Cover and back cover designed by Laurel Hiebert – Drawings modified fromLahille, M.F., 1890. Contributions à l’étude anatomique et taxonomique des Tuniciers. Ph.D. Thesis, Fac. Sciences Paris. Toulouse. ORGANIZING COMMITTEE Stefano Tiozzo Rémi Dumollard Alexandre Alié Janet Chenevert Elisabeth Christians Clare Hudson Alex McDougall Hitoyoshi Yasuo WEBSITE AND COMMUNICATION COMMITTEE Faisal Bekkouche Frédéric Bonino Delphine Dauga SCIENTIFIC COMMITTEE Rémi Dumollard (Sorbonne University, CNRS – France) Stefano Tiozzo (Sorbonne University, CNRS – France) Cristian Cañestro (Universitat de Barcelona – Spain) Bo Dong (Ocean University of China – China) Kaoru Imai (Osaka University – Japan) Kazuo Inaba (University of Tsukuba – Japan) Marie Nydam (Centre College – USA) Rosana Rocha (Universidade Federal do Paraná – Brazil) Ute Rothbächer (Universität Innsbruck – Austria) Antonietta Spagnuolo (Stazione Zoologica Anton Dohrn – Italy) Bob Zeller (San Diego State University – USA) CONTENTS
GENERAL INFORMATION...... pag. 5
INFORMATION ON THE MEETING...... pag. 7
PROGRAM AT GLANCE...... pag. 8
PROGRAM...... pag. 10
TALKS ABSTRACTS...... pag. 15
POSTERS ABSTRACTS...... pag. 85
LIST OF PARTICIPANTS...... pag. 151
SPONSORS AND HOST INSTITUTIONS...... pag. 156 GENERAL INFORMATION How to get here
• From the Airport: Bus service from the airport dedicated airport services, lines 98 start at Termi- Nice Côte d’Azur to Villefranche (15 km). nal 2, before picking up passengers at Terminal • By Train: Frequent service between the stations of 1. An Aéro ticket costs 6€ one-way and can be pur- Nice and Villefranche-sur-Mer (Take bus service or chased on board. The 98 runs frequently, three ti- Taxi, see details below). mes an hour - the first at 05:50 and the last 23:50. • By car: Exit from the autoroute A8 at “Nice - Pro- The ticket is valid for 74 minutes and allows for menade des Anglais” onto the “Promenade des one connecting voyage on any urban bus. To go Anglais” continue along the coast road towards to Villefranche sur Mer, take the bus 98, and get Monaco. Alternatively, exit the autoroute A8 at off at the bus stop “Promenade des Arts”. When “Nice Est”, follow the signs toward the Port (of you arrive at “Promenade des art” take the bus 81 Nice) and from the Port take the “Basse Corniche” (frequently 2 time an hour) in direction “Port de towards Monaco. Saint Jean”. After 15mn you arrive at Villefranche • By Taxi or UBER: You can find many taxis at both sur Mer and get off at the bus stop “Saint Esteve” airport terminals, which can take you to Villefran- if you stay at the Flore Hotel. To go to the Labora- che. Price is around 60€. Remember to have cash tory LBDV, the workshop and your hotel, follow ready to pay the driver (cash points and exchange the Avenue of “Général de Gaulle” and continue bureaux are handily located in both airport termi- along the “Darse port” then towards the “Station nals). For the return is cheaper to call a taxi in Vil- Zoologique”. For the return, take the bus at bus lefranche-sur-Mer. UBER works very well and it is stop “OCTROI” and drop off at the terminal sta- cheaper. tion “Garibaldi”, and take the return bus at the • By Bus: Ligne d’Azur, the city’s bus operator, runs “Lycée Massena” bus stop.
5 Getting around Villefranche sur Mer
The Citadel
6 INFORMATION ON THE MEETING
Registration Lunches and coffee breaks The Registration desk is situated next to the lecture Coffee breaks and lunch boxes will be served outside hall and will be open Sunday 7th from 5pm to 8pm the lecture hall and can be consumed in the surroun- and Monday 8th starting from 8:15am. ding area or at the beach.
Wifi internet access Gala dinner Wifi is available at the venue, you can login without The closing dinner will be served in the citadel at the a password. gazebo.
Oral Presentations Afternoon off 20 minutes: 15 min talk and 5 min discussion (Talks) In the afternoon of Wednesday 10th there won’t be any 40 minutes: 35 min talk and 5 min discussion (Plenary lectures or sessions. Details about the two program- of Session 9) med activities, i.e. the visit to the Monaco Oceano- 1 hour: 45 min talk and 15 min discussion (Plenary graphic Museum and the diving in the Villefranche lectures) bay will be given during the meeting. For those who Speakers will be asked to upload their presentation are not participating at these two events, the area of- from their USB pen on a PC or Mac provided in the fers several amenities: tourist material will be inclu- lecture hall. The speakers are kindly requested to ded in the conference bag, and the organizers will be avoid the use of their computers for the presentation, happy to direct you to points of interests (beaches, if strictly necessary the lecture hall is provided with museums, hiking trails, etc.). VGA and HDMI plugs. 10th ITM T-Shirts Posters The T-shirt of the 10th ITM will be available for pur- The posters (format A0) will be displayed at the ma- chase everyday at the entrance of the lecture hall. rine station IMEV in the Jules Barrois building (see map). Poster boards will be numbered as in the pro- gram. The poster can be hung the same day either during the lunch break or before the poster session. The poster should be removed at the end of each poster session. A list with the names of all the 10th ITM participant will be available by the lecture hall, if you wish so- meone in particular to visit your poster, you have to write the poster number by her/his name.
7 SUNDAY MONDAY TUESDAY JULY 7TH JULY 8TH JULY 9TH
8:15 REGISTRATION
9:00 OPENING REMARKS 9:00
9:15 SESSION 1 ECOLOGY SESSION 5 BEGINNING OF 10:15 COFFEE BREAK EMBRYOGENESIS
10:35 SESSION 2 IMAGING AND MODEL- 10:40 COFFEE BREAK LING 11:00 SESSION 6 EVOLUTION, SYSTEMATI- CS AND TAXONOMY 11:35 SESSION 3 ASEXUAL REPRODUCTION, REGENERATION AND AGING 12:20 LUNCH BREAK 12:35 LUNCH BREAK
SESSION 3 14:00 14:00 14:00 SESSION 6 ASEXUAL REPRODUCTION, EVOLUTION, SYSTEMATI- REGENERATION AND CS AND TAXONOMY AGING
WORKSHOP WASHU 15:20 COFFEE BREAK 15:20 SESSION 7 (MARINE STATION IMEV - LATE EMBRYO AND GALERIENS BLDG.) 15:40 SESSION 4 LARVAE FROM DEVELOPMENT TO GENOMICS 16:00 COFFEE BREAK
16:20 SESSION 7 LATE EMBRYO AND LARVAE 17:00 REGISTRATION POSTER SESSION 1 (CITTADELLE DE VILLE- 17:40 FRANCHE SUR MER) ODD NUMBERS (MARINE STATION IMEV - J.BARROIS BLDG.)
8 WEDNESDAY THURSDAY FRIDAY JULY 10TH JULY 11TH JULY 12TH
PLENARY LECTURE 9:00 9:00 9:00 SESSION 8 PATRICK LEMAIRE NERVOUS SYSTEM SESSION 9 NON TUNICATE AQUATIC DEVELOPMENT 10:00 MODELS COFFEE BREAK 10:15 COFFEE BREAK 10:20 COFFEE BREAK 10:20
SESSION 9 10:40 10:40 SESSION 8 NON TUNICATE AQUATIC NERVOUS SYSTEM MODELS SESSION 12 DEVELOPMENT GENE REGULATION 11:20 SESSION 10 DEVELOPMENTAL 11:40 AND CELL BIOLOGY PLENARY LECTURE STEVE WILSON 12:20 12:20 LUNCH BREAK LUNCH BREAK 12:40 EXCURSION - DIVING
SESSION 12 14:00 14:00 SESSION 10 GENE REGULATION DEVELOPMENTAL AND CELL BIOLOGY 14:40 GROUP PHOTO 15:00 COFFEE BREAK 15:10 ROUND TABLES GENERAL DISCUSSION 15:20 SESSION 11 POST-EMBRYONIC DEVELOPMENT 16:50 CLOSING REMARKS 17:00 POSTER SESSION 2 EVEN NUMBERS (MARINE STATION IMEV - 19:30 J.BARROIS BLDG.) GALA DINNER
9 PROGRAM Sunday, July 7, 2019 Marine Station IMEV (Tregouboff Room);
14:00 - 17:00 Workshop WashU, ANISEED Genome Browser Citadelle de Villefranche-sur-mer 17:00 - 20:00 REGISTRATION
Monday, July 8, 2019 Citadelle de Villefranche-sur-mer
8:15 - 9:00 REGISTRATION 9:00 - 9:15 OPENING REMARKS - Remi Dumollard and Stefano Tiozzo
9:15 - 10:15 Session 1 - ECOLOGY (chair: Marie Nydam) 9:15 - 9:35 T1 Xavier Turon: “Local microbiome of a global invader” 9:35 - 9:55 T2 Gal Vered: “Using solitary ascidians to assess microplastic and phthalate plasticizers pollution in marine environments” 9:55-10:15 T3 Vittoria Roncalli: “Transcriptomics of the developmental response of Oikopleura dioica to diatom-bloom derived biotoxins”
10:15 - 10:35 COFFEE BREAK
10:35 - 11:35 Session 2 - IMAGING AND MODELLING (chair: Alex McDougall) 10:35 - 10:55 T4 Benoit Godard: “Tension driven engulfment of non-mitotic by mitotic tissue determines cell division orientation in ascidian embryos” 10:55 - 11:15 T5 Hidehiko Hashimoto: “Differential expression of Cadherin2 patterns RhoA and Myosin II activity to drive zippering and neural tube closure in a simple chordate.” 11:15 - 11:35 T6 Emmanuel Faure: “MorphoNet: An interactive online morphological browser to explore complex multi-scale data” 11:35 - 12:35 Session 3 - ASEXUAL REPRODUCTION, REGENERATION, AND AGING (chairs: William Jeffery, Anthony De Tomaso) 11:35 - 11:55 T7 Simon Blanchoud: “Investigating the initiation of whole-body regeneration in Botrylloides leachii” 11:55 - 12:15 T8 Megan Wilson: “Epigenetic regulation of Botrylloides leachii whole body regeneration” 12:15 - 12:35 T9 Susannah H. Kassmer: “Primordial Blasts, a population of blood-borne stem cells responsible for whole body regeneration in a basal chordate”
12:35 - 14:00 LUNCH BREAK
14:00 - 15:20 Session 3 - ASEXUAL REPRODUCTION, REGENERATION, AND AGING (chairs: William Jefferey, Anthony De Tomaso) 14:00 - 14:20 T10 Marta Scelzo: “Novel budding mode in Polyandrocarpa zorritensis: a model for comparative studies
10 on asexual development and whole body regeneration” 14:20 - 14:40 T11 Bill Jeffery: “Dying to Regenerate: Apoptosis, Wnt Signaling, and Polarity of Body Regeneration in Ciona intestinalis” 14:40 - 15:00 T12 Delany Rodriguez: “Aging and Senescence Effects on the Extracorporeal Vasculature of Botryllus schlosseri” 15:00 - 15:20 T13 Alexandre Alié: “Evo-Devo approaches to non-embryonic developments in colonial tunicates”
15:20 - 15:40 COFFEE BREAK
15:40 - 17:00 Session 4 - FROM DEVELOPMENT TO GENOMICS (chair: Kaoru Imai) 15:40-16:00 T14 Chiara Anselmi: “Molecular and Morphological Atlas of Botryllus schlosseri Developmental Pathways” 16:00-16:20 T15 Elijah Lowe: “Identification of key morphogenesis genes in Molgulidae with alternate body plans and their interspecific hybrid embryos” 16:20-16:40 T16 Billie Swalla: “Tailless Molgulid Ascidians have a Notochord “Ball” that never converges or extends” 16:40-17:00 T17 Brad Davidson: “Differential drift among enhancer elements in the tunicate cardiopharyngeal gene network”
Tuesday, July 9, 2019 Citadelle de Villefranche-sur-mer
9:00 - 10:40 Session 5 - BEGINNING OF EMBRYOGENESIS (chair: Takahito Nishikata) 9:00 - 9:20 T18 Masaki Matsuo: “Protein phosphatase 2A is essential to maintain meiotic arrest, and to prevent Ca2+ burst at spawning and eventual parthenogenesis in the larvacean Oikopleura dioica” 9:20 - 9:40 T19 Marianne Roca: “The Spindle Assembly Checkpoint in Phallusia mammillata embryos” 9:40 - 10:00 T20 Toshiyuki Goto “Analyses of spatio-temporal interaction between microtubule and ER or maternal mRNAs in Ciona egg using new high resolution double staining methods” 10:00 - 10:20 T21 Silvia Caballero Mancebo “Actin-dependent contraction drives ooplasmic segregation in ascidian oocytes” 10:20 - 10:40 T22 Yana Mikhaleva: “Gap junction-dependent coordination of intercellular calcium signalling during early embryogenesis of Oikopleura dioica”
10:40 - 11:00 COFFEE BREAK
11:00 - 12:20 Session 6 - EVOLUTION, SYSTEMATICS AND TAXONOMY (chairs: Rosana Rocha, Xavier Turon) 11:00 - 11:20 T23 Benyamin Rosenthal: “Evolutionary Origin of the Mammalian Hematopoietic and Immune Systems Found in a Colonial Chordate” 11:20 - 11:40 T24 Federica Montesanto: “Further revelations on Ciona taxonomic complexity” 11:40 - 12:00 T25 Maria Casso: “Single zooids, multiple loci: Population genomics of a global invader”
11 12:00 - 12:20 T26 Jacques Piette: “The thaliacean Salpa fusiformis : the return of sessile ascidians to pelagic life” 12:20 - 14:00 LUNCH BREAK 14:00 - 15:20 Session 6 - EVOLUTION, SYSTEMATICS AND TAXONOMY (chairs: Rosana Rocha, Xavier Turon) 14:00 - 14:20 T27 Marie Nydam: ”Phylogeny of the Botryllid Ascidians” 14:20 - 14:40 T28 Marika Salonna: “The subfamily Botryllinae: species identification and phylogenetic reconstruction” 14:40 - 15:00 T29 Rosana Rocha: “Diplosoma listerianum: an interesting model of cryptic speciation” 15:00 - 15:20 T30 Katrin Braun: “Phylogenetic analysis of phenotypic characters of Tunicata supports basal Appendicularia and monophyletic Ascidiacea” 15:20 - 16:00 Session 7 - LATE EMBRYO AND LARVAE (chairs: Ute Rothbächer, Bo Dong) 15:20 - 15:40 T31 Sebastien Darras: “Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development” 15:40 - 16:00 T32 Bo Dong: “Coordination of asymmetrical notochord contractility and epithelia cell proliferation drives tail bending in Ciona embryogenesis”
16:00 - 16:20 COFFEE BREAK
16:20 - 17:20 Session 7 - LATE EMBRYO AND LARVAE (chairs: Ute Rothbächer, Bo Dong) 16:20 - 16:40 T33 Yuji Mizotani “Pulsatile Transport of basal factors toward apical domain during tubulogenesis, directed by the interaction between 14-3-3εa and ERM 16:40 - 17:00 T34 Hiroki Nishida: “Neurula rotation and left-right asymmetry in ascidian embryos: Ciliary movements and the vitelline membrane signal” 17:00 - 17:20 T35 Steve Irvine: “The effects of high water temperature on developmental processes in C. intestinalis: limits to embryonic robustness”
Marine Station IMEV - (Jules Barrois Bldg.) 17:40 - 19:40 POSTER SESSION 1 - Odd numbers
Wednesday, July 10, 2019 Citadelle de Villefranche-sur-mer
9:00 - 10:20 Session 8 - NERVOUS SYSTEM DEVELOPMENT AND ANATOMY (chairs: Bob Zeller, Clare Hudson) 9:00 - 9:20 T36 Alberto Stolfi: ”Transcriptional states underlying neuron subtype-specific traits in the Motor Ganglion of Ciona” 9:20 - 9:40 T37: Kouhei Oonuma: “Left-right asymmetric development of cells in the larval brain of Ciona” 9:40 - 10:00 T38: Bob Zeller: “Jagged acts as a lateral inducer to specify caudal epidermal sensory neuron fate in Ciona” 10:00 - 10:20 T39: Nanako Okawa: “In vivo calcium-imaging reveals a possible role of the GnRH system in larval swimming of Ciona”
10:20 - 10:40 COFFEE BREAK
10:40 - 11:40 Session 8 - NERVOUS SYSTEM DEVELOPMENT AND ANATOMY (chairs: Bob Zeller, Clare Hudson) 12 10:40 - 11:00 T40 Izumi Oda-Ishii: “Transcriptional regulatory mechanisms of Tbx6b by Zic-r.a (Macho1) through secondary binding motif sites” 11:00 - 11:20 T41 Kerriane Ryan: “Stereotypy and variation among the larval motor circuits” 11:20 - 11:40 T42 Marios Chatzigeorgiou: “Ciona intestinalis as a neuroethology model” 11:40 - 12:40 PLENARY LECTURE T43 Steve Wilson: “Brain asymmetry – from genes to circuits and behavior” 14:00 FREE TIME (EXCURSION and DIVING)
Thursday, July, 11, 2019 Citadelle de Villefranche-sur-mer
9:00 - 10:20 Session 9 - NON TUNICATE AQUATIC MODELS (chairs: William Smith, Alexandre Alie) 9:00 - 9:40 T44 (PLENARY) Carl-Philipp Heisenberg: “Mechanosensation of tight junctions by ZO-1 phase separation and flow” 9:40 - 10:00 T45 Amro Hamdoun: “Patterning of transport function: How transporters are integrated in early developmental programs of the sea urchin” 10:00 - 10:20 T46 Alex Venn: “The Highs and Lows of pH regulation in Corals and Symbiotic Cnidarians”
10:20 - 10:40 COFFEE BREAK
10:40 - 11:20 Session 9 - NON TUNICATE AQUATIC MODELS (chairs: William Smith, Alexandre Alie) 10:40 - 11:00 T47 Eric Rottinger: “Regeneration is a partial redeployment of the embryonic gene regulatory network” 11:00 - 11:20 T48 Ina Arnone: “Gene regulatory network approaches for gut patterning and evolution”
11:20 -12:20 Session 10 -DEVELOPMENTAL AND CELL BIOLOGY (chairs: Hiroki Nishida, Lionel Christiaen) 11:20-11:40 T49 David Lagman: “Evolutionary novelties in tunicates: Pax37B is essential for the development of giant Fol cells in the house secreting epithelium of Oikopleura dioica” 11:40 - 12:00 T50 Geraldine Williaume: “ephrin-mediated “damping” of FGF signaling underlies the spatial precision of ascidian neural induction” 12:00 - 12:20 T51 Rose Jacobson: “Shp2 Phosphatase is Required for Normal Embryogenesis in C. intestinalis”
12:20 - 14:00 LUNCH BREAK
14:00 - 15:00 Session 10 -DEVELOPMENTAL AND CELL BIOLOGY (chairs: Hiroki Nishida, Lionel Christiaen) 14:00 - 14:20 T52 Christina Cota: “Mitotic Kinases choreograph FGF receptor storage and redistribution during cardiopharyngeal cell” 14:20 - 14:40 T53 Alfonso Ferrández-Roldán: “Braveheart, Oikopleura dioica a cardiogenic loser, but not a heartless chordate” 14:40 - 15:00 T54 Fan Zeng: “A fine analysis of the Ciona larval sensory adhesive organ”
15:00 - 15:20 COFFEE BREAK
13 15:20 - 16:40 Session 11 POST-EMBRYONIC DEVELOPMENT (chair: Cristian Cañestro) 15:20 - 15:40 T55 Yasunori Sasakura: “GABA - mediated GnRH release triggers metamorphosis of Ciona” 15:40 - 16:00 T56 Christopher Johnson: “Molecular profile and function of the axial columnar cells of Ciona papillae” 16:00 - 16:20 T57 Xiaoming Zhang: “miR-4055 regulates Ciona sensory organ morphogenesis through AKT-MAPK signaling pathway” 16:20 - 16:40 T58 Maiki Wakai: “Ca2+ signaling contributes to the beginning of metamorphosis via mechanical stimuli in palps”
Marine Station IMEV - (Jules Barrois Bldg.) 17:00 - 19:00 POSTER SESSION 2 - Even numbers
Friday, July 12, 2019 Citadelle de Villefranche-sur-mer
9:00 - 10:00 PLENARY LECTURE T59 Patrick Lemaire: “Extreme morphogenetic canalization of ascidian embryonic development despite high genetic variability”
10:00 - 10:20 COFFEE BREAK
10:20 - 12:20 Session 12 - GENE REGULATION (chairs: Antonietta Spagnuolo, Sebastien Darras) 10:20 - 10:40 T60 Emma Farley: “Highly Conserved Enhancer Grammar” 10:40 - 11:00 T61 Cristelle Dantec: “Comparative genomic analysis: Identification of novel genes in ascidians” 11:00 - 11:20 T62 Kotaro Shimai: “Working towards a comprehensive and quantitative Ciona notochord gene regulatory network” 11:20 - 11:40 T63 Claudia Racioppi: “Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices” 11:40 - 12:00 T64 Shin-ichi Tokuhiro: “Foxd acts as an activator and a repressor for patterning along the animal-vegetal axis in early embryos” 12:00 - 12:20 T65 Boqi Liu: “Foxg is required for the palp formation in ascidian embryos”
12:20 - 14:00 LUNCH BREAK
14:00 - 14:40 Session 12 GENE REGULATION (chairs: Antonietta Spagnuolo, Sebastien Darras) 14:00 - 14:20 T66 Takehiro Kusakabe: “Evolution of developmental programs for the midline structures in chordates: insights from gene regulation in the floor plate and hypochord homologues of Ciona embryos” 14:20 - 14:40 T67 Burcu Vitrinel: “Discovering post-transcriptional regulators of heart development”
14:40 - 15:10 Christophe TROJANI (Major of Villefranche sur Mer) GROUP PHOTO COFFEE BREAK 15:10 - 16:50 ROUND TABLE - GENERAL DISCUSSION 16:50 - 17:10 CLOSING REMARKS - Remi Dumollard and Stefano Tiozzo 19:30 - 24:00 GALA DINNER - Citadelle de Villefranche-sur-mer
14 TALKS ABSTRACTS T1-T67 T1 LOCAL MICROBIOME OF A GLOBAL INVADER
Xavier Turon1,*, Maria Casso 1,2, Marta Turon1, Nuria Marco2 and Marta Pascual2
1. Centre for Advanced Studies of Blanes (CEAB, CSIC), Blanes, Catalonia, Spain 2. Department of Genetics, University of Barcelona, Barcelona, Catalonia, Spain
Ascidians harbour in their tunics a high diver- but low-diversity core microbiome in ascidians, sity of prokaryote symbionts, which may play as only 8 ZOTUs (0.27%) were present in all col- an important role in the species’ biology and onies, while 47 (1.58%) were present in all local- survival. The global invader Didemnum vexillum ities. However, these ZOTUs comprised 71.31 provides a unique opportunity to assess the pat- and 89.60%, respectively, of the total number of terns of variability of ascidian microbiomes and, reads obtained. The variable component is quan- in particular, whether they remain stable as the titatively much less important but comprises a host species colonises new habitats or whether highly diverse assemblage. This component dif- they shift according to local environment. We fers within localities (0.59 average Bray-Curtis have analysed, using 16S amplicon sequencing, distance), but more so between localities (0.70) the microbiome of 60 D. vexillum colonies be- and between recognized genetic groups (0.74). longing to 2 native (Japan) and 10 introduced Each colony had an average of 11.95% exclusive populations spawning the known range of the ZOTUs, while each locality had a mean of 22.05% species. We found 2,984 zero-radius OTUs (ZO- exclusive ZOTUs. The combination of a quanti- TUs) in the ascidians, belonging to 36 bacterial tatively dominant core component and a highly and 3 archaeal phyla. The ascidian microbiome diverse variable fraction in the microbiome of D. had a markedly different composition from sur- vexillum can contribute to the success of this glob- rounding seawater. We found a high-abundance al invader in different environments.
