mature larvae is delimited by the migration of primary mesenchyme cells (PMCs), which integrate local positional cues within the blastocoel and arrange into a stereotypic pattern, along which they secrete calcium carbonate biomineral. From an RNA-Seq screen for transcripts involved in skeletal patterning, we have identified LvLipoxygenase (LOX) as a potent regulator of PMC migration toward and with respect to the bilateral midline. LOX is expressed in the apical and ventrolateral ectoderm, where it converts arachidonic acid to hydroxyeicosatetraenoic acids (HETEs). HETEs and their hormone-like metabolites have been shown to exhibit autocrine and paracrine signaling properties across a range of organisms. In Lytechinus variegatus, knockdown of LOX expression typically produces embryos with missing bilateral midline skeletal elements and/or elements rotated with respect to the midline. Pharmacological inhibition of LOX with MK886 mimics these effects. The spectrum of observed skeletal defects suggests that LOX products signal to PMCs both locally and at a distance. In addition, MK886 treatment during discrete temporal windows produces skeletons with distinct skeletal patterning defects. These results suggest that tight spatiotemporal regulation of migratory cues throughout skeletogenesis is crucial for appropriate positioning of PMCs and patterning of the resulting larval skeleton. Funding provided by the National Science Foundation.
32
Author Index
A Cary, Greg (Carnegie Mellon University, Pittsburgh, PA, USA) ...... 13 Allen, Raymond L. (Duke University, Durham, NC, USA)7 Cary, Greg (Carnegie Mellon University, USA) ...... 8 Andrus, Avery K. (California State University Long Chang, Wei-Lun (1Institute of Cellular and Organismic Beach, Long Beach, CA, USA) ...... 20 Biology, Academia Sinica, Taipei 11529, Taiwan Arenas-Mena, Cesar (CSI-CUNY, STATEN ISLAND, NY, 2Graduate Institute of Life Sciences, National Defense USA) ...... 21 Medical Ce, USA) ...... 2 Arnone, M. Ina (Stazione Zoologica Anton Dohrn, Chang, WeiLun (Institute of Cellular and Organismic Naples, ITA) ...... 11 Biology, Taipei, TWN) ...... 25 Arnone, M. Ina (Stazione Zoologica Anton Dohrn, USA) Chang, Yi-Cheng (Institute of Cellular and Organismic ...... 27 Biology, Academia Sinica, Taipei 11529, Taiwan, Arnone, Maria I. (Stazione Zoologica Anton Dohrn, USA) USA) ...... 2 ...... 14 Cheeseman, Iain (Whitehead Institute for Biomedical Arnone, Maria Ina (Stazione Zoologica Anton Dohrn, Research, USA) ...... 3 USA) ...... 9 Chen, Jen-Hao (Institute of Cellular and Organismic Arnone, Maria Ina (Stazione Zoologica Anton Dohrn, Villa Biology, Academia Sinica, Taipei 11529, Taiwan, Comunale, 80121 Napoli, Italy, USA) ...... 31 USA) ...... 2 B Chessel, Aline (USA) ...... 8 Cocurullo, Maria (Stazione Zoologica Anton Dohrn, Villa Batzel, Grant (Scripps Institution of Oceanography, San Comunale, 80121 Napoli, Italy, USA) ...... 31 Francisco, CA, USA) ...... 24 Cuomo, Claudia (Stazione Zoologica Anton Dohrn, Ben Tabou de Leon, Smadar (University of Haifa, USA) napoli, ITA) ...... 14 ...... 17 Czarkwiani, Anna (USA) ...... 6 Ben-Tabou de-Leon, Smadar (The University of Haifa, D Haifa, ISR) ...... 13 Ben-Tabou de-Leon, Smadar (University of Haifa, USA) De Tomaso, Tony (UC Santa Barbara, Santa Barbara, ...... 25 CA, USA) ...... 5 Bleich, Markus (Physiological Institute, Christian- Dionne, Kristen (Boston University, USA) ...... 32 Albrechts-University, Kiel, Germany, USA) ...... 26 Dojer, Brielle (Boston University, USA) ...... 8 Bonilla, Alejandro (LaGuardia Community College, USA) Dupont, Sam (SLC, University of Gothenburg, Sweden, ...... 29 USA) ...... 19 Bonilla, Alejandro (LaGuardia Community College, USA) Dylus, David (USA) ...... 