16 T2 USING SOLITARY ASCIDIANS TO ASSESS MICROPLASTIC AND PHTHALATE PLASTICIZERS POLLUTION IN MARINE ENVIRONMENTS
Gal Vered1,2, Aviv Kaplan3, Dror Avisar3, Noa Shenkar1,4
1. School of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 2. The Interuniversity Institute for Marine Sciences (IUI), Eilat, 88103, Israel 3. The Hydro-Chemistry Laboratory, Porter School for Environment and Earth Sciences, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 69978, ISRAEL. 4. The Steinhardt Museum of Natural History, Israel National Center for Biodiversity Studies, Tel-Aviv University, Tel Aviv, Israel
Marine plastic pollution has become an emerg- of 5064 ± 1806 ng/g dry weight and 9095 ± 4574 ing threat to marine ecosystems worldwide. ng/g dry weight for DBP and DEHP respective- Studying the interaction of microplastic (MP) ly. MP particles were discovered at all the stud- with marine organisms is crucial for understand- ied sites, with mean concentrations of 1.37 ± 1.29 ing the effect that MP and plastic additives may particles per individual. The high levels of pol- have on oceanic environments. However, knowl- lutants found at the studied sites emphasize the edge regarding the magnitude and effects of these need for further research into the magnitude and pollutants in general, and particularly in the East- effects of MP and PAEs in the Eastern Mediter- ern Mediterranean Sea and the tropical Red Sea, ranean and the Red Sea. The use of widely-dis- is still lacking. Here we examined the levels of tributed ascidian species that are able to create common plastic additives, phthalate acid esters large aggregates and interfere with native biota (PAEs), and MP in two widely-distributed ascid- present fundamental opportunities for their use ians, Herdmania momus and Microcosmus exas- in environmental monitoring. Together with the peratus, sampled along the Mediterranean and developed methodology for PAE quantification Red Sea coasts of Israel. High levels of Dibutyl in marine invertebrates this study demonstrates phthalate (DBP) and Bis (2-ethylhexyl) phthalate a strong potential for the use of ascidians for as- (DEHP), were found in ascidians at the majority sessing microplastic and plastic additives pollu- of sampling sites, reaching mean concentrations tion worldwide.
17 T3 TRANSCRIPTOMICS OF THE DEVELOPMENTAL RESPONSE OF OIKOPLEURA DIOICA TO DIATOM-BLOOM DERIVED BIOTOXINS
Vittoria Roncalli*1, Nuria P. Torres-Águila1, Connie Whiting1, Josep Martí-Solans1, Alfonso Ferrández-Roldán1, Alba Almazán1, Salvatore D’Aniello2, Giovanna Romano3, Anna Palumbo2, Ricard Albalat1 and Cristian Cañestro1
1. Department of Genetics, Microbiology and Statistics, & Biodiversity Research Institute (IRBio), University of Barcelona, Spain 2. Stazione Zoologica Anton Dohrn, Napoli, Italy
Understanding the impact that biotoxins de- midline convergence and tail elongation. The ge- rived from microalgal blooms might have on netic response of embryos exposed to biotoxins the development of appendicularians, one of includes global silencing of zygotic transcription the most abundant components of the mesozo- of developmental genes, and a rapid upregula- oplankton, is of prime ecological interest, espe- tion of some defensome genes. We are currently cially upon a possible future intensification of performing a transcriptomic approach by RNA- blooms associated to ocean warming and acidi- seq to reveal differential gene expression pro- fication. Our work, after performing treatments files underlying the phenotypes caused by these with polyunsaturated-aldehydes and extracts biotoxins. Our results reveals that some of these from oxylipin-producing diatoms, reveals that differences can be already detected at the 8-cell embryo development of the appendicularian stage, much earlier than the onset of most of the Oikopleura dioica is compromised by these bio- zygotic transcription. Finally, our study aims to toxins, even at concentrations in the same range provide a catalogue of gene markers that could than those measured after blooms. Analyses of be used as molecular biosensors to monitor the developmental gene markers reveals a novel genetic stress of natural populations exposed to “golf ball” phenotype caused by diatom biotox- microalgal blooms. ins, which includes blockage of morphogenesis,
Reference 1. Torres-Águila NP, Martí-Solans J, Ferrández-Roldán A, Almazán A, Roncalli V, D’Aniello S, Ro- mano G, Palumbo A, Albalat R, Cañestro C. 2018 Diatom bloom-derived biotoxins cause aberrant development and gene expression in the appendicularian chordate Oikopleura dioica. Commun Biol. 1:121. PMID:30272001 18 T4 TENSION DRIVEN ENGULFMENT OF NON-MITOTIC BY MITOTIC TISSUE DETERMINES CELL DIVISION ORIENTATION IN ASCIDIAN EMBRYOS
Godard BG1,2,*, Dumollard R2, Chenevert J2, Munro E3 McDougall A2 and Heisenberg C-P1
1. Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria 2. Sorbonne Université, CNRS, Laboratoire de Biologie du Dévelopement de Villefranche- sur-mer (LBDV) IMEV, 06230, Villefranche-sur-mer, France. 3. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, United States
Even though the invariant cleavage pattern non-mitotic cells possess higher surface tension of ascidians has been described for more than a than mitotic cells and this is associated with an century [1], the mechanism that generates this accumulation of P-myosin at the apical cortex pattern is still unknown. Clues come from the and at the adjacent apical junctions, which both observations that the invariant cleavage pattern lose P-myosin in mitosis. Next, we show that the relies on mitotic cell shape, which is modulated shape of the mitotic cells is influenced by tension by cell cycle asynchrony between the animal and generated by non-mitotic cells with which they vegetal hemispheres [2]. This suggests the pres- are in contact. Finally, by manipulating the spa- ence of spatial and temporal anisotropic tension tial or temporal distribution of tension within the distribution within the embryo with the mitotic embryo we were able to change the shape of mi- cell as the source of mechanical forces. Indeed, totic cell leading to a change in spindle orienta- extensive studies in various model systems have tion and hence cleavage plane. demonstrated that mitotic cell rounding arises In conclusion, we report the first case of mitotic from an increase of the actomyosin cortical ten- cells that do not obey the classical mechanism of sion and hydrostatic pressure [3,4]. In order to mitotic cell rounding. As a consequence of their decipher the mechanics of ascidian blastula, we lower apical surface tension, the mitotic cells of conducted direct measures of the cell surface ten- the ascidian blastula integrate all the mechanical sion and furthermore we manipulate the tension forces present within the embryo. Therefore the distribution within the embryo. remarkable fine-tuning of tension distribution We found that the apical surface tension de- in space and time sustains the evolutionary con- creases when cells round up in mitosis (without served invariant cleavage pattern of ascidian. change of the cell volume), rather than increas- ing as would have been predicted. Conversely,
References 1. Conklin, J. of the Academy of Naural Sciences of Philadelphia, 1905 2. Dumollard et al., ELife, 2017 3. Thery and Bornens, HSFP J., 2008 4. Champion et al., Trends in Cell Biology, 2017 19 T5 DIFFERENTIAL EXPRESSION OF CADHERIN2 PATTERNS RHOA AND MYOSIN II ACTIVITY TO DRIVE ZIPPERING AND NEURAL TUBE CLOSURE IN A SIMPLE CHORDATE.
Hidehiko Hashimoto1,*, Edwin Munro1
1. Department of Molecular Genetics and Cell Biology, University of Chicago, USA
* Presenting Author: [email protected]
Dynamic control of force generation at tissue where RhoA and Myosin II are locally activated. boundaries underlies many forms of morphogen- Equalizing Cadherin2 expression across the Ne/ esis, but these mechanisms remain poorly under- Epi boundary inhibits RhoA and Myosin II acti- stood. Here, we address this question in the con- vation and zipper progression, while creating ec- text of zippering and neural tube closure in the topic Cadherin2 expression boundaries in neural basal chordate, Ciona robusta. During zippering, or epidermal domains is sufficient to direct RhoA the neural folds meet and fuse from posterior to and Myosin II activity to those boundaries. Cad- anterior, replacing heterotypic junctions between herin2 directs RhoA activity to heterotypic junc- neural and epidermal cells (Ne/Epi junctions) tions by sequestering the Rho GTPase activating with homotypic Ne/Ne and Epi/Epi junctions. protein, GAP21/23, to homotypic junctions and We previously established that zippering in- away from heterotypic junctions. Ahead of the volves spatiotemporal patterning of Myosin II zipper, sequestration of GAP21/23 to Ne/Ne activity: high along the Ne/Epi junctions ahead junctions redirects RhoA and Myosin II activity to of the zipper, and low along Ne/Ne and Epi/Epi heterotypic Ne/Epi junctions; behind the zipper, junctions behind the zipper. Here we show how Cadherin2 recruits GAP-21/23 to newly-formed this pattern of activity is shaped by neural-spe- Ne/Ne junctions to inactivate RhoA and Myosin cific expression and homotypic localization of a II. Together, these actions couple local junction classical cadherin, Cadherin2. Cadherin2 accu- exchange and RhoA/Myosin II activity to dy- mulates along homotypic Ne/Ne junctions, but namically maintain tissue level contractile asym- is absent from heterotypic Ne/Epi junctions, metry that is essential for zipper progression.
20 T6 MORPHONET: AN INTERACTIVE ONLINE MORPHOLOGICAL BROWSER TO EXPLORE COMPLEX MULTI-SCALE DATA
Bruno Leggio1,2,3, Julien Laussu1, Axel Carlier4, Christophe Godin2,3, Patrick Lemaire1,3 and Emmanuel Faure1 ,2,3,4,5,*
1. Centre de Recherche de Biologie cellulaire de Montpellier, CRBM, CNRS, Université de Montpellier, France. 2. Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Inria, F-69342, Lyon, France. 3. Institut de Biologie Computationnelle, IBC, Université de Montpellier, France. 4. Informatics Research Institute of Toulouse (IRIT), CNRS, INPT, ENSEEIHT, University of Toulouse I and III, France. 5. Laboratoire d’Informatique, de Robotique et de Microélectronique de Montpellier (LIRMM), Univ Montpellier, CNRS, Montpellier, France.
The last 20 years have seen astonishing prog- cover a remarkably broad range of phenomena, ress in technological and experimental approach- species, and scales, ranging from the reconstruc- es in life sciences. Novel types of - frequently tion of entire animals or organs from MRI images large-scale - data have brought unprecedented down to the single-cell resolution segmentation insight into fundamental biological processes of developing embryos imaged through, for in- and concepts, and helped bridge scales of anal- stance, light-sheet microscopy.High volumes of ysis.These innovations, however, generate a del- morphological information need be shared, cat- uge of experimentally -produced data whose egorized and quantified in order to achieve a analysis, sharing, and interpretation necessitate better understanding of the fundamental role of the development of advanced informatics and shape and shape changes in biological structures. computational tools.A remarkable technological But the field misses both the equivalent of the ge- revolution in biology has been brought by the nome browsers and of standardized formats to next-generation genome sequencing, which led describe and share morphological and quantita- to the development of the concept of genome tive or qualitative geometrical properties. browsers. These browsers allow research com- We have therefore adapted the philosophy un- munities to share data, findings, to collaborate derlying the creation of genome browsers to im- in the creation of growing online databases, and aging datasets. The result is MorphoNet, a novel above all permit researchers to make sense of tre- class of online morphological browsers, which mendously large amounts of raw data. can be used both for research and dissemination. The last decade has seen a new wave of techno- MorphoNet allows to explore complex segment- logical revolution in life sciences: novel microsco- ed and meshed natural or simulated structures, py and tomography approaches have opened the onto which various morphological or genetic way to quantitative approaches in morphogene- data can be projected. As a companion to the sis and morphodynamic research. Large volumes browser, we also introduce an easy-to-use and of raw and reconstructed 3D or 3D+time data are human-readable universal format to describe currently available, often covering several hours and exchange morphological data and associated in the evolution of dynamic processes. These data information. This format can be used to describe
21 a broad range of datasets (from developing em- overview of MorphoNet (Fig. 1) and discuss a bryos to termite nests) acquired using diverse specific use case of application on tunicates. We imaging technologies and will greatly help re- have efficiently used MorphoNet for multidisci- searchers carry out quantitative analyses on their plinary research within the ascidian community, systems and share their results within and be- with the reconstruction of more than 10 Phallusia tween multidisciplinary scientific communities. mammillata embryos acquired on light sheet mi- croscopy. During this presentation, I will give a brief
Figure 1. MorphoNet framework and its online graphical user interface.
22 T7 INVESTIGATING THE INITIATION OF WHOLE-BODY REGENERATION IN BOTRYLLOIDES LEACHII
Matas Lluis1, Bugada Laura1, and Blanchoud Simon1*
1. Department of Zoology, University of Fribourg
Colonial ascidians are the only known chor- scribed, which facets of this process are required dates that can undergo whole-body regeneration for WBR to succeed remains unknown. In addi- (WBR). This dramatic developmental process al- tion, we have observed some variability in the lows a minute isolated fragment of vascular sys- pace of WBR, potentially reflecting an underly- tem to restore a functional animal in as little as ing disparity in the organization of the vascular 10 days. system. Following the initial injury of the colony, heal- To explore these dependencies, we have set ing of the lesion takes place followed by the re- out to characterize the involvement of the vari- modeling and condensation of the vascular sys- ous components of colony during the initiation tem. After this initiation phase, the establishment of WBR. To study this dramatic process, our lab- and development of multiple regeneration nich- oratory is using an interdisciplinary approach es will invariably lead to the regeneration of one combining biology, microscopy and computer single zooid. science. By perturbing and modulating the re- generating vascular system with targeted mi- Intriguingly, the origin of the stem-like cells re- cro-surgeries, we are dissecting the initiation of sponsible for WBR remain elusive and regener- WBR in Botrylloides leachii. ation establishes exclusively within the vascular system. Similarly, while the remodeling of the vascular system has been morphologically de-
23 T8 EPIGENETIC REGULATION OF BOTRYLLOIDES LEACHII WHOLE BODY REGENERATION
Lisa Zondag , Rebecca Clarke, and Megan J Wilson,.
Developmental Biology and Genomics group. Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin.Affiliation
The colonial tunicate Botrylloides leachii is ex- and quantified the expression levels of HDAC ceptional at regenerating from a piece of vascu- and histone acetyltransferase (HAT) enzymes lar tunic after loss of all adults from the colony. during whole body regeneration (WBR). To de- Previous transcriptome analyses indicate a brief termine if HDAC activity is required for WBR, period of healing before regeneration of a new we inhibited its action using valproic acid (VPA) adult (zooid) in as little as 8-10 days. However, and Trichostatin A (TSA). HDAC inhibition pre- there is little understanding of how the resulting vented the final morphological changes normal- changes to gene expression, required to drive re- ly associated with WBR and resulted in aberrant generation, are initiated and how the overall pro- gene expression. B. leachii genes including Slit2, cess is regulated. Rapid changes to transcription TGF-b, Piwi and Fzd4 all showed altered mRNA often occur in response to chromatin changes, levels upon HDAC inhibition in comparison to mediated by histone modifications such as his- the control samples. Additionally, atypical ex- tone acetylation. Here, we investigated a group pression of Bl_Piwi was found in immunocytes of key epigenetic modifiers, histone deacetylas- upon HDAC inhibition. es (HDAC) that are known to play an important Together, these results show that HDAC func- role in many biological processes such as devel- tion, specifically HDAC I/IIa class enzymes, are opment, healing and regeneration. vital for B. leachii to undergo WBR successfully. Through our transcriptome data, we identified
24 T9 PRIMORDIAL BLASTS, A POPULATION OF BLOOD BORNE STEM CELLS RESPONSIBLE FOR WHOLE BODY REGENERATION IN A BASAL CHORDATE
Susannah H. Kassmer1, Adam Langenbacher2, Anthony W. De Tomaso1
1. Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA USA. 2 Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA USA.
The invertebrate chordate Botrylloides lea- MMC treated recipients, Edu label is detected in chii regenerates entire new bodies from small regenerating tissues. Inhibitors of either Notch or fragments of blood vessels. Here, we show that canonical Wnt signaling block regeneration and during early stages of regeneration, proliferation proliferation of pou3+ stem cells. We propose occurs only in small, blood borne cells. These that ia6+pou3+ cells are pluripotent, proliferat- proliferating blood cells express integrin-alpha-6, ing stem cells that are present in the blood of B. pou3 and vasa and are present in the blood of un- leachii and are responsible for whole body regen- injured colonies. Elimination of these proliferat- eration. We named these cells Primordial Blasts ing blood cells with Mitomycin C (MMC) blocks - based on the fact that they are undifferentiated, regeneration. Injection of 1 single prospectively constantly dividing, express germline multipo- isolated Integrin-alpha-6 (IA6)-positive cell is ca- tency genes and give rise to somatic regenerating pable of rescuing whole body regeneration. Upon tissues. transplantation of Edu-labeled IA6+ cells into
25 T10 NOVEL BUDDING MODE IN POLYANDROCARPA ZORRITENSIS: A MODEL FOR COMPARATIVE STUDIES ON ASEXUAL DEVELOPMENT AND WHOLE BODY REGENERATION
Marta Scelzo1, Alexandre Alié1, Sophie Pagnotta2, Camille Lejeune1, Pauline Henry1, Marie Deschler1, Laurent Gilletta1, Laurel S. Hiebert1,3, Francesco Mastrototaro4, Stefano Tiozzo1
1. Sorbonne Universités, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-Mer (LBDV), 06230 Villefranche-sur-Mer, France 2. Centre Commun de Microscopie Appliquée, UFR Sciences, Faculté des Sciences de l’Université de Nice - Sophia Antipolis, 06108 Nice, France 3. Departamento de Zoologia, Instituto Biociências, Universidade de São Paulo, 05508- 090 São Paulo, Brazil 4. Department of Biology and CoNISMa LRU, University of Bari Aldo Moro, I-70125 Bari, Italy
In colonial ascidians the capacity to build an peratures and harsh environmental conditions. adult body via non-embryonic development The bud arises through a folding of the epithe- (NED), i.e., asexual budding and whole body lia of the vessel with the contribution of undif- regeneration, has been gained and lost sever- ferentiated mesenchymal cells. In order to assess al times across the whole subphylum. A recent the involvement of these two tissues we analyzed phylogeny of the family Styelidae revealed an the proliferation dynamic via EdU labelling. We independent acquisition of NED in the colonial characterized the hemocyte morphotypes and species Polyandrocarpa zorritensis and highlighted identified a population of undifferentiated cells, an undescribed budding mode that we named the hemoblasts, known as putative stem cells in “vasal budding”. other ascidian species. We characterized the asexual life cycle and the In conclusion, to understand the mechanisms morphology of the early stages of P. zorritensis of NED and their evolution we started from a ro- budding. During vasal budding, the new zooids bust phylogenetic framework and selected rele- origin from protrusions of the adult epidermis in vant species to compare. The description of NED vascularized areas defined as budding nests. The in P. zorritensis provides the foundation for future budding nest can alternatively develop into a comparative studies on plasticity of budding and round-shaped structure, the spherule, which rep- whole body regeneration in tunicates. resents a dormant state, able to survive low tem-
26 T11 DYING TO REGENERATE: APOPTOSIS, WNT SIGNALING, AND POLARITY OF BODY REGENERATION IN CIONA INTESTINALIS
William R. Jeffery
Marine Biological Laboratory, Woods Hole, MA. 02543 USA and Station Biologique, Roscoff, 29680 France [email protected]
Regeneration in Ciona intestinalis is polarized interferes with BSC activation and prevents distal according to the body axis: severed basal parts regeneration. Furthermore, apoptosis, blastema regenerate distal structures, such as the siphons formation, and BSC proliferation are themselves and neural complex, but severed distal parts do polarized. When animals are bisected across the not replace basal structures. This phenomenon, middle, apoptosis at the injury site, BSC prolif- termed distal regeneration, involves the activity eration, and blastema formation occur in the re- of pluripotent stem cells residing in branchial sac generating basal parts but not in the non-regen- vasculature. In response to distal injury, branchi- erating distal parts, although both parts share the al sac stem cells (BSC) are induced to proliferate, same injury site and contain a large portion of the the progenitor cells migrate to the sites of injury branchial sac. These results show that apoptosis and form a blastema, which replaces missing dis- is required to induce BSC proliferation and that tal tissues and organs. Progenitor cell migration BSC are necessary but not sufficient for distal re- is specific: only injured body parts are targeted. generation. Microarray-based gene expression, We have used microsurgical, pharmacological, regeneration rescue assays, and gene knock- and molecular analysis to investigate the mecha- downs indicate that apoptosis activates Wnt sig- nisms of distal regeneration. Apoptosis at injury naling at injury sites, which in turn triggers BSC sites is one of the earliest events in distal regener- proliferation and mobilizes the migration and ation. Blocking apoptosis with caspase inhibitors distal targeting of progenitor cells.
Supported by NIH and EMBRC-France
27 T12 AGING AND SENESCENCE EFFECTS ON THE EXTRACORPOREAL VASCULATURE OF BOTRYLLUS SCHLOSSERI
Delany Rodriguez1*, and Anthony W. De Tomaso1
1. Department of Molecular, Cellular and Developmental Biology. University of California Santa Barbara, CA (USA)
Ageing causes a progressive deterioration of developed a new quantitative approach (com- both the structure and function of vascular cells puter program) to track these changes at high that triggers several age-related vascular diseas- resolution. In addition, using these phenotypic es. The extra-corporeal vasculature of the colo- markers we can identify the genetic basis of se- nial ascidian Botryllus schlosseri interconnects all nescence. To initiate these studies we have isolat- individuals, but due to weekly turnover of the ed multiple Botryllus genotypes at specific ages zooids inherent in asexual reproduction in this and carried out mRNA-seq. Using differential species, is the only persistent organ in an indi- expression analyses we have identified key genes vidual, thus the only one to age. Characteristics involved in aging. The products of these genes of vascular aging include narrowing of the vessel are involved in multiple processes, including and decreased blood flow. Interestingly, we also regulation of actin cytoskeleton, apoptosis, ATP see predictable age-related changes in vascular metabolism, DNA/RNA repair, among others. cell morphology, including major remodeling Current results will be discussed. of the cytoskeleton and basement membrane as well as changes in cell shape. We have recently
28 T13 EVO-DEVO APPROACHES TO NON-EMBRYONIC DEVELOPMENTS IN COLONIAL TUNICATES
Alexandre Alié1, Marta Scelzo1, Maria Mandela Prünster1, Sonia Lotito1, Lorenzo Ricci1,2, Manon Boosten1, Stefano Tiozzo1
1. Sorbonne Université, CNRS, Laboratoire de Biologie du Dévelopement de Villefranche- sur-mer (LBDV) IMEV, 06230, Villefranche-sur-mer, France. 2 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.
Beside embryogenesis, to build an adult body tion and symmetry breaking, the differentiation colonial tunicates adopt non-embryonic devel- of territories at the onset of budding, which mim- opments (NED), i.e. asexual propagation and ic the embryonic germ layers domains, and how whole body regeneration. NED were acquired neurogenesis and myogenesis are triggered de independently at least seven times across the novo, partially rewiring embryonic gene modules tunicates. In different species NED starts from in different ontogenetic contexts. non-homologous somatic cells and tissues but To compare independently evolved NED, we nevertheless lead to similar adult body plans. have recently described a novel budding mode The possibility to compare within the same in Polyandrocarpa zorritensis. We are now con- group, non-homologous tissues that converge to ducting transcriptomic and genomic studies to common ontogenetic stages makes tunicates an compare dynamics of gene expression and ge- excellent case study to deeper explore the molec- nomic regulation between different buds in di- ular mechanisms underlying evolution of NED verse colonial species. Our goal is to identify and in metazoans. compare prospective GRNs repeatedly co-opted In the recent years we used the colonial ascid- for NED in species that independently acquired ian Botryllus schlosseri as a model to investigate asexual reproduction. The function of identified the partial co-option of embryonic mechanisms genes will then be tested using a transgenic ap- during NED within the same species. For in- proach recently developed in B. schlosseri. stance, we described the redeployment of early signaling pathways involved in AP axis forma-
29 T14 MOLECULAR AND MORPHOLOGICAL ATLAS OF BOTRYLLUS SCHLOSSERI DEVELOPMENTAL PATHWAYS
Chiara Anselmi1, Mark Kowarsky2, Kohji Hotta3, Paolo Burighel1, Giovanna Zaniolo1, Federico Caicci1, Benyamin Rosental4,5, Norma F Neff6, Katherine J Ishizuka4,5, Karla J Palmeri4,5, Jennifer Okamoto6, Tal Gordon7 Irving L Weissman4,5,6, Stephen R Quake6,8, Ayelet Voskoboynik4,5,6, Lucia Manni1
1. Dipartimento di Biologia, Università degli Studi di Padova, Padova, 35135, Italy 2. Department of Physics, Stanford University, CA 94305, USA 3. Department of Biosciences and Informatics, Keio University, Yokohama, 223-8522, Japan 4. Institute for Stem Cell Biology and Regenerative Medicine, and Ludwig Center, Stanford University School of Medicine, Stanford, CA 94305, USA 5. Department of Biology, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950, USA 6. Departments of Applied Physics and Bioengineering, Stanford University, and Chan Zuckerberg Biohub, San Francisco CA 94158, USA 7. Zoology Department, Tel Aviv University, Tel-Aviv Israel 8. Department of Applied Physics and Bioengineering, Stanford University, CA 94305, USA
Colonial tunicates like Botryllus schlosseri are atlas that links the gene profiles describing the unique among chordates in that they use two entire embryogenesis and blastogenesis process- pathways, embryogenesis and blastogenesis, es with morphological events. With tissue and to produce the adult body. Despite the fact that cell-type specific molecular signatures, we - fur these two developmental pathways are morpho- ther identified the developmental origin of the logically distinct, they both result in almost iden- nervous system, endostyle, blood cells, and germ tical individuals. The ways in which organogen- cells. Moreover, we followed their developmen- esis differs when comparing embryogenesis and tal phases including morphogenesis, cytodiffer- blastogenesis, and if a convergent morphology entiation, and gene expression. This study out- implies convergent molecular mechanisms re- lines the molecular and morphological landscape mains almost unknown. of the two developmental modes and demon- strates that different molecular paths can lead to By combining transcriptome sequencing with the same outcomes. It also suggests that cellular confocal, two-photons, electron microscopy and trajectory is defined early in development and digital 3D reconstructions of major embryonic that the adult tissue specific stem cells and the and blastogenic developmental stages, we char- embryonic precursor populations share the same acterized the molecular and morphological sig- molecular profile. natures along both developmental pathways. We then generated a comprehensive developmental
30 T15 IDENTIFICATION OF KEY MORPHOGENESIS GENES IN MOLGULIDAE WITH ALTERNATE BODY PLANS AND THEIR INTERSPECIFIC HYBRID EMBRYOS
Elijah K Lowe1,2,3,4,5, C. Titus Brown2,3,4,5, Billie J. Swalla2,3,4,5, Alberto Stolfi1,5
1. Georgia Institute of Technology, USA; 2. Univ of Wash, USA; 3. Friday Harbor Labs, USA; 4. BEACON, Michigan State Univ, USA; 5. Station Biologique de Roscoff, France
Typical solitary tunicate development is char- lata a tailed species, and in lab conditions are able acterized by a biphasic life cycle, having a swim- to cross fertilized producing interspecific hybrids ming larva containing 40 notochord cells that with partially rescued motile phenotypes. These converge and elongate to form the larval tail, animals and their hybrid have presented a sys- and a sessile filter feeding adult. After the free tem where we are able to examine the evolution- swim stage, tunicates absorb their larval tail and ary mechanism behind alternate body plans and a metamorphosis into their adult stage. Howev- the rescue of the motile phenotypes. We have se- er, within the Molgulidae clade, several species quenced the genome of both parent species and have independently evolved an alternate “tail- key developmental transcriptome stages (gastru- less” body plan, forgoing the development tailed la, neurula, and tailbud) of the M. occulta, M. oc- swimming larval form and losing the differen- ulata and their hybrid. This analysis has enabled tiation of their notochord and several other fea- us to identify key genes involved in the motile tures such paraxial muscles, motor neurons, and behavior/structures. One of which being ebf, an pigmented cells associated with geotactic and important motor neuron terminal selector gene, light-sensing organs required for swimming and whose expression has been reduced in the immo- settlement. One such tail-less species, Molgula oc- tile M. occulta. culta, is found in close proximity to Molgula ocu-
Funding Provided by: BEACON Center at Michigan State University, and NIH/NICHD
31 T16 TAILLESS MOLGULID ASCIDIANS HAVE A NOTOCHORD “BALL” THAT NEVER CONVERGES OR EXTENDS
Alexander Fodor1,2,3, Elijah Lowe1,3,4, C. Titus Brown1,3,4 Alberto Stolfi4,5 and Billie J. Swalla1,2,3,4
1. Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250 2. Department of Biology, University of Washington, Seattle, WA 98125 3. BEACON, Michigan State University, East Lansing, MI 48824 4. Station Biologique de Roscoff, Roscoff, 29682, France
Transcriptomic and genomic data offer exciting bryos. We show by transcriptome analyses that new approaches to examine the genetic networks the notochord gene network is intact in mol- underlying the origin and evolution of the chor- gulid ascidians, and the expression of most of date body plan. One system for studying chor- the downstream genes is seen in the “notoball”. date body plan evolution is two tunicate species Hybrid embryos made from the sperm of the with radically different larval body plans—the tailed, Molgula oculata and the egg of the tailless tailed ascidian Molgula oculata and the tailless Molgula occulta undergo convergence and exten- M. occulta. Tailed M. oculata embryos, like most sion of the notochord to form a short tail. We are solitary ascidians, have 40 notochord cells that searching for the factors that allow the notochord are converged and extended in the center of the convergence and extension in the hybrid embry- tail of the tadpole larvae. The larvae also have os by computational analyses. We show that the tail muscle cells flanking the notochord in the expression of the downstream notochord genes tail, and, in the head, an otolith, a gravity sen- is normal in the tailless Molgula occulta, with a sory organ. The tailless M. occulta does not form few notable exceptions. We are continuing anal- a tail in their larval stage, and have only 20 no- yses of the notochord convergence and extension tochord cells that do not converge and extend in hybrid embryos in an effort to understand the during larval development. We have sequenced genetic network necessary for convergence and the genomes and obtained developmental tran- extension in ascidian embryos. scriptomes for both species and the hybrid em-
32 T17 DIFFERENTIAL DRIFT AMONG ENHANCER ELEMENTS IN THE TUNICATE CARDIOPHARYNGEAL GENE NETWORK
William Colgan1, Ariel Hwang1, Alexis Leanza1, Isabel Llosa1, Daniel Rodrigues1 and Brad Davidson1*
1. Swarthmore College
Tunicates have retained a stringently conserved inflata enhancers for two key cardiopharyngeal program for embryonic patterning over millions transcription factors (mesp and notrlc/hand-like) of years of evolutionary divergence1. Remark- reveals that upstream trans factors regulating ably, this conservation occurs in the context of these genes have been conserved while associat- extremely high rate of sequence polymorphism ed binding sites have undergone extensive rear- in tunicate genomes2. Thus, tunicates represent rangements. Surprisingly, comparative enhancer an ideal clade for examining how robust devel- analysis for a third cardiopharyngeal transcrip- opmental networks can retain function despite tion factor, foxf, revealed stringent conservation changes in the architecture of underlying de- of binding site number position and spacing. Ex- velopmental gene regulatory networks (GRNs), perimental manipulation of binding site spacing a process referred to as developmental systems compromised the function of the Ciona FoxF en- drift3. We employ the newly sequenced Corella in- hancer. The different level of selective constraint flata genome to explore drift within the well char- acting on these regulatory elements may reflect acterized cardiopharyngeal GRN. Here we show discrete variations in enhancer structure/func- that levels of drift are highly variable across the tion rather the reflecting the hierarchical position regulatory nodes of the cardiopharyngeal GRN. of the genes they regulate in the network. Comparative analysis of Ciona robusta and Corella
References 1. Lemaire, P. (2011). Evolutionary crossroads in developmental biology: the tunicates. Development (Cambridge, England), 138(11), 2143–2152. http://doi.org/10.1242/dev.048975 2. Tsagkogeorga, G., Cahais, V., & Galtier, N. (2012). The Population Genomics of a Fast Evolver: High Levels of Diversity, Functional Constraint, and Molecular Adaptation in the Tunicate Ciona intestinalis. Genome Biology and Evolution, 4(8), 740–749. http://doi.org/10.1093/gbe/evs054 3. Stolfi, A., Lowe, E. K., Racioppi, C., Ristoratore, F., Brown, C. T., Swalla, B. J., & Christiaen, L. (2014). Divergent mechanisms regulate conserved cardiopharyngeal development and gene ex- pression in distantly related ascidians. eLife, 3, 1–64. http://doi.org/10.7554/eLife.03728.032 33 T18 PROTEIN PHOSPHATASE 2A IS ESSENTIAL TO MAINTAIN MEIOTIC ARREST, AND TO PREVENT CA2+ BURST AT SPAWNING AND EVENTUAL PARTHENOGENESIS IN THE LARVACEAN OIKOPLEURA DIOICA
Masaki Matsuo*, Takeshi A. Onuma, and Hiroki Nishida
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
Unfertilized eggs of most animals are arrested brought into the egg by a sperm. Activation of at a certain point in the meiotic cell cycles. Reini- the KD oocytes was triggered by an ambient in- tiation of meiosis and the start of embryogenesis tracellular rise of pH upon their release from the are triggered by fertilization. This is essential for gonad into seawater at spawning. Live recording preventing parthenogenetic activation and for of intracellular calcium concentration indicated promoting proper initiation of development by that the pH rise caused an aberrant Ca2+ burst, fertilization. In the larvacean Oikopleura dioica, which mimicked the Ca2+ burst that occurs at fer- unfertilized eggs are arrested at metaphase-I. We tilization. Then, the aberrant Ca2+ burst triggered show here that protein phosphatase 2A (PP2A) meiosis resumption through Calcium/calmod- is essential for maintenance of meiotic arrest af- ulin-dependent protein kinase (CaMK II). There- ter spawning of oocytes. Knockdown (KD) of fore, PP2A is essential for maintenance of meiotic the maternal PP2A catalytic subunit, which was arrest and prevention of parthenogenesis by sup- found in functional screening of maternal factors, pressing the aberrant Ca2+ burst at spawning. In caused unfertilized eggs to spontaneously re- addition, genomic data suggest the possibility lease polar bodies after spawning, and then start that the molecular mechanism of meiotic arrest pseudo-cleavages without fertilization, name- in O. dioca is similar to that in vertebrates rather ly, parthenogenesis. Parthenogenetic embryos than ascidians. failed to undergo proper mitosis and cytokinesis because of lack of a centrosome, which is to be
34 T19 THE SPINDLE ASSEMBLY CHECKPOINT IN PHALLUSIA MAMMILLATA EMBRYOS
Marianne Roca*, Lydia Besnardeau, Janet Chenevert, Elisabeth Christians, Stefania Castagnetti
Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche- sur-Mer (LBDV), 181 chemin du Lazaret, 06230, Villefranche-sur-Mer, France
Erroneous chromosome segregation leads to mitotic timing during cleavage divisions. How- aneuploidy, a condition deleterious for develop- ever, mitotic progression was delayed, in a SAC ment. To prevent it, the Spindle Assembly Check- dependent manner, starting from the gastrula point (SAC) delays entry into anaphase until all stage. The length of the mitotic delay increased chromosomes are properly attached to spindle further in neurula stage embryos, and SAC ef- microtubules. Our team has shown that where- ficiency varied among different populations of as some embryos, like those of sea urchin, have cells within the embryo. By interfering with em- an active SAC and delay mitotic progression in bryonic patterning by egg microsurgery or ecto- the presence of spindle defects, in chordate em- pic expression of transcription factor, we are cur- bryos, including C. intestinalis and P. mammillata, rently testing the contribution of cell identity to the SAC is inefficient, during cleavage divisions SAC efficiency in ascidian embryos. Indeed our 1. To understand the mechanisms controlling preliminary results suggest that the difference in chromosome segregation during development SAC efficiency is lost when all cells acquire the in chordates, we characterized further how SAC same identity. activity is regulated in embryos of the ascidian P. 1. Chenevert, J. et al. The spindle assembly mammillata. checkpoint functions during early development Using live imaging, we find that treatment of in non-chordate embryos. bioRxiv 582759 (2019). P. mammillata embryos with the microtubule de- doi:10.1101/582759 polymerizing drug nocodazole did not affect
35 T20 ANALYSES OF SPATIO-TEMPORAL INTERACTION BETWEEN MICROTUBULE AND ER OR MATERNAL mRNAs IN CIONA EGG USING NEW HIGH RESOLUTION DOUBLE STAINING METHODS
Toshiyuki Goto1 and Takahito Nishikata1
1. Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe, Japan
In ascidian embryogenesis, antero-posterior myoplasmic movement, we could distinguish axis is predetermined by the egg polarity, which two different compartments in cER, outermost is established by large movements of the myo- region, which directly interact with CAMP, and plasm, cortical endoplasmic reticulum (cER), and inner region. Type I postplasmic RNAs were maternal determinants (i.e. Type I and II postplas- found on both inner and outermost regions of mic/PEM RNAs) during first cell cycle1). We pre- cER, while Type II localized only within inner re- viously reported the CAMP (cortical array of mi- gion of cER. Moreover, Type II postplasmic mR- crotubules in posterior vegetal region)2), which NAs have already started to localize at about 30 resembled to cortical microtubule arrays in frog min postfertilization to the vegetal pole region, and zebrafish eggs3),4). Thus, there is a possibility where the Type I postplasmic RNAs were al- that CAMP contributes to the transportation of ready colocalized with TAF (transiently accumu- cER and maternal mRNAs. However, there has lated microtubule fragment). These observations not been established the appropriate techniques are consistent with the report, in which the vege- for the visualization of these structures and mol- tal localization pattern of both types of postplas- ecules, simultaneously. mic RNAs overlaps with each other during 1st myoplasmic movement5). Our bland new double In this study, we developed new double stain- staining methods and result of this study could ing methods, which consisted of ER immunos- have great impact for understanding the axis for- taining or in situ hybridization and high-resolu- mation in ascidian egg. tion microtubule immunostaining. During 2nd
References 1. Prodon et al., Dev Dyn, 236, 1698-1715 (2007) 2. Ishii et al., Dev Growth Differ, 59, 648-656 (2017) 3. Elinson & Rowning Dev Biol, 128, 185–197 (1988) 4. Tran et al., Development, 139, 3644–3652 (2012) 5. Paix et al., Dev Biol, 336, 96-111 (2009) 36 T21 ACTIN-DEPENDENT CONTRACTION DRIVES OOPLASMIC SEGREGATION IN ASCIDIAN OOCYTES
Silvia Caballero-Mancebo,* and Carl-Philipp Heisenberg
1. Institute of Science and Technology Austria, Am Campus 1, 3400, Klosterneuburg, Austria
Reorganization of the cytoplasm within the are linked to maternal mRNAs segregation, we oocyte (ooplasm) constitutes the first step in extended the flow analysis to other cortical and patterning of the tunicate embryo. Ooplasmic subcortical domains involved in this process: reorganization is initiated by contraction of the plasma membrane, cER and a mitochondria-rich actomyosin cortex along the animal-vegetal axis domain positioned above the actomyosin cortex of the oocyte, leading to the accumulation of cor- termed ‘myoplasm’. The flow velocities indicate tical endoplasmic reticulum (cER)–associated that cER and plasma membrane flow together maternal mRNAs in a region of the vegetal pole with the actomyosin cortex. The myoplasm, in termed ‘contraction pole’ (CP). We used the spe- contrast, shows slower flow velocity and does cies Phallusia mammillata to unravel the mechano- not accumulate at the same degree at the vege- chemical mechanisms underlining CP formation tal pole. It also displays pronounced buckling at and maternal mRNAs segregation. the CP, suggesting that a rigid myoplasm layer becomes compressed at the vegetal pole by the We first have analyzed the dynamic distribu- contracting actomyosin cortex. We are current- tion of actomyosin during the cortical contrac- ly analyzing how these domains are coupled tion process. We found that upon fertilization a to each other and how their differential friction gradient of contractility along the animal-vegetal determines the distinct shape changes and, con- axis drives vegetal-directed flows of the actomy- sequently, the distribution of maternal mRNAs osin cortex leading to the formation of the CP. associated with these different domains. To investigate how cortical actomyosin flows
37 T22 GAP JUNCTION-DEPENDENT COORDINATION OF INTERCELLULAR CALCIUM SIGNALLING DURING EARLY EMBRYOGENESIS OF OIKOPLEURA DIOICA
Yana Mikhaleva1,*, Oleg Tolstenkov 1, and Joel C. Glover1,2
1. Sars International Centre for Marine Molecular Biology, University of Bergen 2. Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo
Calcium signaling is an important feature of crease (about 5% of the main peak amplitude), embryonic development. Here, we characterized which we term the “minipeak”. By gastrulation, spontaneous Ca2+ signals in Oikopleura dioica Ca2+ transients exhibited a stereotyped initiation embryos from pre-fertilization to gastrula stag- site on either side of the embryo, likely in the na- es after injecting GCaMP6 into unfertilized eggs, scent muscle precursor cells, and spread thereaf- and we addressed the potential developmen- ter symmetrically in a stereotyped spatial pattern tal role of intercellular Ca2+ waves by dsRNA from posterior to anterior. knockdown of connexin genes. To assess the role of gap junctions in calcium The unfertilized egg exhibited regular, transient wave spread and coordination, we first charac- elevations in intracellular Ca2+ concentration terized the expression of two Oikopleura con- with an average duration of 4-6 seconds and an nexins, Od-CxA and Od-CxB, both of which are average frequency of about 1 every 2.5 minutes. expressed during pre-gastrulation and gastrula Fertilization was accompanied by a longer Ca2+ stages, and then co-injected dsRNAs to suppress transient that lasted several minutes. Thereaf- connexin expression. Connexin mRNA knock- ter, regular Ca2+ transients were reinstated that down led to a gradual increase in Ca2+ transient spread within seconds among blastomeres and peak width, a decrease of interpeak interval and gradually increased in duration and decreased a marked disruption of wave synchronization. in frequency by gastrulation. Each peak was pre- This desynchronization was accompanied by a ceded by about 15 seconds by a smaller Ca2+ in- disruption of normal development.
38 T23 EVOLUTIONARY ORIGIN OF THE MAMMALIAN HEMATOPOIETIC AND IMMUNE SYSTEMS FOUND IN A COLONIAL CHORDATE
Benyamin Rosental1,2*, Mark Kowarsky3*, Jun Seita2,4, Daniel M. Corey2, Katherine J. Ishizuka2, Karla J. Palmeri2, Shih-Yu Chen5, Rahul Sinha2, Jennifer Okamoto3, Gary Mantalas3, Lucia Manni6, Tal Raveh2, D. Nathaniel Clarke2, Aaron M. Newman2, Norma F. Neff3, Garry P. Nolan5, Stephen R. Quake3&, Irving L. Weissman2& and Ayelet Voskoboynik2&.
1. French Associates Institute for Agriculture and Biotechnology of Drylands, the Jacob Blaustein Institutes for Desert Research, Ben Gurion University of the Negev. 2. Institute for Stem Cell Biology and Regenerative Medicine, and Ludwig Center, Stanford University School of Medicine. 3. Department of Physics, Stanford University. 4. AI based Healthcare and Medical Data Analysis Standardization Unit, Medical Sciences Innovation Hub Program, RIKEN, Japan. 5. Department of Microbiology and Immunology, Stanford University School of Medicine, USA. 6. Dipartimento di Biologia, Università degli Studi di Padova, Italy. *,& Equal contribution.
Hematopoiesis is an essential process that ies by Cytof, lectins, and fluorescent enzymatic evolved in multicellular animals. At the heart reagents, we isolated 34 B. schlosseri cell popu- of this process are hematopoietic stem cells lations. Using whole-transcriptome sequencing (HSCs), which are multipotent, self-renewing of defined cell populations, and diverse func- and generate the entire repertoire of blood and tional assays, we identified HSCs, progenitors, immune cells throughout life. Here we studied immune-effector cells, and the HSC niche. Our the hematopoietic system of Botryllus schlosseri, study implies that the HSC and myeloid lineag- a colonial tunicate that has vasculature, circulat- es emerged in a common ancestor of tunicates ing blood cells, and interesting characteristics of and vertebrates and suggests that hematopoietic stem cell biology and immunity. Self-recognition bone marrow and the B. schlosseri endostyle niche between genetically compatible B. schlosseri colo- evolved from the same origin. Furthermore, we nies leads to the formation of natural parabionts identified a B. schlosseri cytotoxic cell population with shared circulation, whereas incompatible originating from large granular lymphocyte-like colonies reject each other. By means of flow-cy- cells and demonstrated that self-recognition in- tometry in combination with screened antibod- hibits cytotoxic reaction.