6 ...... 24 E Bradham, Cynthia (Boston University, USA) ...... 8 Bradham, Cynthia A. (Boston University, Boston, MA, Edgar, Allison (Duke University, Durham, NC, USA) .... 25 USA) ...... 27, 29, 32 Ellis, Andrea (New Mexico State University, USA) ...... 6 Bradham, Cynthia A. (Boston University, Boston, MA, Elphick, Maurice (School of Biological and Chemical USA) ...... 13, 23 Sciences, Queen Mary, University of London, Mile Browne, William E. (University of Miami, Coral Gables, End Road, London E1 4NS, UK, USA) ...... 31 FL, USA) ...... 5 Erkenbrack, Eric (Yale University, USA) ...... 31 Buckley, Katherine (Department of Immunology, Escriva, Hector (Observatoire Oceanologique de Banyuls University of Toronto, Toronto, ON , USA) ...... 20 sur Mer, Banyuls sur Mer, FRA) ...... 21 Buckley, Katherine M. (University of Toronto, Espinoza, Jose A. (University of California San Diego, Sunnybrook Research Institute, Toronto, ON, CAN) 19 San Diego, CA, USA) ...... 15 Burgess, David (Boston College, Chestnut Hill, MA, USA) F ...... 4 Burgess, David (Boston College, USA) ...... 28 Feuda, Roberto (California Institute of Technology, Burke, Robert (University of Victoria, USA) ...... 10 California, CA, USA) ...... 10 Burke, Robert D. (University of Victoria, Victoria, BC, Flores, Rachel L. (California State University, USA) .... 27 CAN) ...... 10 Fresques, Tara (Brown Univeristy, USA) ...... 24 Byrne, Maria (University of Sydney, USA) ...... 32 Fresques, Tara (Brown University, USA) ...... 7 Byrum, Christine (College of Charleston, Charleston, SC, G USA) ...... 22 Gamache, Courtney (Dickinson College, USA) ...... 3 Byrum, Christine (College of Charleston, USA) ...... 24 C Garm, Anders (University of Copenhagen, USA) ...... 27 Garno, Chelsea (New Mexico State University, USA) .... 3 Cabrera, Claudia (Department of Biology, University of George, Andrew N. (Duke University, Durham, NC, USA) Miami, USA) ...... 30 ...... 15 Cary, Greg (Carnegie Mellon University, Pittsburgh, PA, Gildor, Tsvia (The University of Haifa, USA) ...... 13 USA) ...... 17 Gildor, Tsvia (University of Haifa, USA) ...... 17 Gildor, Tsvia (University of Haifa, USA) ...... 25
Gomez Skarmeta, Jose Luis (Observatoire Livingston, Brian (California State University Long Beach, Oceanologique de Banyuls sur Mer, USA) ...... 21 USA) ...... 20 Grant, Daisy (Mississippi State University, USA) ...... 26 Livingston, Brian T. (Cal State Long Beach, Long Beach, Grunbaum, Daniel (University of Washington, USA) .... 24 CA, USA) ...... 27 Guay, Catherine (Rutgers, USA) ...... 16 Lopez, Lupita (California State University, Fountain H Valley, CA, USA) ...... 27 Lowe, Elijah K. (Stazione Zoologica Anton Dohrn, Napoli, Hadyniak, Sarah (Boston University, USA) ...... 8 ITA) ...... 27 Haillot, Emmanuel (USA) ...... 8 Lowe, Elijah K. (Stazione Zoologica Anton Dohrn, USA) Hamdoun, Amro (Scripps Institution of Oceanography, La ...... 14 Jolla, CA, USA) ...... 15 Lyons, Deirdre (Scripps Institution of Oceanography, Hamdoun, Amro (Scripps Institution of Oceanography, USA) ...... 11 USA) ...... 28, 30 M Hathaway, Devon (College of Charleston, USA) ...... 24 Maboloc, Elizaldy (Hong Kong University of Science, Henry, Jonathan (University of Illinois Urbana- USA) ...... 24 Champaign, USA) ...... 11 Maeso, Nacho (Observatoire Oceanologique de Banyuls Henson, John (Dickinson College, USA) ...... 3, 6 sur Mer, USA) ...... 21 Hernandez, Felipe (Institut Biologie Valrose, USA) ...... 12 Majic, Paco (MMBS, The Univ. of Tokyo, USA) ...... 6 Hernandez, Felipe (USA) ...... 8 Malik, Assaf (USA) ...... 25 Himmerkus, Nina (Physiological Institute, Christian- Manahan, Donal T. (University of Southern California, Albrechts-University, Kiel, Germany, USA) ...... 