39 T24 FURTHER REVELATIONS ON CIONA TAXONOMIC COMPLEXITY
Federica Montesanto1*, Marika Salonna2, Giovanni Chimienti1, Frédèrique Viard3, Egidio Trainito4, Carmela Gissi2,5 and Francesco Mastrototaro1
1. Dept of Biology and CoNISMa LRU, University of Bari “Aldo Moro” Via Orabona, 4 - 70125 Bari, Italy 2. Dept of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Via Orabona, 4 - 70125 Bari, Italy 3. Sorbonne Université, CNRS, Lab. Adaptation & Diversity in Marine Environment (UMR 7144), Station Biologique, Roscoff, France 4. Villaggio I Fari, Loiri Porto San Paolo, Olbia-Tempio, Italy 5. IBIOM, CNR, Bari, Via Amendola 165/A - 70126, Bari, Italy
The genus Ciona Fleming, 1822 constitutes an previously used for discriminating Ciona cryp- interesting “taxonomic case”. Despite the large tic species; and cox1, a reliable DNA barcoding number of studies, its evolutionary history and marker. Phylogenetic trees clearly resolved the complex taxonomy have not been yet completely phylogenetic position of the specimens within resolved. This study presents new elements on the genus. Altogether, our data confirmed that this topic, describing specimens of a non-identi- our Ciona specimens could not be assigned to any fied Ciona species found along the North-eastern molecularly-characterized or already described coasts of Sardinia (Tyrrhenian Sea), through an species within the genus (i.e., Ciona intestinalis integrative taxonomic approach. Morphological (Linnaeus, 1767), Ciona robusta Hoshino & Tokio- analyses first revealed that these Ciona speci- ka, 1967, Ciona savignyi Herdman, 1882, Ciona mens showed intermediate characters compared roulii Lahille, 1887, Ciona edwardsi Roule, 1884, to those of other moderate and shallow-water intestinalis spC and intestinalis spD) and they are species belonging to the Ciona genus, such as a closely related to C. edwardsi. Our findings add smooth tunic surface without tubercles; 6 longi- further complexity to the Ciona taxonomy, un- tudinal muscles bands; a flat branchial sac; trans- derlying the importance of integrative taxonom- versal vessels of about equal sizes; and a marked ic approaches, and the need to gather additional preference for shady sites. Molecular characteri- knowledge regarding the evolutionary history of zation was based on three mitochondrial regions: this still enigmatic genus. cox2-cob fragment, for the first time for a phylo- genetic study in Ciona; cox3-trnK-nad1 fragment,
40 T25 SINGLE ZOOIDS, MULTIPLE LOCI: POPULATION GENOMICS OF A GLOBAL INVADER
Maria Casso1,2,*, Xavier Turon 1, and Marta Pascual 2
1. Center for Advanced Studies of Blanes (CEAB, CSIC), Blanes, Spain. 2. Department of Genetics, Microbiology and Statistics, and IRBio, University of Barcelona, Spain.
Invasive species constitute a major threat to bio- ways within the same locality. At the global level, diversity and can cause high economic and eco- we identified two groups matching Cytochrome logical negative impacts. In the study of non-in- Oxidase I clades (Stefaniak et al. 2009), confirming digenous species, it is essential to assess genomic that only one clade spread worldwide and sug- diversity and population structure for develop- gesting reproductive isolation between the two ing adequate management strategies. We com- clades in the native area, Japan, and a speciation bined whole genome amplification (WGA) and process. We identified three independent coloni- genotyping-by-sequencing (GBS) techniques to zation events determining the presently known set a protocol for samples with small DNA quan- distribution of the species, although significant tities, and we applied it to single zooids of col- differentiation was found in all population pair- onies of the worldwide invasive colonial ascid- wise comparisons. The lack of isolation by dis- ian Didemnum vexillum. WGA-GBS performance tance within introduced areas indicates that pas- was tested using half zooids, providing empiri- sive transportation would drive the distribution cal demonstration for genotyping reliability. We pattern of this species worldwide and regionally. analysed 296 colonies from 12 localities world- Diverse and well differentiated populations in wide including native and main invaded areas, the introduced and native areas point to a high generating polymorphic loci datasets by locality, expansion potential of this worrisome world- region and globally. We detected some clones, al- wide invader.
References 1. Stefaniak, L., Lambert, G., Gittenberger, A., Zhang, H., Lin, S., & Whitlatch, R. B. (2009). Genetic conspecificity of the worldwide populations of Didemnum vexillum Kott, 2002. Aquat. Invasions 4, 29–44. 41 T26 THE THALIACEAN SALPA FUSIFORMIS : THE RETURN OF SESSILE ASCIDIANS TO PELAGIC LIFE
Jacques Piette*, Paul Simion2 , Frédéric Delsuc2, Christelle Dantec1, Justine Dardaillon1, Céline Scornavacca2 , Stefano Tiozzo3, Emmanuel Douzery2 and Patrick Lemaire
1. Centre de Recherche en Biologie Cellulaire, Montpellier 2. Institut des Sciences de l’Evolution, Montpellier 3. Laboratoire de Biologie du Développement, Villefranche-sur-Mer
Thaliaceans are a fascinating group of pelagic ian relatives. In particular, the early invasion of tunicates with complex life cycles, encompass- blastomeres by surrounding follicle cells, also ing doliolids, pyrosomes and salps (Piette and called calymmocytes, led to a profound modifi- Lemaire 2015). Their proximity to ascidians was cation of salp embryogenesis when compared to noted already by Cuvier (Cuvier 1804). By an ex- that of ascidians. Calymmocytes actively partic- tensive phylogenomic analysis based on a large ipate in building the embryo, possibly by bring- number of coding sequences we confirmed that ing to their final position the blastomeres, which thaliaceans are the sister group of phlebobranchs will eventually form the definitive adult organs. and aplousobranchs inside the polyphylic as- Most intriguingly, the patterned expression of cidians (Delsuc et al. 2018). Moreover, our data developmental regulators such as Otx in calym- position doliolids, some of which still possess mocytes, suggests that these cells also exert some a tadpole larva, as the sister-group of all other morphogenetic role. Finally, the striking diver- thaliaceans. Thaliaceans, and especially salps, gence of the early embryogenesis between two are characterized by a rapid molecular evolution, salp species, Salpa fusiformis and Thalia democrati- even when compared to ascidians. We will pres- ca, suggests a possible relaxation of developmen- ent a more detailed analysis of the tempo and tal constraints in salps. mode of evolution of ca. 3000 proteins, and dis- We are convinced that despite the experimen- cuss a possible role in thaliacean evolution. tal challenge of studying these intriguing class of We chose the salp Salpa fusiformis as a model organisms, a better understanding of their devel- organism to better understand thaliacean de- opment and evolution will be an essential contri- velopment, which is very different from the bution to tunicate biology. stereotyped mosaic development of their ascid-
References 1. Cuvier, G. 1804. “Mémoire Sur Les Thalides (Thalia Brown) et Sur Les Biphores (Salpa Forskaohl).” Annales Du Muséum National d’Histoire Naturelle 4: 360–82. 2. Delsuc, F., H. Philippe, G. Tsagkogeorga, P. Simion, M.-K. Tilak, X. Turon, S. López-Legentil, J. Piette, P. Lemaire, and E.J.P. Douzery. 2018. “A Phylogenomic Framework and Timescale for Com- parative Studies of Tunicates.” BMC Biology 16 (1): 39. 3. Piette, J., and P. Lemaire. 2015. “Thaliaceans, the Neglected Pelagic Relatives of Ascidians: A De- velopmental and Evolutionary Enigma.” The Quarterly Review of Biology 90 (2): 117–45. 42 T27 PHYLOGENY OF THE BOTRYLLID ASCIDIANS
Nydam M.L.1*, Lemmon, A.R.2, Cherry, J.R.2, Hernandez, C.3, Cohen, C.S.3
1. Division of Science and Mathematics, Centre College, 600 W. Walnut Street, Danville, KY 40422, USA 2. Department of Scientific Computing, Florida State University, Dirac Science Library, Tallahassee, FL 32306, USA 3. Estuary and Ocean Science Center, San Francisco State University, 3150 Paradise Dr, Tiburon, CA 94920, USA
The botryllid ascidians are a clade within the genome and Botrylloides leachii transcriptome to Family Styelidae (Class Ascidiacea, Order Sto- identify phylogenetically informative loci. The lidobranchia) comprising 50 species. Botryllids current phylogeny contains 17 taxa, with addi- are used as models in several biological fields, tional taxa to be added in 2020. A Botryllus pri- including allorecognition, aging, development, migenus clade occupies the basal position in the invasion genetics, mitochondrial genome evo- phylogeny. The sister group to Botryllus schlosseri lution, and regeneration. However, the majori- is Botryllus horridus. There are four major sub- ty of botryllid species are not well known to the clades within the Botrylloides clade: a B. fuscus/B. ascidian research community. Lack of genetic giganteum/B. violaceus clade, a B. nigrum/B. prae- resources and distinguishing morphological longus/B. diegensis clade, a Caribbean clade con- characters have made species identifications dif- taining two species, and a Pacific clade contain- ficult, and a multi-locus phylogeny of the group ing two species. B. praelongus has been described has never been published. We have generated a from Japan, and B. diegensis from many temper- phylogeny based on 100 nuclear loci developed ate locations. From a phylogenetic perspective, through an anchored enrichment protocol. Five these two taxa are likely the same species. The genomes (Botrylloides praelongus, Botrylloides vio- genus Botryllus is paraphyletic with respect to the laceus, Botrylloides sp., Botryllus horridus, Botryllus genus Botrylloides, so the taxonomic distinction schlosseri Clade E, and Symplegma brakenhielmi) between the two genera may not be appropriate. were aligned with the Botryllus schlosseri Clade A
43 T28 THE SUBFAMILY BOTRYLLINAE: SPECIES IDENTIFICATION AND PHYLOGENETIC RECONSTRUCTION
Marika Salonna1,* Federica Montesanto2, Francesco Mastrototaro2, Merrick Ekins3, Fabio Gasparini4, Carmela Gissi1,5
1. Dept of Biosciences, Biotechnologies and Biopharmaceutic, University of Bari “Aldo Moro”, Via Orabona, 4 – 70125 Bari, Italy 2. Dept of Biology and CoNISMa LRU, University of Bari “Aldo Moro”, Via Orabona, 4 – 70125 Bari, Italy 3. Biodiversity & Geosciences Program, Queensland Museum, PO Box 3300, South Brisbane 4101, Brisbane, Queensland, Australia; Adjunct Fellow School of Biological Sciences, University of Queensland, St Lucia, 4072, Queensland, Australia 4. Dept of Biology, University of Padova, Via U. Bassi, 58/B – 35131, Padova, Italy 5. IBIOM, CNR, Via G Amendola 165/A - 70126 Bari Bari, Italy
The subfamily Botryllinae comprises small co- cies. The species delimitation analyses confirmed lonial ascidians of the genera Botryllus and Bot- the existence of a clear barcode gap and identified rylloides, whose morphological identification at 18 different OTUs (i.e., Operational Taxonomic species level is very problematic due to the high Units, corresponding to molecularly-delineat- phenotypic variability and the few discriminant ed species), each containing already-described, characters. Furthermore, the molecular phyloge- cryptic or putative new species. The phylogenet- ny of the group is poorly resolved. Even in the ic reconstructions recognized as statistically sig- model species Botryllus schlosseri (Pallas, 1766), nificant the clades: all known species; putative the existence of cryptic species (i.e., clades from new species; clades A-E; further subclades with- A to E) was suggested by molecular data but not in B. schlosseri. Moreover, these trees effectively yet supported by morphological analyses. resolved the relationships among the B. schlosseri clades, but unfortunately left unresolved the bas- The aim of this study is to validate a molecular al nodes of the tree, i.e., the main relationships protocol for the discrimination of Botryllinae spe- among Botryllinae. In conclusion, our results in- cies, and to clarify the phylogenetic relationships dicate that the Botryllinae species can be easily within Botryllinae. Using a nested PCR strategy, discriminated based on COI data but that a dif- we successfully amplified an elongated COI frag- ferent molecular marker is needed for resolving ment of about 860 bp in more than 120 Botryllus/ the Botryllinae phylogeny. Botrylloides worldwide-sampled colonies, mainly with uncertain or unknown morphological iden- tification or belonging to B. schlosseri cryptic spe-
44 T29 DIPLOSOMA LISTERIANUM: AN INTERESTING MODEL OF CRYPTIC SPECIATION
Joyce Ana Teixeira1, Rosana Moreira da Rocha2, *
1. Graduate program in Zoology, Universidade Federal do Paraná, Brazil 2. Zoology Departament, Universidade Federal do Paraná, Brazil
The ascidian Diplosoma listerianum is distrib- clades, and divided the former Clade A into two uted worldwide, and has been used as a model clades: clade A1 exclusively found in the west At- for a variety of biological studies. Recently, mi- lantic with one population at the Pacific side of tochondrial DNA (mtDNA) evidence suggests Panama and another in South Africa, and clade that the species may be a cryptic species complex A2, found worldwide. Two new clades are ex- of four distinct clades (Pérez-Portela et al. 2013). clusively from Mexico and another is from Pan- That study, while global in scope, included few ama. In combination, the presence of exclusive samples from the western Atlantic, where D. liste- clades in sympatry with widespread clades, the rianum is widespread. Here we add an addition- high diversity found in the tropical west Atlantic, al 55 samples from coastal Brazil, Panama (both and the strong morphological similarity among sides), Mexico (Yucatan) and the United States clades suggests that D. listerianum is a fast-evolv- (Florida) to test whether those populations are ing complex of cryptic species, with indications part of world-wide clades or are native species of ongoing speciation. This species complex is hidden within this species complex. We found also easily maintained in the laboratory and thus 33 new COI haplotypes. Phylogenetic analysis it is an interesting candidate as a model for stud- resulted in seven clades of which three were re- ies of speciation covered before. Species delimitation analyses (ABGD, GMYC and bPTP) corroborated these
References 1. Pérez-Portela R, Arranz V, Rius M & Turon X (2013) Cryptic speciation or global spread? The case of a cosmopolitan marine invertebrate with limited dispersal capabilities. Scientific Reports, 3, 3197. DOI: 10.1038/srep03197 2. Notes: This study was financed by National Council of Technological and Scientific Development − CNPq (200914/2008-1, 305201/2014-0, 440429/2015-3) and Fundação Araucária (PROTAX – 267/2015), with research grants to RMR and by Coordenação de Aperfeiçoamento de Pessoal de Nível 3. Superior - Brazil (Capes) with scholarship to JAT. 45 T30 PHYLOGENETIC ANALYSIS OF PHENOTYPIC CHARACTERS OF TUNICATA SUPPORTS BASAL APPENDICULARIA AND MONOPHYLETIC ASCIDIACEA
Katrin Braun1*, Fanny Leubner2, Thomas Stach1 1. Humboldt-Universität zu Berlin, Institut für Biologie, Philippstrasse 13, 10115 Berlin, Germany 2. Georg-August-University Göttingen, Animal Evolution and Biodiversity, J-F-Blumenbach Institute for Zoology & Anthropology, Untere Karspüle 2, 37073 Göttingen, Germany
With approximately 3000 marine species Tuni- ers monophyletic Appendicularia as sister tax- cata represents the most disparate subtaxon of on to the remaining tunicate taxa. “Thaliacea” is Chordata. Molecular phylogenetic studies sup- found paraphyletic with Pyrosomatida as sister port Tunicata as sister taxon to Craniota render- taxon to Ascidiacea and the relationship between ing it pivotal to understand vertebrate evolution. Doliolida and Salpida unresolved. Thus, the phe- While successively more molecular data became notypic data support the hypothesis that the last available to resolve internal tunicate phyloge- common ancestor of Tunicata was free-living, netic relationships, phenotypic data have not that ascidian sessility is a derived character and been utilized consistently. Here we address these that the last common ancestor of Ascidiacea was shortcomings by cladistically analyzing 116 phe- colonial. The combined analysis, congruent with notypic characters for 49 tunicate species com- published purely molecular analyses, recovers prising all higher tunicate taxa, and 5 craniate Thaliacea monophyletic nested within paraphy- and cephalochordate outgroup species. In addi- letic “Ascidiacea”. Successively up-weighting tion, we performed a combined analysis of the phenotypic data indicates that phenotypic data phenotypic characters with 18S rDNA-sequence contribute disproportionally more to the result- data in 32 OTUs. The strict consensus tree from ing phylogenetic hypothesis. the analysis of the phenotypic characters recov-
46 T31 ANTERO-POSTERIOR ECTODERM PATTERNING BY CANONICAL WNT SIGNALING DURING ASCIDIAN DEVELOPMENT
Stacy Feinberg¶, Agnès Roure¶, Julie Piron and Sébastien Darras*
Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), F-66650, Banyuls/Mer, France ¶: equal contribution
Wnt/β-catenin signaling is an ancient pathway rocal anteriorization of the embryo with a loss of in metazoans and controls various developmen- more posterior identities like in vertebrates and tal processes, in particular the establishment and hemichordate. Epidermis patterning was overall patterning of the embryonic primary axis. In ver- unchanged. Only the identity of two discrete re- tebrates, a graded Wnt activity from posterior to gions of the central nervous system, the anterior- anterior endows cells with positional informa- most and the posteriormost regions, were under tion in the central nervous system. Recent stud- the control of Wnt. Finally, the caudal peripheral ies in hemichordates support a conserved role nervous system, while being initially Wnt depen- for Wnt/β-catenin in ectoderm antero-posterior dent, formed normally. Our results show that the patterning at the base of the deuterostomes. As- Ciona embryonic ectoderm responds to Wnt ac- cidians are marine invertebrates and the closest tivation in a manner that is compatible with the relatives of vertebrates. By combining gain- and proposed function for this pathway at the base loss-of-function approaches, we have deter- of the deuterostomes. However, possibly because mined the role of Wnt/β-catenin in patterning of its fast and divergent mode of development the three ectoderm derivatives of the ascidian that includes extensive use of maternal deter- Ciona intestinalis, central nervous system, periph- minants, the overall antero-posterior patterning eral nervous system and epidermis. Activating of the Ciona ectoderm is Wnt independent, and Wnt/β-catenin signaling from gastrulation led to Wnt/β-catenin signaling controls the formation a dramatic transformation of the ectoderm with of some sub-domains. Our results thus indicate a loss of anterior identities and a reciprocal an- that there has likely been a drift in the develop- terior extension of posterior identities, consistent mental systems controlling ectoderm patterning with studies in other metazoans. Surprisingly, in the lineage leading to ascidians. inhibiting Wnt signaling did not produce a recip-
47 T32 COORDINATION OF ASYMMETRICAL NOTOCHORD CONTRACTILITY AND EPITHELIA CELL PROLIFERATION DRIVES TAIL BENDING IN CIONA EMBRYOGENESIS
Qiongxuan Lu1, Yuan Gao2, Xiqiao Feng2, Bo Dong1,*
1. Laboratory of Morphogenesis & Evolution, Ocean University of China, Qingdao 266003, China 2. Laboratory of mechanical biology, Tsinghua University, Beijing, China
In early embryogenesis, a bending tail sur- the tail bending. We further characterized the cell rounding the body within the chorion is evolu- proliferation in tail tissues during tail bending by tionally conserved feature in most of invertebrate BrdU staining. The results showed that the dorsal and vertebrate. However, whether this process midline of tail epidermis divided faster than the is genetic control and how the embryo acquires corresponding ventral domain, suggesting that a bending shape remain completely unknown. the discrepancy of the tail epidermis cell division Here, using Ciona embryogenesis, we first may produce asymmetrical force to drive the tail demonstrated that tail-bending process is a genet- bending. To understand further the mechanical ic control program. To reveal the cellular process roles of each tissue and their coordination, we during bending process, we next examined the develop a physical model to simulate the bend- distribution of actomyosin in Ciona embryogen- ing process and predict the key parameters that esis and found that they were mainly accumu- determine bending degree and position. Thus, lated along the ventral, but not dorsal side of the we revealed that ventral actomyosin contractility notochord during tail bending. The quantitative in notochord coordinated with the faster dorsal results reveled that dorsal-ventral asymmetry epithelial cell proliferation to drive tail bending distribution of actomyosin was more prominent in Ciona embryo. Our results thus provide an in- at the tail-bending site, indicating that asymmet- sight on how the different tissues coordinate to rical actomyosin contractility in notochord drives determine the embryo morphogenesis.
48 T33 PULSATILE TRANSPORT OF BASAL FACTORS TOWARD APICAL DOMAIN DURING TUBULOGENESIS, DIRECTED BY THE INTERACTION BETWEEN 14-3-3ΕA AND ERM
Yuji Mizotani1,2*, Mayu Suzuki1, Kohji Hotta1, Hidenori Watanabe3, Kogiku Shiba4, Kazuo Inaba4, Etsu Tashiro1, Kotaro Oka1, and Masaya Imoto1,
1. Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Japan 2. Department of Molecular Genetics and Cell Biology, University of Chicago, USA 3. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan 4. Shimoda Marine Research Center, University of Tsukuba, Japan
Ascidian notochord provides a simple and Interestingly, the disappearance of 14-3-3εa and highly tractable model for tubulogenesis. Com- ERM during each cycle is tightly correlated with bining chemical and genetic perturbations with a transient flow of 14-3-3εa, ERM, myosin II, and live cell imaging, we show that 14-3-3εa plays other cytoplasmic elements from the basal cor- a crucial role for tubulogenesis. Phenotypic tex to the lumen-facing apical domain, which is screening from our in-house chemical library often accompanied by visible changes in lumen led us to identify UTKO1, a human cancer cell architecture. Pulsatile behavior, transient flow, migration inhibitor we previously reported, as and lumen formation were abrogated in larvae a selective inhibitor of Ciona notochord tubulo- treated with UTKO1, depleted of either 14-3-3εa genesis. UTKO1 directly bound to 14-3-3εa and or ERM, or expressed with truncated form of 14- prevented 14-3-3εa from interacting with ezrin/ 3-3εa that lack the ability to interact with ERM. radixin/moesin (ERM), another regulator of Ci- These results suggest that 14-3-3εa and ERM in- ona notochord tubulogenesis. Therefore, we fo- teract at the basal cortex to direct pulsatile bas- cused on how interactions between 14-3-3εa and al-apical transport of factors that are important ERM could contribute to tubulogenesis in space for lumen formation. Because many core compo- and time. During tubulogenesis, 14-3-3εa and nents of this Ciona system are highly conserved, ERM colocalize at the basal cortex of notochord we propose that similar mechanisms may under- cells. As the lumen begins to open, they under- lie tubulogenesis in other systems. go cycles of accumulation and disappearance.
49 T34 NEURULA ROTATION AND LEFT-RIGHT ASYMMETRY IN ASCIDIAN EMBRYOS: CILIARY MOVEMENTS AND THE VITELLINE MEMBRANE SIGNAL
Shiori Yamada1, Yuka Tanaka1, Kaoru S. Imai1, Motohiko Saigou1, Takeshi A. Onuma1, Samantha L. Connop1, Noritaka Hashii2, Hitoshi Sawada3, Hiroki Nishida1*
1. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan 2. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Kawasaki, Kanagawa 210-9501, Japan 3. Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Sugashima, Toba 517-0004, Japan
Tadpole larvae of the ascidian, Halocynthia was also observed in Ciona robusta embryos. Our roretzi, show morphological left–right asymme- observations suggest that the driving force of ro- try in the brain structures and the orientation tation is generated using the vitelline membrane of tail bending within the vitelline membrane. as a substrate but not by making a water current Neurula embryos rotate along the anterior–pos- around the embryo. terior axis in a counterclockwise direction, and We also showed that a chemical signal from the then this rotation stops when the left side of the vitelline membrane promotes nodal gene expres- embryo is oriented downwards. Contact of the sion since the treatment of devitellinated neuru- left-side epidermis with the vitelline membrane lae with an extract of the vitelline membrane promotes nodal gene expression in the left-side promoted nodal expression on both sides. These epidermis. We show that epidermal monocilia, signal molecules are proteins but not sugars. Spe- which appear at the neurula rotation stage, gen- cific fractions in gel filtration chromatography erate the driving force for rotation. Epidermis had the nodal promoting activity. By mass spec- cilia moved in a serpentine way like sperm fla- trometry, we selected 48 candidate proteins. It is gella but not in a rotational way or beating way also shown that epidermal cilia drive the neurula with effective stroke and recovery stroke. They rotation but are dispensable for sensing the sig- moved very slowly, at 1/7 Hertz. Similar motility nal from the vitelline membrane.