26 Los Angeles, CA, USA) ...... 20 Hinman, Veronica (Carnegie Mellon U, Pittsburgh, PA, Marletaz, Ferdinand (Observatoire Oceanologique de USA) ...... 17 Banyuls sur Mer, USA) ...... 21 Hinman, Veronica (Carnegie Mellon University, USA)8, 9, Martinez-Bartolome, Marina (Mississippi State University, 13 USA) ...... 16 Ho, Eric (University of Toronto, Toronto, ON, CAN) ..... 19 Mashanov, Vladimir (University of North Florida, Rio Hu, Marian Y. (Institute of Physiology, Christian- Piedras, PR, USA) ...... 28 Albrechts University of Kiel, Germany, USA) ...... 26 Mattiello, Teresa (Department of Genetics, Evolution and Hu, Marian Y. (Institute of Physiology, Christian- Environment, University College London, Gower Albrechts-University of Kiel, Germany, USA) ...... 19 Street, London, WC1E 6BT, UK, USA) ...... 31 Huang, Katherine (USA) ...... 13 McCauley, Brenna (USA) ...... 13 Huth, James (Boston University, Boston, MA, USA) .... 27 McClay, David (Duke University, USA) ...... 7, 14, 15 I McDougall, Alex (UPMC Villefranche sur mer, USA) ..... 4 McFadden, Greg (College of Charleston, USA) ...... 24 Irimia, Manu (Observatoire Oceanologique de Banyuls sur Mer, USA) ...... 21 Meaders, Johanathan (Boston College, USA) ...... 4 Irons, Zoe (Dickinson College, USA) ...... 3 Meaders, Johnathan (Boston College, USA) ...... 28 K Molina Jimenez, Maria Dolores (Institut Biologie Valrose, Nice, FRA) ...... 8 Kensuke, Takatani (MMBS, The Univ. of Tokyo, USA) .. 6 Molina, Maria Dolores (Insitut Biologie Valrose, USA) . 12 Khalaily, Lama (University of Haifa, Haifa, ISR) ...... 17 Mollett, Dan (University of Victoria, USA) ...... 10 Khalialy, Lama (The University of Haifa, USA) ...... 13 Morgulis, Miri (The University of Haifa, USA) ...... 13 Kitchloo, Shweta (Boston University, USA) ...... 8 Morton-Curl, Sarah (University of California San Diego, Kondo, Mariko (The Univ. of Tokyo, Miura, Kanagawa, USA) ...... 15 JPN) ...... 6 N Koop, Demian (University of Sydney, USA) ...... 32 Nam, Jongmin (Rutgers University-Camden, USA) ...... 21 L Nam, Jongmin (Rutgers, USA) ...... 16 Lawton, Matthew (Boston University, USA) ...... 8 Nesbit, Katherine (Scripps Institution of Oceanography, Lee, Jay-Ron (ICOB, Academia Sinica, Taiwan, USA) 19 La Jolla, CA, USA) ...... 28 Lee, Pei Yun (University of California Los Angeles, Los Nicklisch, Sascha (University of California San Diego, Angeles, CA, USA) ...... 22 USA) ...... 15 Lein, Etienne (Helmholtz Centre for Ocean Research Kiel Nizhnik, Anastasia (Boston University, Boston, MA, USA) (GEOMAR), Kiel, Germany, USA) ...... 19 ...... 29 Lénárt, Peter (USA) ...... 29 O Lepage, Thierry (CNRS, Nice, FRA) ...... 12 Okada, Akari (MMBS, The Univ. of Tokyo, USA) ...... 6 Lepage, Thierry (USA) ...... 8 Oliveri, Paola (Department of Genetics, Evolution and Li, Han-Ru (Institute of Cellular and Organismic Biology, Environment, University College London, Gower Academia Sinica, Taipei 11529, Taiwan, USA) ...... 2 Street, London, WC1E 6BT, UK, USA) ...... 31 Lin, Kuan-Ting (Institute of Cellular and Organismic Oliveri, Paola (University College London, London, GBR) Biology, Academia Sinica, Taipei 11529, Taiwan, ...... 6 USA) ...... 2 Omori, Akihito (MMBS, The Univ. of Tokyo, USA) ...... 6
Onorato, Thomas M. (LaGuardia Community College, Seaver, Elaine (University of Florida Whitney Marine Lab, Long Island City, NY, USA) ...... 24, 29 Saint Augustine, FL, USA) ...... 11 Onorato, Thomas M. (LaGuardia Community College, Sepulveda, Silvia (New Mexico State University, USA) .. 6 Long Island City, NY, USA) ...... 23 Sher, Noa (USA) ...... 25 Oulhen, Nathalie (Brown University, Providence, RI, Shirai, Miwa (USA) ...... 13 USA) ...... 5 Shuster, C. Bradley (New Mexico State University, USA) Oulhen, Nathalie (Brown University, USA) ...... 23, 24 ...... 3 Overcash, Melanie (College of Charleston, USA) ...... 24 Shuster, Charles b. (New Mexico State University, Las P Cruces, NM, USA) ...... 6 Siegwald, Paul (College of Charleston, USA) ...... 24 Pai, Chih-Yu (Institute of Cellular and Organismic Slota, Leslie (Duke University, Durham, NC, USA) ...... 14 Biology, Academia Sinica, Taipei 11529, Taiwan, USA) ...... 2 Smith, Lauren (Department of Biology, University of Miami, USA) ...... 7 Perillo, Margherita (Boston College, chestnut hill, MA, USA) ...... 9 Somogyi, Kalman (USA) ...... 29 Perry, Kimberly (University of Illinois Urbana-Champaign, Song, Jia L. (University of Delaware, Newark, DE, USA) USA) ...... 11 ...... 19 Peter, Isabelle (California Institute of Technology, Song, Jia L. (University of Delaware, USA) ...... 30 USA)10 Stepicheva, Nadezda (University of Delaware, USA) .. 19, Peter, Isabelle (Caltech, Pasadena, CA, USA) ...... 12 30 Peter, Isabelle (Caltech, USA) ...... 14 Stocks, Christian D. (Mississippi State University, USA) ...... 26 Piacentino, Michael (Boston University, Boston, MA, USA) ...... 32 Stumpp, Meike (Zoological Institute, Taipei, TWN) ...... 26 Piñeiro López, Cristina (EMBL, HEIDELBERG, DEU) .. 29 Stumpp, Meike (Zoological Institute, Taipei, TWN) ...... 19 Piovani, Laura (USA) ...... 6 Su, Yi-Hsien (ICOB, Academia Sinica, Taiwan, USA) .. 19 Q Su, Yi-Hsien (Institute of Cellular and Organismic Biology Academia Sinica, Taipei, TWN) ...... 2 Quirin, Magali (Insitut Biologie Valrose, USA) ...... 12 Sugni, Michela (University of Milano, USA) ...... 6 Quirin, Magali (USA) ...... 8 Sun, Hongyan (University of Miami, Coral Gables, FL, R USA) ...... 30 Swalla, Billie J. (University of Washington, Seattle, WA, Range, Ryan (Mississippi State University, USA) .. 16, 26 USA) ...... 17 Range, Ryan (University of Miami, USA) ...... 12 Swartz, S. Zachary (USA) ...... 5 Range, Ryan C. (Mississippi State University, Mississippi Swartz, Zak (Whitehead Institute for Biomedical State, MS, USA) ...... 9 Research, Cambridge, MA, USA) ...... 3 Rast, Jonathan (Department of Immunology, University T of Toronto, Toronto, ON , USA) ...... 20 Rast, Jonathan (University of Toronto, Toronto, ON, Tadros, Albert (LaGuardia Community College, USA) . 24 CAN) ...... 19 Tanaka, Hiroyuki (Hokkaido University, USA) ...... 12 Reardon, Riley (Duke University, USA) ...... 7 Technau, Ulrich (University of Vienna, Vienna, AUT) ..... 1 Reidy, Patrick (Boston University, USA)...... 8 Thompson, Jeffrey R. (University of Southern California, Romano, Laura (Denison University, Granville, OH, USA) Los Angeles, CA, USA) ...... 31 ...... 22 Toledo, Leslie (New Mexico State University, USA) ...... 6 Roopin, Modi (The university of Haifa, USA) ...... 13 Tseng, Yung-Che (Institute of Cellular and Organismic Rose, Sviatlana (Boston University, USA) ...... 32 Biology, Academia Sinica, Taiwan, USA) ...... 19 Rosenblatt, Hannah (Scripps Institution of U Oceanography, USA) ...... 30 Ullrich-Lüter, Esther (Museum fuer Naturkunde Berlin, Rowe, Matt (School of Biological and Chemical Sciences, USA) ...... 27 Queen Mary, University of London, Mile End Road, London E1 4NS, UK 3Stazione Zoologica Anton V Dohrn, Villa, USA) ...... 31 Valencia, Jon (California Institute of Technology, USA) 10 S Valencia, Jon E. (Caltech, Pasadena, CA, USA)...... 14 Salgado, Torey (New Mexico State University, USA) ..... 6 Vargas, Henry (LaGuardia Community College, USA) 24, Sampilo, Nina Faye (University of Delaware, Newark, 29 DE, USA) ...... 30 Veras, Ingrid D. (LaGuardia Community College, USA) Schatzberg, Daphne (Boston University, Boston, MA, ...... 23, 24 USA) ...... 8 Veras, Ingrid D. (LaGuardia Community College, USA) 29 Schrankel, Catherine S. (University of Toronto, Toronto, W ON, CAN) ...... 19 Schrankel, Catherine S. (University of Toronto, Toronto, Wang, Lingyu (Duke University, Durham, NC, USA) ...... 7 ON, CAN) ...... 20 Wang, Lingyu (Duke University, Durham, NC, USA) .... 32 Schuh, Nicholas (University of Toronto, Toronto, ON, Wang, Lingyu (University of Miami, USA) ...... 30 CAN) ...... 19 Wang, Lingyu (USA) ...... 5