References 1. Yamada, S., Tanaka, Y., Imai, K.S., Saigou, M., Onuma, T.A., and Nishida, H. Wavy movements of epidermis monocilia drive the neurula rotation that determines left–right asymmetry in ascidian embryos. Dev. Biol. (2019) in press. 2. Tanaka, Y., Yamada, S., Connop, S. L., Hashii, N., Sawada, H., Shih, Y., and Nishida, H. Vitel- line membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi. Dev. Biol. (2019) in press.
50 T35 THE EFFECTS OF HIGH WATER TEMPERATURE ON DEVELOPMENTAL PROCESSES IN C. INTESTINALIS: LIMITS TO EMBRYONIC ROBUSTNESS.
Steven Q. Irvine
University of Rhode Island, Kingston, RI, USA
The normal embryogenesis of marine animals more pronounced as embryogenesis proceeds. is typically confined to a species-specific range We used live imaging to study which morphoge- of temperatures. Within that temperature range netic processes are most susceptible to high tem- development results in a consistent, or canalized, perature. We also examined the expression of cell phenotype, whereas above and below the range type specific markers to see if cell specification is abnormal phenotypes are produced. This study affected by high temperature. The results suggest reveals a high temperature threshold, occurring that morphogenesis is more sensitive to high tem- over a 1-2°C range, for normal embryonic devel- perature than cell type specification. This work- opment in C. intestinalis. Above that threshold ing hypothesis points to further approaches to morphological abnormalities in the notochord the study of the mechanisms enabling embryonic and other organs are observed, beginning with robustness in face of environmental challenges. cleavage and gastrula stages, and becoming
51 T36 TRANSCRIPTIONAL STATES UNDERLYING NEURON SUBTYPE-SPECIFIC TRAITS IN THE MOTOR GANGLION OF CIONA
Susanne Gibboney1, Jameson Orvis1, Kwantae Kim1, Elijah K. Lowe1, and Alberto Stolfi*1
1. School of Biological Sciences, Georgia Institute of Technology, Atlanta, USA
The central nervous system (CNS) of the Ciona are ipsilaterally-projecting descending interneu- intestinalis larva has only 177 neurons, making it rons that form conspicuous electrical synapses the smallest described in any animal. The CNS with ipsilateral MN2s, but receive synaptic in- compartment that has been studied in greatest de- puts mainly from photoreceptor relay neurons tail is the Motor Ganglion (MG), a simple pattern and other interneurons of the brain, where the generator that drives the swimming behaviors of larval light- and gravity-sensing organs are locat- the larva. Within the MG, 7 bilaterally symmet- ed. Thus, these two MG neuron subtypes might ric left/right pairs of neurons from the majority modulate asymmetric swimming behaviors in of the synaptic connectivity of the MG, and can response to sensory cues processed by distinct all be traced to the A7.8 pair of blastomeres of thigmotactic (ddNs) and phototactic/geotactic the 64-cell stage embryo. Here we focus on the (MGIN2s) pathways. By analyzing and compar- comparison between two very different core MG ing the transcriptional profiles of isolated ddNs interneuron types: the descending decussating and MGIN2s, we identified and validated differ- neuron (ddN) and MG Interneuron 2 (MGIN2). entially-expressed transcripts enriched in either As their name implies, ddNs are the only neu- neuron type. This revealed candidate effectors rons whose axons cross the midline before de- of ddN/MGIN2 morphology, connectivity, and scending towards the tail. They receive synaptic function. Probing the subcellular localization inputs from peripheral nervous system (PNS) re- and functions of key ddN-enriched candidate ef- lay neurons and in turn synapse onto other MG fectors revealed a potential role for microtubule neurons, each in particular forming electrical stabilization and centrosome migration in estab- synapses with their respective contralateral Mo- lishing the unique contralateral projection of the tor Neuron 2 (MN2). On the other hand, MGIN2s ddNs.
52 T37 LEFT-RIGHT ASYMMETRIC DEVELOPMENT OF CELLS IN THE LARVAL BRAIN OF CIONA
Kouhei Oonuma* and Takehiro G. Kusakabe
Department of Biology, Konan University, Japan
To understand development of the brain at sin- gic/glycinergic, cholinergic and dopaminergic. gle-cell resolution in chordates, Ciona is an ideal Although the left-right asymmetric distribution model organism. However, both cell lineage and of neurons in the Ciona brain has been reported, developmental mechanisms of the brain vesicle how each neuron subtype is generated during of the Ciona larva remains unclear because re- development remains unknown. To elucidate the moval of the chorion, which is a commonly used precise cell lineage of the four neuron subtypes technique for manipulation of Ciona embryos, in the brain vesicle, we chased development of disturbs the structure and the left-right asymme- each cell of the neural plate using FITC and the try of the brain vesicle. By improving the micro- photo-convertible fluorescent protein Kaede. We injection technique and developing a method to revealed that the dopaminergic neurons (coronet label a single cell of Ciona embryos without re- cells) located at the left side of the brain vesicle moving the chorion, we have studied the cell lin- are derived equally from the left and right a9.37 eage and the molecular mechanisms of the brain cells. We also found left-right asymmetric devel- vesicle [1, 2]. opment of other neuron subtypes. We discuss the dynamic movement of brain vesicle cells during Neurons of the Ciona larval brain can be clas- the period between the late tailbud and larval sified into four subtypes by neurotransmitter stages. phenotypes; namely, glutamatergic, GABAer-
References 1. Oonuma, K. et al. (2016) Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation. Dev. Biol. 420, 178–185. 2. Oonuma, K. and Kusakabe, T. G. (2019) Spatio-temporal regulation of Rx and mitotic patterns shape the eye-cup of the photoreceptor cells in Ciona. Dev. Biol. 445, 245–255. 53 T38 JAGGED ACTS AS A LATERAL INDUCER TO SPECIFY CAUDAL EPIDERMAL SENSORY NEURON FATE IN CIONA
Stacy L. Feinberg1, and Robert W. Zeller1*
1. Department of Biology, San Diego State University, San Diego CA, USA
Notch signaling is utilized in numerous ways cells form. Here we show that the Jagged ligand to specify cells during metazoan development. mediates the lateral induction of caudal epider- The Notch receptor and its ligands Delta and mal sensory neurons (ESNs) in the tail of the Cio- Jagged are transmembrane proteins that under- na robusta embryo. Jagged is expressed in midline go specific processing events during the signal stripes in early embryos and is likely activated by transduction process. In many cases, Notch/ Msxb, one of the earliest-acting caudal midline Delta signaling mediates the selection of one cell transcription factors. Previous work from several type from a group of cells that have the potential labs has shown that ectopic epidermal expres- to develop into two different types of cells, a pro- sion of Delta prevents the specification of ESNs; cess referred to as lateral inhibition. Notch sig- ectopic epidermal expression of Jagged has the naling has also been found to operate in a com- opposite effect and expands ESNs along the mid- pletely opposite manner with regards to cell type line. Caudal ESN patterning is thus much more specification; rather than inhibit, Notch signaling complex than current models suggest, requiring can also laterally induce, as has been shown in both inductive and inhibitory Notch signaling the specification of sensory patches in the verte- to specify and position these cells along the tail brate inner ear from which mechanosensory hair midline.
54 T39 IN VIVO CALCIUM-IMAGING REVEALS A POSSIBLE ROLE OF THE GNRH SYSTEM IN LARVAL SWIMMING OF CIONA
Nanako Okawa1,*, Kotaro Shimai1, Masamichi Ohkura2, Junichi Nakai2, Takeo Horie3,4, Atsushi Kuhara1, and Takehiro G. Kusakabe1
1. Institute for Integrative Neurobiology & Department of Biology, Konan University, Kobe, Japan 2. Brain and Body System Science Institute, Saitama University, Saitama, Japan 3. Shimoda Marine Research Center, University of Tsukuba, Shimoda, Japan 4. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, USA
Gonadotropin-releasing hormone (GnRH) is a In this study, to obtain insights into physiologi- neuroendocrine peptide that plays a central role cal roles of GnRH in the Ciona larva, we identified in reproductive control of vertebrates. Non-re- the types of cells expressing gnrh2. Furthermore, productive function of GnRH has been suggest- we visualized activity of gnrh2-expressing cells ed but poorly understood. The Ciona larva, a in the larva by Ca2+ imaging using a calcium sen- non-reproductive stage of the simple chordate, sor protein, G-CaMP8 (Ohkura et al., 2012). Some has a prominent GnRH system spanning the en- cholinergic motor neurons as well as unique tire length of the nervous system (Kusakabe et cholinergic cells along the nerve cord express al., 2012). One of the gnrh genes, gnrh2, is con- gnrh2, suggesting a role of GnRH in the control spicuously expressed in the motor ganglion and of swimming behavior. By contrast, none of the nerve cord, which are homologous structures gnrh2-expressing cells overlap with glycinergic to the hindbrain and spinal cord of vertebrates. or GABAergic neurons. A role in the motor con- Correspondingly, GnRH receptor genes are ex- trol is also suggested by simultaneous activation pressed in the tail muscle and notochord, both of of some gnrh2-expressing cells with tail move- which are phylotypic axial structures along the ments. Active Ca2+ transients were also observed nerve cord. The gnrh2 gene is also expressed in in the proto-placodal sensory neurons. Interest- the proto-placodal sensory neurons, which are ingly, the nerve cord ependymal cells produced the proposed homologue of vertebrate olfactory Ca2+ transients, suggesting a novel role of glial neurons (Abitua et al., 2015). The tunicate larvae ependymal cells in the control of tail movement. occupy a non-reproductive dispersal stage, yet the roles of their GnRH system remain elusive.
References: 1. Kusakabe, T. G. et al.: A conserved non-reproductive GnRH system in chordates. PLoS One 7, e41955 (2012) 2. Abitua, P. B. et al.: The pre-vertebrate origins of neurogenic placodes. Nature 524, 462-465 (2015) 3. Ohkura, M. et al.: Genetically encoded green fluorescent Ca2+ indicators with improved detectabil- ity for neuronal Ca2+ signals. PLoS One 7, e51286 (2012) 55 T40 TRANSCRIPTIONAL REGULATORY MECHANISMS OF TBX6B BY ZIC-R.A (MACHO1) THROUGH SECONDARY BINDING MOTIF SITES
Izumi Oda-Ishii1,*, Deli Yu1, and Yutaka Satou1
1. Department of Zoology, Graduate School of Science, Kyoto University
Specific binding of transcription factors toa bound directly to a regulatory region of Tbx6-r.b specific DNA sequence, the binding motif, is to activate Tbx6-r.b in B6.4 cells of 32-cell em- the primary step by which cells express genetic bryos. Importantly, this region contained two information held within their genome. Recent Zic-r.a binding sites, which were similar to the development of high-throughput profiling of secondary binding motif of Zic transcription transcription factor binding specificities has re- factors (UniprobeUP00102) 2 but not similar to vealed that many transcription factors can recog- the primary motif for Zic transcription factors nize multiple binding motifs, rather than a single (UniprobeUP00102) 2 or a motif for Ciona Zic-r.a binding motif 1. However, their developmental 3. The secondary motif sites bound Zic-r.a more roles and mechanisms of the choice of binding weakly than the primary motif sites in vivo. Our motifs by transcription factors are largely un- reporter assays showed the significance of sec- known. ondary binding motif. Specifically, when the secondary binding motif was replaced with the A Zic transcription factor Zic-r.a (Macho1) is primary binding motif, a reporter gene was ex- a maternal muscle determinant and activates pressed ectopically in non-muscle lineages. The Tbx6-r.b zygotically. At the 16-cell stage, Zic-r.a data indicates that secondary binding motif sites dose not necessarily need to bind directly to the contribute to restricting the Zic-r.a-dependent ac- regulatory region to activate Tbx6-r.b 1. How- tivation of Tbx6-r.b to muscle-lineage cells. ever, in the present study, we found that Zic-r.a
References 1. Oda-Ishii et al. 2016, PLoS Genetics. 12 (10): e1006392, 2. Badis et al. 2009, Science. 324:1720-1723. 2. Uniprobe (http://the_brain.bwh.harvard.edu/uniprobe/) 3. Yagi et al. 2004, Developmental Biology. 274: 478-489. 56 T41 STEREOTYPY AND VARIATION AMONG THE LARVAL MOTOR CIRCUITS
Kerrianne Ryan*, Zhiyuan Lu2, and Ian A. Meinertzhagen
1. Department of Psychology and Neurosciance Dalhousie University 1355 Oxford Street, Rm 3263 3rd Floor Life Sciences Centre (Psychology Wing) P.O. Box 15000 Halifax, Nova Scotia B3H 4R2Canada
Small nervous systems allow us to examine the have undertaken comparative cell-by-cell analy- entirety of a nervous system in detail, revealing sis of the morphology and synaptic connections both ultrastructural and network features. We between cells of the motor networks of two indi- have used anatomical connectomic approaches vidual sibling larvae at the same stage. The val- by means of serial-section EM (ssEM) to analyze idation of cell types, and their synaptic proper- the number and distribution of synapses within ties and connections are revealed through these the networks of the larval brain of Ciona intesti- analyses, as well as some putative distinctions in nalis (Ryan et al., 2016; Ryan et al., 2017; Ryan et important network components. These include al. 2018). individual differences in some motor neurons and the ACINs. These comparisons also empha- Larval responses to environmental cues rely size the location of synaptic contacts and reveal on the network of underlying neuronal connec- the variable stereotypy of axonal bundling. Com- tions that translates these cues into motion. The paring network properties helps to identify ro- anatomical connectome reveals specific details bustness of substrates for more complex larval of motor networks at a snapshot during devel- behaviours based on interactions between path- opment, which are involved in central pattern ways that converge on the motor generating net- generation and a putative escape response. To work of the Ciona intestinalis larva. examine the consistency of these networks, we
References 1. Ryan, K., Lu, Z., Meinertzhagen, I.A. 2016. Sidedness in the brain of a chordate sibling, the tadpole larva of Ciona intestinalis. ELife. http://dx.doi.org/10.7554/eLife.16962 2. Ryan, K., Lu, Z., Meinertzhagen, I.A. 2017. Circuit homology between decussating pathways in the Ciona larval CNS and the vertebrate startle-response pathway. Current Biology. http://dx.doi.org/10.1016/j. cub.2017.01.026 3. Ryan, K., Lu, Z., & Meinertzhagen, I. A. 2018. The peripheral nervous system of the ascidian tad- pole larva: Types of neurons and their synaptic networks. Journal of Comparative Neurology, 526(4), 583-608. 57 T42 CIONA INTESTINALIS AS A NEUROETHOLOGY MODEL
Jerneja Rudolf, Kushal Kolar, Daniel Dondorp, Marios Chatzigeorgiou*
Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen 5006, Norway
te towards the establishment of the tunicate havioural modes using agglomerative clustering. Ciona intestinalis as a neuroethology model. We Here we will be presenting new data on the role are trying to match the growing insights into the of biogenic amines in modulating C. intestinalis structure1-3 and development of the larval ner- larval behaviour. vous system with a detailed characterization of Furthermore, we will be presenting our on-go- its neural circuits and behavioural output. ing efforts to perform functional imaging of the As a starting point, we have been developing larval nervous system. Our dataset includes, open-source hardware and software in order to both spontaneous neuronal activity at a brain- perform quantitative behavioural analysis of wide level as well as sensory stimulus evoked re- swimming larvae and functional imaging of the sponses from a small set of sensory receptor cells tadpole nervous system. and downstream neurons. Previously, using our behavioural setups, we Finally, we will be discussing some of the chal- collected large datasets of free-swimming C. in- lenges and possible solutions towards a systems testinalis larvae and employed machine-learning level mapping of neuronal function onto the wir- methods to create an objective ethogram of the ing diagram of C. intestinalis. larvae4. In addition, we went on to identify be-
References: 1. Ryan, K., Lu, Z. & Meinertzhagen, I. A. in Elife Vol. 5 (2016). 2. Ryan, K., Lu, Z. & Meinertzhagen, I. A. Circuit Homology between Decussating Pathways in the Ci- ona Larval CNS and the Vertebrate Startle-Response Pathway. Curr Biol 27, 721-728, doi:10.1016/j. cub.2017.01.026 (2017). 3. Ryan, K., Lu, Z. & Meinertzhagen, I. A. The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks. J Comp Neurol, doi:10.1002/cne.24353 (2017). 4. Rudolf, J., Dondorp, D., Canon, L., Tieo, S. & Chatzigeorgiou, M. Automated behavioural analy- sis reveals the basic behavioural repertoire of the urochordate Ciona intestinalis. Sci Rep 9, 2416, doi:10.1038/s41598-019-38791-5 (2019). 58 T43 BRAIN ASYMMETRY – FROM GENES TO CIRCUITS AND BEHAVIOUR
Steve Wilson
University College London, UK
It is likely that the nervous systems of all bilat- acter independent of signals from the parapineal erally symmetric animals are left-right asymmet- nucleus (which are normally required to impart ric with respect to processing of information and left-sided character). The rorschach mutation is in control of behaviour. However, we know very a gene which encodes an orphan transmembrane little about how asymmetries arise in develop- receptor that we find interacts with Wnt path- ment, how they are encoded in circuits and what way receptors. To explore the behavioural conse- their importance is for nervous system function. quences of disrupting habenular asymmetry, we We are using developmental, genetic, imaging are assessing how habenular circuitry impacts and behavioural approaches to study habenular an innate exploration strategy. We find that ex- asymmetry in zebrafish to address these issues. ploration strategy is modulated by levels of light One focus is to determine the mechanisms that and the ability to switch strategy upon changes lead to neurons on the left and the right acquiring in illumination is lost in embryos in which both different character and establishing different cir- habenulae develop with right-sided character. cuit connectivity between left and right sides of The people who have done this work and our the brain. Through a forward genetic screen, we many collaborators will be acknowledged in the identified the rorschach mutant in which neurons presentation. This research is supported by the on both left and right sides adopt left-sided char- Wellcome Trust.
59 T44 MECHANOSENSATION OF TIGHT JUNCTIONS BY ZO-1 PHASE SEPARATION AND FLOW
Carl-Philipp Heisenberg
IST Austria, Klosterneuburg, Austria
Cell-cell junctions respond to mechanical forc- ZO-1 junctional accumulation by driving retro- es by changing their organization and function. grade actomyosin flow within the YSL that trans- Tension-dependent conformational changes of port non-junctional ZO-1 clusters towards the junctional proteins are thought to underlie this TJ. Non-junctional ZO-1 clusters form by phase junctional mechanosensitivity. Here we show separation, and their effective formation is de- that in the gastrulating zebrafish embryo, tight pendent on the actin binding region (ABR) with- junctions (TJ) mechanosensitivity is mediated in the C-terminus of ZO-1. If the non-junctional by actomyosin-driven flow of phase separated ZO-1 pool is absent, TJ lose their mechanosen- Zonula occludens-1 (ZO-1) clusters. We found sitivity, and, consequently, EVL-YSL movement that ZO-1 junctional accumulation at the contact is impaired. Thus, phase separation and flow of between the Enveloping Layer (EVL) and the nonjunctional ZO-1 confer mechanosensitivity to Yolk Syncytial Layer (YSL) closely scales with TJ. actomyosin tension. Actomyosin tension triggers
60 T45 PATTERNING OF TRANSPORT FUNCTION: HOW TRANSPORTERS ARE INTEGRATED IN EARLY DEVELOPMENTAL PROGRAMS OF THE SEA URCHIN.
Amro Hamdoun, Catherine Schrankel, Himanshu Vyas, Travis Fleming ([email protected])
Scripps Institution of Oceanography, 8750 Biological Grade Road La Jolla CA, 92037.
Membrane transporters play essential roles in cell nanobodies, and in-vivo membrane transport development, including protection of the embryo assays. The results have revealed protective and and control of cell differentiation and motility. homeostatic genes such as ABCB1, ABCC1 and ATP-Binding Cassette (ABC) transporters are a ABCC9 which are expressed ubiquitously, as well major family of small molecule transporters that as developmental transporters such as ABCC4 include proteins that handle xenobiotics, signal- and ABCC5 which are expressed in distinct terri- ing molecules and metabolites. These transport- tories within the embryo. ABCC4, a lipid-derived ers are already well-appreciated for their roles in signal molecule transporter, is expressed in ring drug disposition of adults, however less appre- of mesodermal cells of the early blastula, and ciated for their diverse functions in the embryo. later becomes restricted to mesoderm and germ- Here I will summarize our recent results eluci- line-fated tissues. In addition, we have identified dating how these proteins are integrated into the two genes, including ABCB4 and ABCG2, that are program of early embryogenesis. These include expressed exclusively in the hindgut at the onset conserved developmental roles in the formation of gut development, and eventually expand to of primordial germ cells, and in differentiation of the midgut region. Both proteins are further ac- the gut. To place ABC transporter genes within tivated in larval guts post-feeding. These are the the regulatory networks of development, we have most comprehensive systems-level descriptions systematically identified the major ABC trans- of transporter expression during embryogene- porters of the sea urchin embryo, which include sis of any embryo, and lay the groundwork for ABCB1, ABCB4, ABCC1, ABCC4, ABCC5, ABCC9, defining the regulatory programs that specialize and ABCG2, and aligned their patterns of activity membrane function. and expression, using in-situ hybridization, live-
61 T46 THE HIGHS AND LOWS OF PH REGULATION IN CORALS AND SYMBIOTIC CNIDARIANS
Alexander Venn
Centre Scientifique de Monaco
Corals are world-class ecosystem engineers we have investigated how corals decrease pH in that rely on dissolved inorganic carbon (DIC) to the immediate vicinity of the symbionts to maxi-
perform important ecological roles: CO2 for pho- mise CO2 uptake. Coral calcification occurs in an 2- tosynthesis, and CO3 for calcification. Low pH extracellular calcifying medium under the calci-
promotes formation of CO2 from other forms of fying cells, where pH is elevated. This pH “up- 2- DIC, whereas higher pH favours CO3 , but at nor- regulation” potentially promotes both the flux - mal intracellular pH most DIC exists as HCO3 . of DIC to the calcification site and the formation As such, corals are presented with an internal pH of calcium carbonate. Additionally, we have in- regulatory challenge in order to support photo- vestigated if corals can sustain pH upregulation synthesis and calcification. One objective of our against changes in seawater pH expected to oc- work has been to characterise the extent to which cur under ocean acidification. Our findings sug- internal pH varies in corals at sites of photosyn- gest that pH regulation may be key physiological thesis and calcification. Coral photosynthesis is trait shaping the response of these organisms to a carried out by intracellular algal symbionts and rapidly changing ocean environment.