Wessel, Gary (Brown University, USA) ...... 7 Wessel, Gary M. (Brown University, USA) ...... 23, 24 Wessel, Gary M. (USA) ...... 5 Wikramanayake, Athula (Department of Biology, University of Miami, USA) ...... 30 Wikramanayake, Athula (USA) ...... 5 Wikramanayake, Athula H. (University of Miami, Miami, FL, USA) ...... 7 Wikramanayake, Athula H. (University of Miami, Miami, FL, USA) ...... 1 Wikramanayake, Athula H. (University of Miami, USA) 30 Williams, Erik (Dickinson College, USA) ...... 3 Wolff, Andrew (Carnegie Mellon University, Pittsburgh, PA, USA) ...... 17 Wolff, Andrew (Carnegie Mellon University, Pittsburgh, PA, USA) ...... 9 Wolff, Andrew (Carnegie Mellon University, USA)...... 8 Wood, Natalie (University College London, Gower Street, GBR) ...... 31 Wray, Greg (Duke University, USA) ...... 25 Wray, Gregory (Duke University, USA) ...... 32 Wray, Gregory (Duke University, USA) ...... 18 Wu, Wei (University of Miami, Coral Gables, FL, USA) .. 7 Wu, Wei (University of Miami, USA) ...... 30 Y
Yaguchi, Junko (University of Tsukuba, USA) ...... 12 Yaguchi, Shunsuke (University of Tsukuba, USA) ...... 12 Yajima, Mamiko (Brown University, Providence, RI, USA) ...... 4 Yan, Jia-Jiun (ICOB, Academia Sinica, Taipei, Taiwan, USA) ...... 26 Z
Zaidi, Syed A. (University of Delaware, USA) ...... 30 Zheng, Minyan (Carnegie Mellon University, Pittsburgh, PA, USA) ...... 17 Zheng, Minyan (Carnegie Mellon University, Pittsburgh, PA, USA) ...... 8 Zuch, Daniel (Boston University, Boston, MA, USA) .... 29 Zuch, Daniel (Boston University, Boston, MA, USA) .... 32 Zuch, Daniel T. (Boston University, Boston, MA, USA) 27
Raymond Allen Keith Alvares Duke University Northwestern University [email protected] [email protected]
Avery Andrus Cesar Arenas-Mena California State University Long Beach CSI-CUNY [email protected] [email protected]
M. Ina Arnone Grant Batzel Stazione Zoologica Anton Dohrn Scripps Institution of Oceanography [email protected] [email protected]
Smadar Ben-Tabou de-Leon Alejandro Bonilla The University of Haifa LaGuardia Community College [email protected] [email protected]
Cynthia Bradham William Browne Boston University University of Miami [email protected] [email protected]
David Burgess Robert Burke Boston College University of Victoria [email protected] [email protected]
Christine Byrum R. Andrew Cameron College of Charleston California Institute of Technology [email protected] [email protected]
Greg Cary WeiLun Chang Carnegie Mellon University Institute of Cellular and Organismic Biology [email protected] [email protected]
James Coulombe Claudia Cuomo NATIONAL INSTITUTES OF HEALTH Stazione Zoologica Anton Dohrn [email protected] [email protected]
Tony De Tomaso Allison Edgar UC Santa Barbara Duke University [email protected] [email protected]
Susan Ernst Hector Escriva Tufts University Observatoire Oceanologique de Banyuls sur Mer [email protected] [email protected]
Jose Espinoza Roberto Feuda University of California San Diego California Institute of Technology [email protected] [email protected]
Tara Fresques Courtney Gamache Brown University, Wessel Lab Dickinson College [email protected] [email protected]
Andrew George Tsvia Gildor Duke University University of Haifa [email protected] [email protected]
Daisy Grant Catherine Guay Mississippi State University Rutgers, The State University of NJ [email protected] [email protected]
Amro Hamdoun Devon Hathaway Scripps Institution of Oceanography College of Charleston [email protected] [email protected]
John Henson Veronica Hinman Dickinson College Carnegie Mellon U [email protected] [email protected]
James Huth Richard Hynes Boston University MIT [email protected] [email protected]
Eric Ingersoll Naoki Irie Penn State Abington University of Tokyo [email protected] [email protected]
Zoe Irons Derk Joester Dickinson College Northwestern University [email protected] [email protected]
Lama Khalaily Mariko Kondo University of Haifa The Univ. of Tokyo [email protected] [email protected]
Pei Yun Lee Thierry Lepage University of California Los Angeles CNRS [email protected] [email protected]
Brian Livingston Lupita Lopez Cal State Long Beach California State University [email protected] [email protected]
Elijah Lowe Deirdre Lyons Stazione Zoologica Anton Dohrn Scripps Institution of Oceanography [email protected] [email protected]
Donal Manahan Marina Martinez-Bartolome University of Southern California Mississippi State University [email protected] [email protected]
Vladimir Mashanov David McClay University of North Florida Duke University [email protected] [email protected]
Johnathan Meaders Maria Dolores Molina Jimenez Boston College Institut Biologie Valrose [email protected] [email protected]
Jongmin Nam Katherine Nesbit Rutgers-Camden Scripps Institution of Oceanography [email protected] [email protected]
Anastasia Nizhnik Paola Oliveri Boston University University College London [email protected] [email protected]
Thomas Onorato Nathalie Oulhen LaGuardia Community College Brown University [email protected] [email protected]
Margherita Perillo Isabelle Peter Boston College Caltech [email protected] [email protected]
Cristina Piñeiro López Ryan Range EMBL Mississippi State University [email protected] [email protected]
Jonathan Rast Laura Romano University of Toronto Denison University [email protected] [email protected]
Hannah Rosenblatt Nina Faye Sampilo Scripps Institute of Oceanography University of Delaware [email protected] [email protected]
Daphne Schatzberg Catherine Schrankel Boston University University of Toronto [email protected] [email protected]
Elaine Seaver Silvia Sepulveda University of Florida Whitney Marine Lab New Mexico State University [email protected] [email protected]
Charles Shuster Leslie Slota New Mexico State University Duke University [email protected] [email protected]
Jia Song Meike Stumpp University of Delaware Zoological Institute [email protected] [email protected]
Yi-Hsien Su Hongyan Sun Institute of Cellular and Organismic Biology University of Miami Academia Sinica [email protected] [email protected]
Billie Swalla Zak Swartz University of Washington Whitehead Institute for Biomedical Research [email protected] [email protected]
ALBERT TADROS Ulrich Technau LAGUARDIA COMMUNITY COLLEGE University of Vienna [email protected] [email protected]
Jeffrey Thompson Jon Valencia University of Southern California Caltech [email protected] [email protected]
Ingrid Veras Lingyu Wang LaGuardia Community College Duke University [email protected] [email protected]
Gary Wessel Athula Wikramanayake Brown University University of Miami [email protected] [email protected]
Andrew Wolff Natalie Wood Carnegie Mellon University University College London [email protected] [email protected]
Gregory Wray Wei Wu Duke University University of Miami [email protected] [email protected]
Shunsuke Yaguchi Mamiko Yajima University of Tsukuba Brown University [email protected] [email protected]
Minyan Zheng Daniel Zuch Carnegie Mellon University Boston University [email protected] [email protected]