62 T47 REGENERATION IS A PARTIAL REDEPLOYMENT OF THE EMBRYONIC GENE REGULATORY NETWORK
J. Warner, A. Amiel, H. Johnston & E. Röttinger
Université Côte d’Azur, CNRS, INSERM, Institute for Research on Cancer and Aging, Nice, France
Regenerated body parts are similar to the ones seq time-course for oral regeneration. Latter that were developed during embryonic develop- was compared to available and novel embryon- ment. This observation is at the origin of a cen- ic RNAseq data sets, enabling us to determine tury old hypothesis proposing that regeneration genes that are shared between embryogenesis reutilizes developmental processes. If this is true, and regeneration but importantly also, to high- the genetic interactions driving these two pro- light genes whose dynamic expression is specific cesses are predicted to be largely overlapping. to the regeneration process. Our global compar- One organism that is perfectly suited to compare ison revealed temporal modules corresponding the gene regulatory networks (GRNs) underlying to core biological processes that are highly con- embryogenesis and regeneration is the anthozo- served between both developmental trajectories. an cnidarian Nematostella vectensis. Not only Remarkably, this analysis also unveiled several a global GRN underlying embryogenesis has ‘regeneration specific’ modules that govern dis- established for this cnidarian but also, this sea tinct cellular processes, such as apoptosis. Taken anemone is able to undergo whole body regener- together with the molecular analysis of pathway ation and reform missing body part in only five specific perturbation experiments, we show that days after amputation. After characterizing in regeneration is a partial and rewired re-deploy- detail the regenerative capacity and the morpho- ment of the embryonic GRN rather than a com- logical and cellular events underlying the regen- plete recapitulation of the embryonic program. eration process of Nematostella, we performed a large scale and high-resolution temporal RNA-
63 T48 GENE REGULATORY NETWORK APPROACHES FOR GUT PATTERNING AND EVOLUTION
Maria Ina Arnone
Department of Biology and Evolution of Marine Organisms Stazione Zoologica Anton Dohrn, Napoli -Italy
Comparative gene regulatory network (GRN) controlling exocrine pancreatic-like cell type for- approaches have been proven to be very useful mation), these developmental GRNs appear to be in studying evolution of specification process- subject of considerable rewiring even when they es. Using the sea urchin as main model system, share very similar transcription factor toolkits. we are studying the GRNs that control the for- We are currently using an approach integrating mation of feeding related cell types and organs: multiple NGS applications, including Assay for in particular, the circum-esophageal muscles, transposase-accessible chromatin sequencing the pancreatic cell type and the posterior gut, (ATAC-Seq) and single cell RNA-Seq, for the pre- the latter differentiating into stomach, pyloric diction and validation of gene interactions oper- sphincter and intestine. The comparison of these ating in these GRNs. We expect to gain insight in different GRNs with their putative homologs in the development and evolution of such a crucial other echinoderm (sea star), vertebrate and also innovation for the evolution of multicellular or- protostome animals highlighted striking com- ganisms: the emergence of a specialized system monalities: except for the use of some recurrent for food digestion and nutrient absorption. sub-circuits (such as the hnf1-ptf1a sub-circuit
64 T49 EVOLUTIONARY NOVELTIES IN TUNICATES: PAX37B IS ESSENTIAL FOR THE DEVELOPMENT OF GIANT FOL CELLS IN THE HOUSE SECRETING EPITHELIUM OF OIKOPLEURA DIOICA
David Lagman1,*, Yana Mikhaleva1, Wei Deng1 and Daniel Chourrout1
1. Sars International Centre for Marine Molecular Biology, University of Bergen, Norway
Considering their phylogenetic position among Functional studies using RNA interference chordates, tunicates may be the outcome of an- showed that two of these genes, propA and propB, atomical simplification. While only a few gene are involved in the development of a region situ- regulatory pathways have been uncovered for ated around the dorsal midline of the epithelium tunicates, they have been shown to have lost a and ultimately in the production of Oikosin41a number of developmental genes, including tran- (a protein of the house) [3]. Here the function of scription factors present in the ancestor of chor- the pax37B homeobox gene, strongly expressed dates. Gene loss appears more pronounced in in the putative precursors of the Fol cell territory larvaceans than in ascidians. However, evolu- [3] (producing the food concentrating filter of the tionary novelties have also appeared such as the house in adults), were examined by introducing important house of larvaceans, a complex extra- a 4 bp deletion using CRISPR-Cas9. When exam- cellular structure for filtering seawater and con- ined during development, it was clear that the centrating food particles. It is secreted by multi- mutants lack all giant Fol cells. Thus, they most ple territories of a highly modified epithelium of likely fail to produce a functioning food concen- the trunk, whose development is of high inter- tration filter and probably die due to starvation. est. Sequencing of the O. dioica genome revealed This work shows an important co-option of mul- many duplicated homeobox transcription factor tiple ancient transcription factors for the genesis genes compared to other tunicates [1], many of of a novelty that is essential for the planktonic which are expressed in the developing epitheli- life of tunicate larvaceans. um soon after hatching, indicating importance for its patterning [2,3].
References 1. Edvardsen RB., et al. Remodelling of the homeobox gene complement in the tunicate Oikopleu- ra dioica. Curr Biol. 2005 Jan 11;15(1):R12-3. 2. Denoeud F., et al. Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate. Science. 2010 Dec 3;330(6009):1381-5. doi: 10.1126/science.1194167. Epub 2010 Nov 18. 3. Mikhaleva Y., et al. Development of the house secreting epithelium, a major innovation of tu- nicate larvaceans, involves multiple homeodomain transcription factors. Dev Biol. 2018 Nov 15;443(2):117-126. doi: 10.1016/j.ydbio.2018.09.006. Epub 2018 Sep 11.s 65 T50 EPHRIN-MEDIATED “DAMPING” OF FGF SIGNALLING UNDERLIES THE SPATIAL PRECISION OF ASCIDIAN NEURAL INDUCTION
Géraldine Williaume, Cathy Sirour, Clare Hudson, Hitoyoshi Yasuo
Laboratoire de Biologie du Développement de Villefranche-sur-Mer, CNRS, Sorbonne Université, France
We study the initial step of ascidian neu- in a bimodal manner. During neural induction, ral induction to address how a cell interprets a ephrin signals, acting between the ectoderm graded signal to generate a threshold response. cells themselves, antagonise FGF signalling. In During this process, four ectoderm cells among embryos inhibited for ephrin/Eph signals, ERK sixteen are selected as neural precursors. Mes- activation levels increase in all ectoderm cells. endoderm-derived FGF9/16/20 acts as a neural Under these conditions, the spatial precision of inducer and directly activates Otx expression Otx expression is lost with additional ectoderm though the RTK-ERK-Ets pathway (Bertrand et cells exhibiting the ‘ON’ status of Otx expression. al, 2003, Cell). Each ectoderm cell is in direct con- This suggests that ephrin/Eph signals act to re- tact with FGF-expressing cells, with neural pre- duce the overall levels of ERK activation, such cursors having the largest surface contact (Tassy that the non-neural ectoderm cells remain below et al, 2006, Curr. Biol). the threshold required for Otx gene activation. We have confirmed this by re-establishing the Our quantitative analyses have revealed that normal Otx expression profile in ephrin-blocked each ectoderm cell exhibits a level of ERK acti- embryos with low doses of a MEK inhibitor. Our vation in proportion to its area of surface con- study has thus uncovered a mechanism whereby tact with FGF-expressing cells. In contrast, the signal damping underlies the spatial precision of transcriptional response of Otx is restricted to threshold response to graded signal inputs. only the four neural precursors and operates
66 T51 SHP2 PHOSPHATASE IS REQUIRED FOR NORMAL EMBRYOGENESIS IN C. INTESTINALIS
Rose E. Jacobson1 & Steven Q. Irvine1
1. University of Rhode Island, Kingston, RI USA
During temperature stress, Shp2, a protein ty- high temperatures. It has been shown that Shp2 rosine phosphatase, is highly upregulated in the is an upstream activator of the MapK pathway in ovaries of C. intestinalis. Shp2 has been implicat- zebrafish, dephosphorylating MapK to allow for ed in cell proliferation, migration, stem cell re- the recycling of the protein. The goal of this study newal, and differentiation and can also act as an is to use CRISPR/Cas9 knockouts and transgene adaptor protein. In order to investigate the role overexpression to determine if Shp2 in C. intesti- of Shp2 in embryogenesis we used an inhibitor nalis also functions in the MapK pathway to me- to block the dephosphorylation site of Shp2. This diate the animal’s ability to develop normally at treatment results in embryos that mimic the phe- high temperatures. notypic abnormalities seen in embryos grown at
67 T52 MITOTIC KINASES CHOREOGRAPH FGF RECEPTOR STORAGE AND REDISTRIBUTION DURING CARDIOPHARYNGEAL CELL
Christina D Cota1*, Matthew S. Dreier1, William Colgan1, Anna Cha1 and Brad Davidson1
1. Department of Biology, Swarthmore College, Swarthmore PA. 19081, USA
The response of embryonic cells to inductive AurK further promote receptor storage by ad- signals is strongly influenced by endosomal ditionally suppressing short and long-loop re- trafficking of receptors and associated proteins1. cycling respectively. Through perturbation of a Despite this, the regulation of receptor traffick- conserved CDK1 phosphorylation site3, we also ing in dividing cells remains poorly character- show that CDK1 suppresses short loop recycling ized. We study inductive signal processing in through direct phosphorylation of a key regula- the cardiopharyngeal founder cells of the mod- tor of short-loop recycling, RAB4. As cells prog- el chordate Ciona robusta. In dividing founder ress through metaphase, loss of kinase activity cells, biased mitotic redistribution of Fibroblast permits differential degradation and targeted Growth Factor Receptors (FGFRs) drives differ- recycling of stored receptors, leading to asym- ential induction of cardiopharyngeal progeni- metric induction. Mitotic FGF receptor storage, tor cell (Trunk Ventral Cell, TVC) fate2. Here we as delineated in this study, may facilitate rapid delineate the role of the mitotic kinases Cyclin reestablishment of signaling competence in na- Dependent Kinase 1 (CDK1) and Aurora Kinase scent daughter cells. However, mutations that (AurK) in FGF receptor trafficking during asym- limit or enhance the release of mitotically stored metric founder cell division. In vivo perturbation signaling components could alter daughter cell of kinase activity revealed that CDK1 promotes fate or behavior, disrupting development or tis- mitotic storage of FGFRs by suppressing endo- sue homeostasis. somal degradation of these receptors. CDK1 and
References 1. Irannejad, R., Tsvetanova, N. G., Lobingier, B. T. & von Zastrow, M. Direct effects of endocytosis on receptor-mediated signaling. Current Opinion in Cell Biology 35, 137–143 (2015). 2. Cota, C. D. & Davidson, B. Mitotic membrane turnover coordinates differential induction of the heart progenitor lineage. Developmental Cell 34, 505–519 (2015). 3. van der Sluijs, P. et al. Reversible phosphorylation-dephosphorylation determines the localization of Rab4 during the cell cycle. The EMBO Journal 11, 4379–4389 (1992). 68 T53 BRAVEHEART, OIKOPLEURA DIOICA A CARDIOGENIC LOSER, BUT NOT A HEARTLESS CHORDATE
Alfonso Ferrández-Roldán*, Enya Duran-Bello, Marc Fàbrega-Torrus, Gaspar Sánchez- Serna, Alba Almazán-Almazán, Marcos Plana-Carmona, Ricard Albalat, Cristian Cañestro
Department of Genetics, Microbiology and Statistics, & Biodiversity Research Institute (IRBio), University of Barcelona, Spain
Gene loss has been a significant source of genet- grate from the anterior part of the tail into the ic variation during animal evolution, and among trunk, in a similar way as described in ascidi- chordates, appendicularians such as Oikopleura ans. Despite the cardio-ontogenic similarities dioica are probably the most successful losers. As between O. dioica and ascidians, our exhaustive a case study, we investigate the impact of gene in silico survey for cardiogenic factors conserved loss on the evolution of the mechanisms of heart in other chordates reveals striking differences in development in O. dioica. The heart of O. dioica is O. dioica regarding its early signaling pathways likely the simplest of all chordates with a bi-di- as well as cardiac transcription factors involved mensional chamber-less structure made of only in migration and differentiation. Thus, our work two layers, the pericardium and myocardium, unveils that the braveheart story of O. dioica has the latter beating against the stomach wall. Our been shaped by a process of deconstruction of work provides the first modern developmental the cardiac genetic toolkit including prominent atlas of the heart of O. dioica and describes the gene losses, loss of cardiac expression domains, cell lineage fate map of cardiac progenitors up and the abolishment of developmental signaling to tailbud stage. Our data results are consistent pathways that are fundamental to make a heart with the idea that the cardiac precursors derive in other chordates. from the most anterior muscular cells and mi-
69 T54 A FINE ANALYSIS OF THE CIONA LARVAL SENSORY ADHESIVE ORGAN
Fan Zeng1*, and Ute Rothbächer1
1. Dpt. Evol. and Dev., Institute of Zoology, University Innsbruck, Austria
Ascidian papillae (palps) constitute a transient for collocytes. Interestingly, we detect two dif- sensory adhesive organ that assures larval settle- ferent types of collocyte granules, one of them ment and the onset of metamorphosis. For Cio- containing fibrous material and larger quantities na, cell numbers and discriminative molecular of polysaccharides. We further propose CCs to markers for the different cell types were missing. derive from an evolutionary ancient neurosecre- Most attention was given to neural cell types tory cell type. We have further explored the com- only. We converge serial-section electron micros- ponents of adhesive secretions and screened for copy and confocal imaging with various marker additional specificities of initial larval adhesion combinations to document the 3D organization [2]. Our findings contribute to understanding the of the Ciona papillae. We show the papillar de- development of the anterior (‘new head’) region velopment with 4 axial columnar cells (ACCs), 4 of the Ciona larva and notably the adhesive se- lateral primary sensory neurons (PSNs) and 12 creting cells important for developmental biolo- central collocytes (CCs). We propose molecular gy, evolution and bioadhesion. markers for each cell type including novel ones
References 1. Zeng et al. (2018). Papillae revisited and the nature of the adhesive secreting collocytes. Dev Biol. epub Nov 22. 2. Zeng et al. (in review). Identifying adhesive components in a model tunicate. 70 T55 GABA-MEDIATED GNRH RELEASE TRIGGERS METAMORPHOSIS OF CIONA
Akiko Hozumi1, Shohei Matsunobu1, Kaoru Mita-Yoshida1, Nicholas Treen1‡, Takaho Sugihara2, Takeo Horie1, Tetsushi Sakuma3, Takashi Yamamoto3, Akira Shiraishi4, Mayuko Hamada5, Nori Satoh6, Keisuke Sakurai2, Honoo Satake4, Yasunori Sasakura1*
1. Shimoda Marine Research Center, University of Tsukuba 2. Department of Biology, Graduate School of Life and Environmental Sciences, University of Tsukuba 3. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University 4. Bioorganic Research Institute, Suntory Foundation for Life Sciences 5. Ushimado Marine Institute, Okayama University 6. Marine Genomics Unit, Okinawa Insitute of Science and Technology Graduate University
The larva of ascidians performs perhaps the tor of GABA. Our epistasis analysis suggests that most dramatic metamorphosis of any animal, GABA positively regulates secretion of GnRH changing from a swimming tadpole to a vase-like, for inducing metamorphosis, although GABA is sessile adult. The mechanisms of metamorphosis a well-known inhibitory neurotransmitter that remain mysterious, despite many attempts to generally relaxes postsynaptic neurons. GABA characterize them. Here we report the neuronal is an important regulator of GnRH neurons in signaling essential for metamorphosis of Ciona. vertebrate hypothalamus for promoting matura- Pharmacological analyses and gene knockout/ tion during puberty. In the hypothalamus, both knockdown experiments indicated that GABA inhibitory and excitatory activities of GABA on is the neurotransmitter required for starting all GnRH have been reported. Our study suggests metamorphic events. GABA uses its metabo- an unexpected similarity between metamorpho- tropic receptor to relay metamorphic signals to sis of ascidians and reproductive maturation of the post-synaptic cells. The neuropeptide Gonad- vertebrates, both of which are the major events otropin-releasing hormone (GnRH) is the factor pushing animals toward adulthood. inducing tail regression as the downstream fac-
71 T56 MOLECULAR PROFILE AND FUNCTION OF THE AXIAL COLUMNAR CELLS OF CIONA PAPILLAE
Christopher V. Johnson*1, Florian Razy-Krajka1, and Alberto Stolfi1
1. School of Biological Sciences, Georgia Institute of Technology, Atlanta, USA
Recent scRNAseq data from our laboratory traction could also be a signal for metamorpho- (Sharma et al. 2018) revealed the transcriptome sis. Calcium signaling, important for sensory and profile of the axial columnar cells (ACCs) of contractile processes, was visualized in the ACCs the Ciona robusta papilla. Unexpectedly, vari- using CryBG>Gcamp6s, which revealed waves of ous genes encoding smooth muscle-like func- Ca2+ flux prior to settlement and metamorpho- tion were identified as enriched in ACCs, in- sis. These waves of Ca2+ could be resulting from cluding Myosin heavy chain 10/11/14, Myosin a chemo- or mechanosensory mechanism, which light chain kinase, Calponin, IP3 receptor, Atp2a could in turn be triggering papilla retraction (SERCA), and various calmodulins. Given this and metamorphosis. We have begun to use tis- profile, we are probing the morphological and sue-specific CRISPR/Cas9-mediated mutagene- physiological functions of the ACCs. A putative sis to disrupt the function of candidate effector contractile function would suggest the ACCs genes in the papillae to see if it they play any may have a role in retracting the papillae once a role in Ca2+ signaling, papilla retraction, and/or suitable location to settle is established. This re- metamorphosis.
72 T57 MIR-4055 REGULATES CIONA SENSORY ORGAN MORPHOGENESIS THROUGH AKT-MAPK SIGNALING PATHWAY
Xiaoming Zhang1,*, Xiaozhuo Liu1, Bo Dong1
1. Laboratory of Morphogenesis & Evolution, Ocean University of China, Qingdao, 266003, China.
The sensory organs such as ocellus and otolith the regulation of ocellus formation and devel- of ascidian larva localize within a sensory vesicle, opment. Furthermore, we screened and verified which is homologous with the vertebrate’s fore- the RAC-alpha serine/threonine-protein kinase brain. Formation and the regulatory mechanisms (AKT) as one target of miR-4055 through bioin- for ascidian sensory vesicle are largely unknown. formatics and luciferase reporter gene analysis. In this study, we aim to screen small miRNA that Immunostaining results showed that AKT and regulate sensory morphogenesis through the its downstream gene mitogen-activated protein construction of three small RNA libraries in Cio- kinase (MAPK1) localized in photoreceptor cells na savignyi. After screening, miR-4055 was iden- (Arrestin-positive cells). The expression level of tified and validated to highly express in ascidian AKT was significantly higher in miR-4055 KO sensory organ in 28, 32 and 36 hpf’s larvae. Loss cells than that in the wild-type ones. MAPK1 was of miR-4055 through CRISPR-Cas9 knocking out down-regulated in sensory vesicle in those AKT (KO) induced abnormal morphology of ocel- overexpressed larvae. Overexpression of AKT lus, and the increased distance between ocellus or inhibition of MAPK1 produced the consistent and otoliths than that in wild type. Behavior- phenotypes in sensory vesicles as observed in al studies conducted on those mutants showed miR-4055 KO larvae. These results indicated that that the photo-response to dim light stimuli was miR-4055 regulated the development and func- abolished in miR-4055 KO larvae. The results tion of ocellus through AKT-MAPK signaling demonstrated that miR-4055 were involved in pathway.
73 T58 CA2+ SIGNALING CONTRIBUTES TO THE BEGINNING OF METAMORPHOSIS VIA MECHANICAL STIMULI IN PALPS
Maiki Wakai1,*, Kohji Hotta1, and Kotaro Oka1
1. Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Kouhoku-ku, Yokohama 223-8522, Japan
Calcium ion (Ca2+) is one of the major second meta-period with a Ca2+ sensor, GCaMP6s. The messengers and involved in many important bi- timing of metamorphosis was precisely deter- ological phenomena. We have reported the dy- mined by a newly observed initiation point of namics of Ca2+ in the Ciona embryo from gastrula metamorphosis. As a result, it was observed that to tailbud stages (Akahoshi et al., 2017). Howev- Ca2+ concentration at specific organs transiently er, Ca2+ dynamics in later developmental stages increased before metamorphosis. In adhesion pe- have never been reported. In the metamorpho- riod, Ca2+ transients in palps were discovered just sis meta-period, the larvae start metamorphosis after their mechanical stimulation. In addition, with settlement by the adhesive papillae (palps), the Ca2+ dynamics in palps consisted of several but its mechanisms are still unknown. We don’t temporally distinct components. These findings know even the detailed role of ascidian palps, indicated that Ca2+ signaling in palps contributes which is necessary to induce tail absorption. to the beginning of metamorphosis. To answer these questions, we first developed a This is first report showing direct evidence that novel experimental system to observe individual mechanical stimulation induces metamorphosis. larva from swimming to metamorphosis stages We also discovered the role of palps as a mech- including the moment of adhesion. This system ano-sensory organ. Our study will help further enables us to artificially induce metamorphosis understanding of mechanisms of mechanical and we successfully observed Ca2+ dynamics of sensor and metamorphosis. the larva through pre- and post-metamorphosis
74 T59 EXTREME MORPHOGENETIC CANALIZATION OF ASCIDIAN EMBRYONIC DEVELOPMENT DESPITE HIGH GENETIC VARIABILITY
Patrick Lemaire
Centre de Recherche en Biologie Cellulaire, Montpellier
Canalization of developmental processes en- between embryos. This extreme reproducibility sures the reproducibility and robustness of em- may be linked to the control of fate specification bryogenesis within each species. In its extreme by local cell inductions. While in vertebrates the form, found in ascidians, early embryonic cell outcome of cell inductions is usually controlled lineages are invariant between embryos within by the concentration of diffusible extracellular li- and between species, despite rapid genomic di- gands, ascidian cell inductions appear to be con- vergence. To resolve this paradox, we used live trolled by the area of contacts between signalling light-sheet imaging and developed automat- and responding cells, rather than by differential ed single-cell segmentation and tracking pro- concentrations of ligands. We propose that the cedures to quantify individual embryonic cell duality between genetic and geometric control behaviors. This quantitative approach revealed of inductions contributes to the counter-intuitive that individual cell lineages, cell geometries, po- inverse correlation between geometric and ge- sitions and even contacts are highly reproducible netic variability during embryogenesis.
75 T60 HIGHLY CONSERVED ENHANCER GRAMMAR
Fabian Lim, Jim Posakony, Emma Farley
Fabian Lim, Roger Revelle College, Molecular Biology major
Jim Posakony, PhD., Division of Biological Sciences, Section of Cell and Developmental Biology
PI: Emma Farley, Ph.D., Division of Biological Sciences, Section of Molecular Biology and School of Health Sciences, Department of Medicine.
Enhancers are elements within the genome that 500 million years across all deuterostomes and act as switches to control the location and timing some protostomes. We tested this enhancer from of gene expression. Thus, enhancers are funda- 4 protostomes, including scorpion, and 6 deu- mental for successful development. The func- terostomes for activity in Ciona intestinalis embry- tional features of an enhancer are “binding sites”, os. The conserved enhancer grammar encodes a sequences that recruit specific transcription fac- conserved expression pattern in the developing tors to allow activation of gene expression. The nervous system. Further studies of this rare con- order, orientation, and spacing of these sites — servation could uncover important principles collectively known as “enhancer grammar” — governing enhancer grammar and help identify is thought to be important for the activity of an how grammar confers fitness during evolution. enhancer. We bioinformatically identified an en- hancer that has conserved its grammar for over
76 T61 COMPARATIVE GENOMIC ANALYSIS : IDENTIFICATION OF NOVEL GENES IN ASCIDIANS
Christelle Dantec1,*, Paul Simion2, Magali Naville3 , Justine Dardaillon1 , Remo Sanges4, Céline Scornavacca2 , Frédéric Delsuc2 , Jean-Nicolas Volff3, Hiroki Nishida5, Emmanuel Douzery2 and Patrick Lemaire1
1. Centre de Recherche en Biologie Cellulaire, CNRS-U. Montpellier, Montpellier, France 2. Institut des Sciences de l’Evolution, CNRS-IRD-EPHE-U. Montpellier, Montpellier, France 3. Institut de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, CNRS UMR5242, Lyon, France 4. Neuroscience Area, International School for Advanced Studies, Trieste, Italy 5. Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan
Although ascidians are the sister group of Ver- orthologs outside the tunicate group, of which tebrates, they have much faster evolving coding about a quarter are completely novel genes genes (Delsuc et al., 2018). We have sequenced, showing no protein sequence similarity with assembled and annotated 4 solitary ascidians ge- other bilaterians. We found that the majority of nomes (2 Phallusia and 2 Halocynthia) and per- these novel genes are not genus- or family-spe- formed a comparative analysis of our data along cific but emerged at the root of the tunicata sub- with 4 other solitary ascidians (2 Ciona and 2 phylum. The majority of these genes showed no Molgula) and a colonial ascidian (Botrylloïdes lea- evidence for horizontal transfer, suggesting they chii, Blanchoud et al., 2018). originated de novo. We will report the results of our global analysis of the evolution of the tuni- New genes are considered to significantly con- cate gene repertoire and compare the regulation tribute to adaptive evolutionary innovation. Our and chromosome environment of novel and an- analysis identified approximately twenty - per cient genes. cent of tunicate-specific genes with no detectable
References 1. Blanchoud, S., Rutherford, K., Zondag, L., Gemmell, N.J., Wilson, M.J., 2018. De novo draft assem- bly of the Botrylloides leachii genome provides further insight into tunicate evolution. Sci. Rep. 8, 5518. https://doi.org/10.1038/s41598-018-23749-w 2. Delsuc, F., Philippe, H., Tsagkogeorga, G., Simion, P., Tilak, M.-K., Turon, X., López-Legen- til, S., Piette, J., Lemaire, P., Douzery, E.J.P., 2018. A phylogenomic framework and timescale for comparative studies of tunicates. BMC Biol. 16. https://doi.org/10.1186/s12915-018-0499-
77 T62 WORKING TOWARDS A COMPREHENSIVE AND QUANTITATIVE CIONA NOTOCHORD GENE REGULATORY NETWORK.
Kotaro Shimai1, *, Wendy Reeves1 and Michael T. Veeman1
1. Division of Biology, Kansas State University, 1717 Claflin Rd. Manhattan KS 66506, USA
The gene regulatory network for Ciona noto- of CRISPR/Cas ribonucleoprotein complexes. chord fate is thought to resemble an hourglass, We have identified effective guide RNAs for both with upstream regulators such as FoxA, ZicL and Bra and FoxA and analyzed multiple replicates FGF converging to induce Brachyury (Bra) ex- with RNAseq on crispant embryos. Major effects pression in the notochord founder cells, and Bra are seen on the expression of notochord-enriched directly or indirectly inducing the expression of genes, but the results are not consistent with a downstream notochord-enriched genes (1-4). It is simple model in which Bra is strictly down- unclear, however, if the current GRN models in- stream of FoxA and other notochord genes are clude all the key players, and many of the poten- strictly downstream of Bra. In parallel, we are tial network relationships have not been directly also systematically mutating putative transcrip- tested. To build a comprehensive and quantita- tion factor binding sites in the main Bra enhancer tive notochord GRN model, we are both tran- and shadow Bra enhancer to test the dependence scriptionally profiling embryos in which import- of these cis-regulatory modules on predicted reg- ant notochord transcription factors have been ulators. Results from our ongoing experiments perturbed, and also dissecting selected cis-regu- will be integrated into a model of early noto- latory regions. We have optimized a protocol for chord gene regulation. somatic CRISPR gene disruption by egg injection
References 1. Imai et al. (2006) Science, 312, 1183–1187. 2. Jose-Edwards et al. (2011) Dev Dyn, 240, 1793–1805. 3. Takahashi et al. (1999) Genes Dev, 13, 1519–1523. 4. Hotta et al. (2000) Dev Biol, 224, 69–80. 78 T63 COMBINATORIAL CHROMATIN DYNAMICS FOSTER ACCURATE CARDIOPHARYNGEAL FATE CHOICES
Claudia Racioppi*1, Keira A. Wiechecki1, Lionel Christiaen1
1. New York University, Center for Developmental Genetics, Department of Biology
In embryogenesis, chromatin accessibility plays We built an atlas of accessible regions to ana- a fundamental role in the establishment of cell lyze differential accessibility and integrated sin- types controlling gene expression and modulat- gle cell and bulk RNA-seq data to compare the ing their transcription. The interaction between chromatin states and gene expression in cardio- genes and the multiple distal and accessible reg- pharyngeal precursors. We revealed an FGF-Foxf ulatory elements impacts multipotent progen- pathway acting in multipotent progenitors to itor states and subsequent fate choices. Subsets establish cardiopharyngeal-specific chromatin of cardiac and pharyngeal/head muscles share accessibility. This pathway governs later heart a common origin in the cardiopharyngeal meso- vs. pharyngeal muscle-specific expression pro- derm, but the chromatin landscapes that govern files, demonstrating spatiotemporal decoupling multipotent progenitors’ competence and early between early enhancer accessibility and late fate choices remain largely elusive. cell-type-specific activity. We further show that combinations of accessible cis-regulatory ele- Here, we used the simple chordate model Ciona ments with distinct chromatin accessibility pro- robusta to profile chromatin accessibility through files are required to activate key determinants of stereotyped transitions from naive Mesp+ meso- cardiopharyngeal fate choices, such as Tbx1/10 derm to distinct fate-restricted heart and pharyn- and Ebf/COE. geal muscle precursors. To characterize chroma- tin dynamics in cardiopharyngeal development, We propose that this combinatorial logic in- we induced targeted molecular perturbations creases the repertoire of regulatory inputs that that altered heart vs. pharyngeal muscle fate control gene expression, thus fostering spatially specification and then profiled chromatin acces- and temporally accurate fate choices. sibility on FACS-purified cells using ATAC-seq.
79 T64 FOXD ACTS AS AN ACTIVATOR AND A REPRESSOR FOR PATTERNING ALONG THE ANIMAL-VEGETAL AXIS IN EARLY EMBRYOS
Shin-ichi Tokuhiro1,* and Yutaka Satou1
1. Department of Zoology, Graduate School of Science, Kyoto University, Kyoto, Japan
In many animal embryos, localized maternal acts as an activator and repressor stimultaneous- factors induce specific gene expression along ly, and stabilizes the first patterning along the the animal-vegetal axis at early developmental animal-vegetal axis established by β-catenin and stages(1-3). In Ciona embryos, soon after the ani- Gata.a. mal and vegetal hemispheres divide to different In the present study, to understand how Foxd blastomeres, two maternal factors Gata.a and acts as an activator and repressor, we analyzed β-catenin activate specific genes in the animal the upstream regulatory regions of the target and vegetal hemispheres, respectively(4-8). Foxd genes. We examined a possibility that cis-regu- is one of β-catenin targets and activated in the latory sequences for Foxd binding are different vegetal hemisphere at the 16-cell stage(9). In the between genes activated and repressed by Foxd. vegetal hemisphere of 32-cell embryos, Foxd re- We also examined a possibility that transcription presses animal-hemisphere specific genes includ- factors acting with Foxd are different between ing Dmrt1 and Dlx.b, and activates vegetal-hemi- these two groups of genes. sphere specific genes including Neurogenin and Lhx3/4(10-11). Thus, Foxd transcription factors
References 1. White JA et al. J Exp Zool Part B. 2008;310b(1):73-84. 2. Ma J et al. Wiley Interdiscip Rev Dev Biol. 2016; 5(5):562-581. 3. Stein DS et al. Wires Dev Biol. 2014; 3(5):301-330. 4. Imai K et al. Development. 2000; 127(14):3009-3020. 5. Bertrand V et al. Cell. 2003; 115(5):615-627. 6. Rothbächer U et al. Development. 2007; 134(22):4023-4032. 7. Horikawa Y et al. Dev Growth Differ. 2013; 55(9):776-785. 8. Oda-Ishii I et al. PLoS genetics. 2016; 12(5):e1006045. 9. Imai KS et al. Development. 2002; 129(14):3441-3453. 10. Hudson C et al. Elife. 2016;5. 11. Tokuhiro S et al. PLoS Genetics. 2017; 13(5):e1006741. 80 T65 FOXG IS REQUIRED FOR THE PALP FORMATION IN ASCIDIAN EMBRYOS
Boqi Liu1,* and Yutaka Satou1
1. Department of Zoology, Graduate School of Science, Kyoto University, Japan
The ascidian larva has three palps, which are the MAPK signaling pathway. Ephrina.d was ex- adhesive structures required for attaching to sub- pressed in the entire presumptive palp and oral strates before metamorphosis. Previous studies siphon primordium region, and negatively reg- have shown that the anterior region of the neural ulated the MAPK signaling pathway to restrict plate contributes to the palps under the control of Foxg expression domain. Because Foxg is import- Foxc and Islet[1-4]. In the present study, we found ant for specification of cranial placodes in verte- that Foxg was expressed in the presumptive palp brate embryos[5], our results provide additional and oral siphon primordium region at the ear- evidence suggesting that the ascidian palps and ly neurula stage. Knockdown of Foxg resulted vertebrate cranial placodes share an evolutionary in loss of expression of Islet and loss of the palp origin[1-4,6,7]. Specifically, Foxg may have played protrusions, but we found no obvious effects in an important role in specification of placode-like the formation of oral siphon and sensory vesi- structures in the last common ancestor of verte- cles. We also found that Foxg expression at the brates and ascidians. early neurula stage was controlled by Foxc and
References 1. Ikeda T, Matsuoka T, Satou Y. A time delay gene circuit is required for palp formation in the ascid- ian embryo[J]. Development. 2013, 140(23):4703-4708 2. Wagner E, Levine M. FGF signaling establishes the anterior border of the Ciona neural tube[J]. Development. 2012, 139(13):2351-2359 3. Wagner E, Stolfi A, Gi Choi Y, et al. Islet is a key determinant of ascidian palp morphogenesis[J]. Development. 2014, 141(15):3084-3092 4. Horie R, Hazbun A, Chen K, et al. Shared evolutionary origin of vertebrate neural crest and cranial placodes[J]. Nature. 2018, 560(7717):228-232 5. Duggan C D, DeMaria S, Baudhuin A, et al. Foxg1 is required for development of the vertebrate olfactory system[J]. J Neurosci. 2008, 28(20):5229-5239 6. Abitua P B, Gainous T B, Kaczmarczyk A N, et al. The pre-vertebrate origins of neurogenic plac- odes[J]. Nature. 2015, 524(7566):462-465 7. Mazet F, Hutt J A, Milloz J, et al. Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes[J]. Dev Biol. 2005, 282(2):494-508 81 T66 EVOLUTION OF DEVELOPMENTAL PROGRAMS FOR THE MIDLINE STRUCTURES IN CHORDATES: INSIGHTS FROM GENE REGULATION IN THE FLOOR PLATE AND HYPOCHORD HOMOLOGUES OF CIONA EMBRYOS
Shuto Tsunemi1, Naho Moritsugu1, Kouhei Oonuma1, Mike Levine2, and Takeo Horie2,3, and Takehiro G. Kusakabe1*
1. Institute for Integrative Neurobiology & Department of Biology, Konan University, Kobe, Japan 2. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, USA 3. Shimoda Marine Research Center, University of Tsukuba, Shimoda, Japan
In vertebrate embryos, Sonic hedgehog is reporter gene in the endodermal strand, suggest- produced in midline tissues, including the no- ing that the ventral nerve cord and the endoder- tochord, prechordal plate, and floor plate, and mal strand share a part of their gene regulatory plays important roles in patterning of the central programs. The endodermal strand occupies the nervous system and somites. The Ciona genome same topographic position of the embryo as does contains two genes encoding hedgehog (hedge- the vertebrate hypochord. In vertebrate embryos, hog.a and hedgehog.b). Transcripts of hedgehog.a the hypochord shares expression of several genes are only present as maternal RNA in embryos, with the floor plate, includingsonic hedgehog (Yan while hedgehog.b is expressed in the ventral row et al., 1995; Peyrot et al., 2011). A number of genes of embryonic nerve cord cells, which is the ho- specifically expressed in both the floor plate and molog of the vertebrate floor plate, at the tailbud the endodermal strand in Ciona tailbud embry- stage (Takatori et al., 2002; Islam et al., 2010). Nei- os were predicted by whole-embryo single-cell ther hedgehog.a nor hedgehog.b is expressed in the transcriptomics and confirmed by whole-mount notochord. We have identified acis -regulatory re- in situ hybridization. These genes and their tran- gion that were sufficient to drive a reporter gene scriptional regulation suggest an ancient evolu- expression in the ventral nerve cord (floor plate). tionary origin of a common developmental pro- Interestingly, the hedgehog.b cis-regulatory region gram for the midline structures in Olfactores. was shown to confer the ectopic expression of the
References 1. Takatori, N., Satou, Y., and Satoh N. (2002) Expression of hedgehog genes in Ciona intestinalis em- bryos. Mech. Dev. 116, 235-238. 2. Islam, A. F. M. T., Moly, P. K., Miyamoto, Y., and Kusakabe, T. G. (2010) Distinctive expression pat- terns of Hedgehog pathway genes in the Ciona intestinalis. Zool. Sci. 27, 84-90. 3. Yan, Y. L., Hatta, K., Riggleman, B., and Postlethwait, J. H. (1995) Expression of a type II collagen gene in the zebrafish embryonic axis. Dev. Dyn. 203, 363-376. 4. Peyrot, S. M., Wallingford, J. B., and Harland, R. M. (2011) A revised model of Xenopus dorsal mid- line development: differential and separable requirements for Notch and Shh signaling. Dev. Biol. 352, 254-266. 82 T67 DISCOVERING POST-TRANSCRIPTIONAL REGULATORS OF HEART DEVELOPMENT
Burcu Vitrinel*,1,2, Christine Vogel2, Lionel Christiaen1
1. Center for Developmental Genetics, Department of Biology, New York University, New York, NY, USA 2. Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA
In vertebrate embryos, both head muscles and post-transcriptional activity in these progenitors the chambered heart arise from a common pool by using proteomic approaches, however bench- of progenitor cells in the cardiopharyngeal field. marked proteomic using tandem mass spectrom- Similarly, in the simple chordate model Ciona etry workflows require >100 times the number robusta, multipotent cardiopharyngeal progen- of cells we can obtain from one experiment. itors go through subsequent asymmetric divi- Therefore I have developed a low-input work- sions giving rise to early Atrial Siphon Muscle flow that allows robust protein identifications (ASM) and heart precursors. These multipotent using only 1,000 cells with high reproducibility. progenitors display multi-lineage transcription- Using this new approach, we are investigating al priming: they express a combination of early the post-transcriptional regulation of cell speci- ASM- and heart-specific regulators that become fication in heart and ASM by profiling their pro- restricted to their corresponding precursors fol- teomes. In addition, we performed a small-scale lowing oriented asymmetric divisions. This sug- CRISPR screen on post-transcriptional regulation gests that heart progenitors need to clear gene candidates, assaying cardiopharyngeal fate spec- products that pertain to the ASM program rapid- ification phenotypes. Knock-out of the candidate ly after the asymmetric divisions, and vice versa. Rnf149 resulted in early ASM specification and Consistent with the hypothesis that post-tran- cell behavior defects. I will present progress in scriptional regulation controls fate specification, understanding post-transcriptional regulation of single-cell RNA-seq identified ubiquitination-re- heart specification and Rnf149’s role in regulat- lated and RNA-binding proteins among ASM ing pharyngeal muscle specification. and heart specific markers. We sought to observe
83 84 POSTERS ABSTRACTS P1-P65
85 P1 EVOLUTION OF COLONIALITY IN TUNICATA: ANALYSIS OF PRIMORDIAL GERM STEM CELLS
David dos Santos Soares1; Federico David Brown Almeida1
1. Laboratory of Evolutionary Developmental Biology, Department of Zoology, Instituto de Biociências, Universidade de São Paulo
Tunicata is a group of marine invertebrate histological sections and carry out both immuno- chordates with both solitary and colonial species. cytochemistry and western blot using the germ- Transcriptomic phylogenies and embryological line markers mentioned above. According to ex- evidence suggests that colonial species that re- pectations, was observed expression of Vasa and produce asexually in the Styelidae evolved from Piwi in ovocytes and strong expression of PL10 a solitary ancestor that reproduced only sexually. and CiBY1 in follicular cells; however, we docu- Progenitor Germ Stem Cells (PGSC) are locat- mented several cells outside the gonads of colo- ed in the gonads and cells outside the gonads nial and solitary ascidians that expressed these of extant colonial styelids. Vasa, Piwi, PL10 and proteins, including at least one type of blood cell CiYB1 are protein markers that have been previ- and expression in epithelia surrounding primor- ously been used in the identification of PGSCs of dial gonads and mantle. If Piwi+, Vasa+, PL10+ ascidians. Our objective is to compare the occur- and CiBY1 cells are confirmed outside the go- rence of PGCs in colonial and solitary styelids nads of solitary ascidians, we can hypothesize to understand how the evolution of coloniality that these genes may have previously unknown occurred. We cut specimens of Styela plicata and somatic roles in solitary ascidians, or that PGSCs Symplegma rubra (solitary and colonial Styelidae are present in the blood and other tissues of soli- ascidians, respectively) in order to characterize tary ascidians for yet unknown reasons.
86 P2 EXPERIMENTAL EVALUATION OF THE RELEVANCE OF SPATIO-TEMPORAL VARIATIONS IN THE RECRUITMENT OF CRYPTIC ASCIDIANS OF CORAL REEFS IN THE SOUTH OF THE GULF OF MEXICO AND THE CARIBBEAN SEA IN AUTONOMUS REEF MONITORING STRUCTURES (ARM).
*Lilian A. Palomino-Alvarez*, Edlin Guerra Castro2, Rosana Rocha Moreira3 and Nuno Simoes4
1. PhD Student, Postgraduate of Marine al Limnology Sciencies, Multidisciplinary Unit of Teaching and Sisal Research (UMDI-SISAL), Faculty of Sciences, National Autonomous University of Mexico, Puerto de abrigo s/n, Sisal, CP 97356 Yucatán, Mexico. 2. Professor full time, Department of Ecology and Conservation, National School of Higher Studies, Merida Unit, National Autonomous University of Mexico, Merida, Mexico. 3. Profesor full time. Zoology Department, Universidade Federal do Parana – UFPR, CP 19020, CEP 81531-980, Curitiba, PR, Brazil, cLaboratorio Nacional de Resiliencia Costera Laboratorios Nacionales, CONACYT, Mexico City, Mexico. 4. Professor full time Multidisciplinary Unit of Teaching and Sisal Research (UMDI-SISAL), Faculty of Sciences, National Autonomous University of Mexico, Puerto de abrigo s/n, Sisal, CP 97356 Yucatán, Mexico., National Laboratory of Coastal Resilience National Laboratories, CONACYT, Mexico City, Mexico and International Chair for Coastal and Marine Studies, Harte Research Institute for Gulf of Mexico Studies, Texas A&M University–Corpus Christi, Texas, U.S.A.
The spatio-temporal variation patterns of spe- study, models that could explain spatial variation cies diversity reflect how environmental, biolog- at a few meters scale are evaluated, considering ical, anthropic and stochastic processes influence the effect of the regions on species of ascidians in ecosystems (Legendre et al., 2005). It has been the south GMx of Mexico and the Mexican Carib- described that the processes that contribute most bean Sea on autonomous structures of reef mon- to the variation in the distribution patterns of itoring (ARM) through sampling trimesters over marine benthic species operate at smaller scales 18 months. Preliminary results after 3 months of of a few meters and hundreds of kilometers (Fra- settlement, indicate the presence of at least 15 schetti et al., 2005, Guerra-Castro et al., 2016) and species of ascidians, corresponding to six fami- that the availability of habitat and temporal vari- lies and 15 genera, where species of the family ations in recruits allow explaining different types Styelidae and Dimenidae had a greater doman- of succession in substrates separated by just a cy in terms of coverage per plate and presenting few meters, and such interaction of small spa- differences between the orentation of each of the tio-temporal processes is independent of region- plates. al processes (Guerra-Castro et al., 2018). In this
References Fraschetti S, Terlizzi A, Benedetti-Cecchi L. 2005. Patterns of distribution of marine assemblages from rocky shores: evidence of relevant scales of variation. Mar Ecol Prog Ser 296:13 Legendre, P., Borcard, D. & Peres-Neto, P.R., 2005. Analysing beta diversity: partitioning the spatial variation of community composition data. Ecological Monographs, 75(4), pp.435–450. Guerra-Castro EJ, Conde JE, Cruz-Motta JJ. 2016. Scales of spatial variation in tropical benthic assem- blages and their ecological relevance: epibionts on Caribbean mangrove roots as a model system. Mar Ecol Prog Ser 548:97-110 doi:https://doi.org/10.3354/meps11693 87 P3 ADDITIONAL NOTES AND RECORDS OF THE ASCIDIAN SYMBIONT SHRIMP ASCIDONIA MISERABILIS (HOLTHUIS, 1951) (CARIDEA: PALAEMONIDAE) FROM THE SOUTHERN GULF OF MEXICO
Duarte J.1, Palomino-Alvarez L.1 A., Simões N.1,2,3
1. Unidad Multidisciplinaria de Docencia e Investigación Sisal (UMDI-SISAL), Facultadde Ciencias, Universidad Nacional Autónoma de México, Puerto de abrigo s/n, Sisal, CP 97356 Yucatán, Mexico. 2. Laboratorio Nacional de Resiliencia Costera Laboratorios Nacionales, CONACYT, Mexico City, Mexico. 3. International Chair for Coastal and Marine Studies, Harte Research Institute for Gulf of Mexico Studies, Texas A&M University–Corpus Christi, Corpus Christi, Texas, USA
Currently, ascidians are recognized as the main living alone or in pairs specifically in the pharynx hosts of many marine organisms, either as a re- of the ascidians; It is suggested that the prefer- production or food refuge. A total of 169 solitary ence for the genus is due to the habitat type of ascidians were collected corresponding to four the cryptic ascidians (hidden from predators), families in seven genera and 15 species where to which other ascidians already had symbiosis only shrimp corresponding to the species of As- with other organisms (availability of the host) cidonia miserabilis (Holthuis, 1951) were found in and for the morphology of the tunic and pharynx cryptic ascidians of the genus Ascidia in the reef proper to the order. On the other hand, the inci- systems of Alacranes, Cayo Arenas and Cayo dence in the male / female relationship within Arcas in the southeast of the Gulf of Mexico be- the same ascidia is assumed to be directly related tween 2015-2018. A total of twenty-one shrimps to reproductive purposes.
88 P4 RESOURCE PROJECT OF CIONA INTESTINALIS FOR SUPPORTING TUNICATE COMMUNITY
Yutaka Satou1, Manabu Yoshida2 and Yasunori Sasakura3* 1. Department of Zoology, Graduate School of Science, Kyoto University 2. Misaki Marine Biological Station, Graduate School of Science, University of Tokyo 3. Shimoda Marine Research Center, University of Tsukuba
We have developed the systematic material sharing system lations maintained by crossing within the populations. This for facilitating your experiments. This project, named Na- restricts genetic variations appearing in the families, thus as- tional BioResource Project (NBRP) provides transgenic and suring more reproducible experiments than those done with mutant lines, DNA constructs, and wild types of Ciona intesti- unknown populations collected from the ocean every time nalis. This presentation is purposed to evoke your interest in you do experiments. We are sequencing the genomes of the this project and promote your requests. closed colonies in order to characterize the variations in the families. The genome sequences will allow us to know the Transgenic and mutant lines. Transgenic lines are valuable potential variations in your genes-of-interest to deduce its markers for tissues and organs because they express fluores- effect on your experiments, and to design oligonucleotides cent protein in the non-mosaic fashion. In Ciona, many trans- and genome editing constructs that can bind to the genome genic lines have been created with transposon-based technol- without mismatches. Living, matured wild type (closed colo- ogies, and these lines are available from NBRP. In addition ny) adults can be delivered to abroad countries in a few days to the marker lines for larval tissues, organ markers in the while keeping their health. We wish your use of our wild juvenile/adult body are present whose labeling has not been type (closed colony) to standardize experiments in Ciona. achieved by electroporations of DNA constructs. For abroad laboratories, we can provide alive dry sperm of transgenic Order system and information. You can order resources lines that can be easily used by mixing it with wild type eggs. through our online shopping system and can pay fees with Mutants are particularly useful for elucidating functions of a credit card. Materials Transfer Agreement is necessary for genes. In Ciona, some mutants have been made based on your first orders of the materials. The URL for the shopping transposon-based mutagenesis and genome editing technol- and databases are as follows: ogies. These mutants are also available from NBRP. Yutaka Satou1, Manabu Yoshida2 and Yasunori Sasakura3* DNA constructs. Expression vectors are routinely used 1 Department of Zoology, Graduate School of Science, Kyoto for analyzing gene functions and labeling organelles, cells, University tissues, and organs. These DNA constructs are invaluable resources for Ciona because we can introduce them easily 2 Misaki Marine Biological Station, Graduate School of Sci- by electroporation. NBRP have collected expression vectors ence, University of Tokyo associated with the transgenic lines, and the vector informa- tion has been databased that includes their sequences and 3 Shimoda Marine Research Center, University of Tsukuba references. Recently, knockouts of Ciona genes with TALENs and CRISPR/Cas9 were reported. The technologies have en- Transgenic lines, mutants and DNA constructs; abled us to address gene functions quickly in G0 generation. http://marinebio.nbrp.jp/ciona/ TALEN and CRISPR/Cas9 expression vectors created in Ja- Genome Editing; pan have also been collected in our resource, and their basic http://marinebio.nbrp.jp/ciona/forwardToKnockOutAc- information including the mutation frequencies has been da- tion.do tabased. We are waiting for your requests for these materials. Wild type (closed colony); Wild type (closed colony). Unfortunately, tunicate com- http://marinebio.nbrp.jp/wild.jsp munity does not have a standard wild type strain that as- Genome sequence; sures the reproducibility of experiments. NBRP has started http://ghost.zool.kyoto-u.ac.jp/cgi-bin/gb2/gbrowse/kh/ a new project for circumventing this issue. First, we cultivate closed colonies of wild types. Closed colonies are the popu-
89 P5 INVESTIGATING THE ROLE OF FLRT IN NEURAL TUBE CLOSURE
Haley Smith*, Erin-Newman Smith and William C. Smith Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara
The chordate central nervous system starts out transcript for FLRT. In wildtype Ciona, FLRT is as a flat neural plate. Through a morphogenet- expressed during NTC and then declines pre- ic process known as neural tube closure (NTC) cipitously. However, in bug embryos FLRT does this flat plate will transform into a hollow neu- not appear to be properly downregulated fol- ral tube which gives rise to the brain and spinal lowing this process. FLRT is best known for its cord. Defects in NTC are some of the most prev- role in neurite outgrowth and can act to encour- alent human birth defects and affect as many as age either adhesion or repulsion depending on 1 out of every 1000 births. We have discovered a its interaction partners. One of those partners novel role for a T-Type Calcium Channel (CAV3) is Latrophilin. We show that Latrophilin has a in this process that is conserved among chor- dynamic expression pattern around the closing dates. In the basal chordate Ciona, loss of CAV3 neural folds and opposes the FLRT expression leads to defective NTC characterized by an open domain. Taken together, we speculate that FLRT brain. RNA-Seq analysis of a CAV3 mutant, bug- plays a transient role in tissue separation and eye (bug), has revealed misregulation in a number adhesion during neural tube closure and that of transcripts including ones that are involved failure to downregulate FLRT leads to an open in cell-cell recognition and adhesion. Of partic- neural tube. ular interest is the aberrant upregulation of the
90 P6 PARALLEL FOLD-CHANGE DETECTION VISUAL CIRCUITS IN CIONA L A RVA
Cezar Borba1,*, Matthew J. Kourakis2, William C. Smith1,2
1. Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, CA 93106 2. Neuroscience Research Institute, University of California, Santa Barbara, CA 93106
The Ciona tadpole larval central nervous sys- posed of glutamatergic photoreceptors, projects tem (CNS) has a common structure with the CNS to two groups of relay neurons: cholinergic relay of its close chordate relatives, the vertebrates. An neurons to signal the motor ganglion for navi- advantage to using Ciona as a model organism gational swimming and GABAergic relay neu- is that it has one of the simplest CNS and has a rons that also projects to the cholinergic neurons complete connectome available. Beginning with to modulate the signal. Group 2, composed of visual circuits predicted from its connectome in GABAergic photoreceptors, appears to project combination with past studies, we used behav- to GABAergic relay neurons to signal, through ioral assays, pharmacology, and mutant lines to a likely disinhibitory pathway, shadow response further assess their visual circuits. Ciona have swimming. This is then modulated by the same two groups of photoreceptors: group 1 respon- cholinergic relay neurons as group 1. Tadpoles sible for detecting directional light and group 2 exhibit a shadow response and adaptation time responsible for detecting ambient light. We find that is dependent on the fold-change of light as that both circuits appear to form overlapping but opposed to absolute change. The fold-change de- different fold-change detection networks: group tection processing is shown to be in the relay neu- 1 showing an incoherent type-1 feedforward rons themselves as pharmacologically increasing loop (I1-FFL) and group 2 showing a nonlinear or decreasing their inputs lead to an altered or integral feedback loop (NLIFL). Group 1, com- extinguished response.
References Ryan, K., Lu, Z., and Meinertzhagen, I.A., The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling. Elife, 2016. 5. Salas, P., Vinaithirthan, V., Newman-Smith, E., Kourakis, M.J., and Smith, W.C., Photoreceptor specializa- tion and the visuomotor repertoire of the primitive chordate Ciona. J Exp Biol, 2018. 221(Pt 7). Adler, M. and Alon, U, Fold-change detection in biological systems. Current Opinion in Systems Biology, 2018. 8.
91 P7 CHARACTERIZATION OF THE COMPLEMENT SYSTEM IN A COLONIAL TUNICATE: C3 COMPLEMENT RECEPTORS AND OPSONIC ROLE OF C3
A. Peronato, N. Franchi, L. Schiavon, L. Ballarin
Department of Biology, University of Padova, Padova, Italy
The complement system is one of the most scription in the course of the colonial blastogenet- ancient immune modulator mechanism of bila- ic cycle. In addition, we continued our analysis of terian metazoans. In vertebrates, three comple- the role of C3 in Botryllus immunity by studying ment-activation pathways are known: the classi- the modulation of BsC3 transcription during the cal, the alternative and the lectin pathways: all of colonial blastogenetic cycle and the effect of bsc3 them converge on the cleavage of C3. knockdown on immune responses. The compound ascidian Botryllus schlosseri is a Results indicate that only morula cells, and no reliable model organism for the study of immu- other immunocytes type, are labelled by the an- nobiology. As an invertebrate, B. schlosseri relies tisense probe for BsC3aR, whereas phagocytes only on innate immunity for its defense and im- and young, undifferentiated cells, known as he- munocytes. We already demonstrated the pres- moblasts, are the cells stained by the probe for ence, in Botryllus, of homologues of mammalian BsCR1. This suggests the presence of an import- C3, Bf, MBL and MASP1, referred to as BsC3, ant cross-talk between these two immunocytes BsBf, BsMBL and BsMASP, respectively. All the types. complement components identified so far, are ex- Both the bsc3ar and bscr1 genes are constitu- pressed by morula cells, the most abundant cir- tively transcribed as almost all morula cells and culating hemocytes. phagocytes, respectively, resulted labelled by the In mammals, once the complement system is antisense probe in the ISH assay, independent- activated, a cascade of reactions that involves ly of their previous challenge with zymosan, a proteolysis and polymerization occurs resulting known activator of B. schlosseri hemocytes. How- in the cleavage of the third complement compo- ever, a modulation in the extent of transcription nent (C3) to C3a and C3b, the former exerting occurs during the colonial blastogenetic cycle a chemotactic activity, the latter acting as opso- as the amount of BsC3aR mRNA abruptly de- nin and, ultimately, activating the lytic pathway. creased at TO, whereas no differences were ob- The best-known receptor for C3a in mammals is served when EC and MC were compared. This C3aR, whereas CR1 is the receptor able to recog- is probably related to the renewing of circulating nize and bind C3b on the microbial surfaces. cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, Here, we describe, in B. schlosseri, two new differentiating cells entering the circulation in the genes showing homology with vertebrate C3aR same period. and CR1, respectively, and studied their tran-
92 P8 ASYMMETRIC SPINDLE POSITIONING IN THE GERM LINE OF THE ASCIDIAN EMBRYO
Janet Chenevert, Vlad Costache, Anne Rosfelter, Celine Hebras, Lydia Besnardeau, Remi Dumollard, Alex McDougall
Ascidian BioCell Group, Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), UMR7009, 181 chemin du Lazaret, 06230 Villefranche-sur-Mer, France
In the early ascidian embryo, a series of unequal a cortical pulling force may be localized to this cleavages shape the posterior cleavage pattern posterior midline site. In order to visualize force and segregate the germ line. These asymmetric generators, we used cytochalasin to weaken the cell divisions are regulated by a defined cortical actin cortex and observed strong membrane in- structure termed the centrosome attracting body vaginations at the cortical site toward which the (CAB). The CAB is formed in the zygote, when centrosome migrates. We analyzed the localiza- sperm aster microtubules concentrate a domain tion of the dynein partners NuMa and LGN and rich in endoplasmic reticulum and mRNA deter- found both proteins to be enriched at the midline minants. We have found that the microtubule de- site during anaphase. Inhibition of LGN leads polymerase Kinesin 13 (Kif2/MCAK) is localized to defective spindle positioning ; the CAB-con- to the cortical endoplasmic reticulum domain taining cells continue to divide but in the wrong and released during each mitosis, causing the direction and such embryos fail to form swim- proximal aster to shrink in size and the spindle to ming tadpole larvae. We propose a mechanism shift off-center (Costache et al 2017). At the 16 cell whereby the spindle is both pushed and pulled stage, one centrosome migrates toward the mid- during sequential phases of the cell cycle to gen- line cortex adjacent to the CAB, suggesting that erate unequal cleavage in the ascidian embryo.
93 P9 NEURAL TUBE CLOSURE IN CIONA
Erin D. Newman-Smith*, Haley Smith, and William C. Smith Molecular, Cellular and Developmental Biology, University of California, Santa Barbara
We have found that T-type Calcium channels epidermis, and then adhere to each other. Flrt, (Cav3) play a crucial role in neural tube closure Selectin and neural crest cells all have adherent (NTC) in chordates. This requirement is seen in and repulsive properties and we are interested in the Ciona mutant line bugeye which carries a mu- how these common attributes are contributing to tation in Cav3 and which has incomplete anterior NTC. We have found that these genes (as well NTC. We hypothesize that Cav3 serves a regula- as others identified as DE in the RNAseq analy- tory role in NTC, monitoring cell fusion and con- sis) are expressed in the right tissues (neural) and trolling a suite of genes necessary for NTC. We the right time (during neural stages) to facilitate have performed RNAseq to identify genes dif- NTC. Crispr/Cas9 knock out of Flrt or its bind- ferentially expressed (DE) in Cav3 mutants. We ing partner Latrophilin (but not another interact- have found upregulated genes belonging to three ing protein, Unc5) results in a failure of NTC, as signaling pathways; neurite outgrowth (e.g., does CRISPR of Selectin. We are looking at other Flrt), wound healing (e.g., Selectin) and neural members of these pathways (such as Grainey- crest development (e.g., SoxE). NTC involves head-like which has a role in wound healing), a sequence of tissue separation and adhesion DE transcription factors, SoxE and EGR-1, and events as neural plate cells must first detach from the role of a long non-coding RNA.
94 P10 ROLE OF MIDBODY REMNANT IN MEIOSIS II CREATING TETHERED POLAR BODIES
Alex McDougall1*, Celine Hebras1, Gerard Pruliere1, David Burgess2, Vlad Costache1, Remi Dumollard1, and Janet Chenevert1
1. Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), 06230 Villefranche sur-mer, France 2. Boston College, Biology Department, 528 Higgins Hall, 140 Commonwealth Ave, Chestnut Hill, MA 0246, USA
Polar body (PB) formation is an extreme form follow the dynamics of the PB1 midbody remnant of unequal cell division that occurs in oocytes during meiosis II. Plk1::Ven strongly labeled the due to the eccentric position of the small mei- small polar corps that formed on the surface of otic spindle near the oocyte cortex. Prior to PB the cortical outpocket that created PB2. Follow- formation, a chromatin-centered process causes ing emission of PB2, this polar corps was rich in the cortex overlying the meiotic chromosomes to Plk1::Ven and linked PB2 to PB1. By labelling ac- become polarized. This polarized cortical subdo- tin (with LifeAct::mCherry/GFP or TRITC-Phal- main marks the site where a cortical protrusion loidin) we also demonstrated that actin accumu- or outpocket forms at the oocyte surface creating lates at the midbody remnant and also forms a the future PBs. We observed that PB1 becomes cortical cap around the midbody remnant in mei- tethered to the egg via PB2, indicating that the osis II that prefigured the precise site of cortical site of PB1 cytokinesis directed the precise site outpocketing during PB2 emission. Phalloidin for PB2 emission. We therefore studied whether staining of actin and immunolabelling of an- the midbody remnant left behind following PB1 ti-phospho aPKC during meiosis II in eggs that emission was involved, together with the egg had PB1 removed showed that the midbody rem- chromatin, in defining the precise cortical site for nant remained within the egg following emis- PB2 emission. During outpocketing of PB2 in as- sion of PB1. Dynamic imaging of microtubules cidians, we discovered that a small corps around labelled with Ens::3GFP, MAP7::GFP or EB3::3G- 1µm in diameter protruded from the center of the FP showed that one pole of the second meiotic cortical outpocket that will form the future PB2, spindle was located near the midbody remnant which we call the “polar corps”. During emission while the other pole rotated away from the cor- of PB2, this small polar corps became localized tex during outpocketing. Finally, we report that between PB2 and PB1 and appeared to link PB2 failure of the second meiotic spindle to rotate can to PB1. We tested the hypothesis that this small lead to the formation of two cortical outpockets polar corps on the surface of the forming PB2 was at anaphase II, one above each set of chromatids. the midbody remnant from the previous round It is not known whether the midbody remnant of of PB1 cytokinesis. We had previously discov- PB1 is involved in directing the precise location ered that Plk1::Ven labeled midbody remnants in of PB2 in other species as in ascidians. ascidian embryos. We therefore used Plk1::Ven to
95 P11 A CHORDATE SPECIES LACKING NODAL UTILIZES CALCIUM OSCILLATION AND BMP4 FOR LEFT-RIGHT PATTERNING
Takeshi A. Onuma*, Momoko Hayashi, Fumi Gyojya, Kanae Kishi, Kai Wang and Hiroki Nishida
Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
Larvacean is a chordate that has tadpole mor- that shows left-sided expression in all chordates phology. By contrast to most chordates of which studied to date, was absent in the genome. Sur- early embryonic morphology is bilaterally sym- vey of TGFb superfamily genes uncovered that metric, invariant left-right (L-R) asymmetry be- Bmp4 shows right-sided expression in tailbud comes morphologically visible in the arrange- larva. The right-sided Bmp4 expression was oc- ment of blastomeres as early as four-cell stage. curred in descendants of the R-blastomere. The Eventually, the tail rotates in 90˚ counter-clock- right-side Bmp4 expression was abrogated by wise direction relative to the trunk, and thus the disruption of the Ca2+ oscillation using pharma- neural tube locates on the left side in the tail. Here cological inhibitors of inositol triphosphate and we unraveled three types of nonconventional L-R ryanodine receptors. Blockage of BMP signaling asymmetries in regard to L- and R-blastomeres of by Dorsomorphin induced ectopic expression of the two-cell embryo. First, tracing of descendant a brain marker gene, supporting BMP’s suppres- cells of the L- and R-blastomeres demonstrated sive function in nervous system differentiation that their distribution was largely asymmetrical on the right side. in the functional body. For instance, bilateral gi- These results raised an atypical L-R pattern- ant cell layers in the endostyle are exclusively de- ing mechanism that Ca2+ oscillation generates rived from the L-blastomere. Second, repetitive embryonic L-R asymmetry. The right-side Bmp4 Ca2+ waves were observed in asymmetric direc- expression may have arisen via co-option of con- tion along the L-R axis through embryogenesis. ventional role of BMP signaling in order to re- Third, Nodal, an evolutionary conserved gene strict neural tube formation to the left side.
96 P12 CELLS AND TISSUES OF DORMANCY IN ASCIDIANS: UNDERLYING DEVELOPMENTAL MECHANISMS AND EVOLUTIONARY ORIGINS
Laurel Sky Hiebert12*, Marta Scelzo1, Federico Brown2, and Stefano Tiozzo1
1. Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-Mer (LBDV), 06230 Villefranche-sur-Mer, France 2. Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, 05508-090 São Paulo, Brazil
Many colonial species survive adverse con- ical, and molecular characteristics of dormancy ditions by going dormant, where modules or in two ascidian species: the stolidobranch Polyan- whole colonies enter a state of physiological and drocarpa zorritensis and the aplousobranch Clave- morphological inactivity. Coloniality arose in- lina lepadiformis. These species have evolved bud- dependently many times in the metazoans, and ding and dormancy modes independently. To dormancy has evolved in all classes of colonial understand the dynamics of budding and dor- animals. Yet the link between coloniality and mancy, I determined which temperatures cause dormancy remains unexplored. Colonial species induction and release of dormancy in the lab and rely on populations of circulatory stem cells that the proportions of colonies in dormant states in drive asexual development. It is possible that the the field over the year. I have examined dormant origins of dormancy in colonial species involved tissues and cells using histology and microscopy. a cytological recruitment of these stem cells (or Finally, I am comparing transcriptome profiles of visa versa). However, the cellular mechanisms different stages of dormant tissues to determine underlying dormancy in colonial species are which genes are expressed in the induction and poorly known. Ascidians provide a fitting mod- release of dormancy. This project will provide in- el for understanding the origins/mechanisms sight into the mechanisms underlying dormancy of dormancy because both coloniality and dor- in tunicates, and may provide clues to the links mancy evolved multiple times independently. I between dormancy and coloniality. am examining the environmental, morpholog-
97 P13 NEUROTRANSMITTER USE AT CELL RESOLUTION IN THE CIONA CNS: INTEGRATING IN SITU LABELING WITH THE CONNECTOME FOR BETTER MODELS OF LARVAL BEHAVIOR
Matthew J. Kourakis1*, Angela Zhang2, Cezar Borba3, Erin Newman-Smith1, Priscilla Salas3, Samuel K. Wilson4, B. Manjunath2, and William C. Smith1,3
1. Neuroscience Research Institute, University of California, Santa Barbara, California, USA 2. Department of Electrical and Computer Engineering, UC Santa Barbara 3. Department of Molecular, Cell and Developmental Biology, UC Santa Barbara 4. Biomolecular Science and Engineering, UC Santa Barbara
Many colonial species survive adverse con- ical, and molecular characteristics of dormancy ditions by going dormant, where modules or in two ascidian species: the stolidobranch Polyan- whole colonies enter a state of physiological and drocarpa zorritensis and the aplousobranch Clave- morphological inactivity. Coloniality arose in- lina lepadiformis. These species have evolved bud- dependently many times in the metazoans, and ding and dormancy modes independently. To dormancy has evolved in all classes of colonial understand the dynamics of budding and dor- animals. Yet the link between coloniality and mancy, I determined which temperatures cause dormancy remains unexplored. Colonial species induction and release of dormancy in the lab and rely on populations of circulatory stem cells that the proportions of colonies in dormant states in drive asexual development. It is possible that the the field over the year. I have examined dormant origins of dormancy in colonial species involved tissues and cells using histology and microscopy. a cytological recruitment of these stem cells (or Finally, I am comparing transcriptome profiles of visa versa). However, the cellular mechanisms different stages of dormant tissues to determine underlying dormancy in colonial species are which genes are expressed in the induction and poorly known. Ascidians provide a fitting mod- release of dormancy. This project will provide in- el for understanding the origins/mechanisms sight into the mechanisms underlying dormancy of dormancy because both coloniality and dor- in tunicates, and may provide clues to the links mancy evolved multiple times independently. I between dormancy and coloniality. am examining the environmental, morpholog-
98 P14 MORE THAN ONE-TO-FOUR VIA 2R: EVIDENCE OF AN INDEPENDENT AMPHIOXUS EXPANSION AND TWO-GENE ANCESTRAL STATE IN VERTEBRATES FOR CHORDATE MYOD-RELATED MRFS
Madeleine E. Aase-Remedios, Clara Coll-Lladó, Ian A. Johnston, David E.K. Ferrier
The Scottish Oceans Institute, Gatty Marine Laboratory, University of St Andrews, East Sands, St Andrews, Fife, KY16 8LB, UK.
MyoD (Myogenic differentiation) has long for MRFs in the vertebrates, where MyoD and been recognized as a master developmental con- Myf5 act early in myogenic determination while trol gene and a key element of the bilaterian de- Myog and Myf6 are expressed later, in differenti- velopmental toolkit. The MyoD family of bHLH ating myoblasts. Comparing chordate MRFs, we transcription factors (Myogenic regulatory fac- find an independent expansion of MRFs in the tors, MRFs) drives myogenesis across the bilate- invertebrate chordate amphioxus, with evidence rians, though these comparisons are complicated for a parallel instance of subfunctionalisation by multiple instances of gene duplication and relative to that of vertebrates. Conserved syn- loss in several lineages. Following duplications, teny amongst chordate MRF loci supports the for instance the two rounds of whole genome 2R WGD events as a major force in shaping the duplication (2R WGD) at the origin of the verte- evolution of vertebrate MRFs. We also resolve brates, duplicate regulatory genes like the MRFs vertebrate MRF complements and organization often subfunctionalise, whereby the function of and infer an ancestral two-gene state in the verte- the ancestral gene is partitioned amongst the brates which corresponds to the creation of early- daughter genes, a process which is frequently and late-acting types of MRFs. This necessitates a focused on the complex regulatory regions char- revision of previous conclusions about the sim- acteristic of developmental transcription factors. ple one-to-four origin of vertebrate MRFs. Subfunctionalisation has been well-documented
99 P15 A REDESCRIPTION OF SYNCARPA COMPOSITA (ASCIDIACEA, STOLIDOBRANCHIA) WITH AN INFERENCE OF ITS PHYLOGENETIC POSITION WITHIN STYELIDAE
Naohiro Hasegawa1* and Hiroshi Kajihara2
1. Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido 060-0810, Japan 2. Faculty of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido 060- 0810, Japan
Two species of styelid colonial ascidians in the phylogenetic position within Styelidae based on genus Syncarpa Redikorzev, 1913 are known from the 18S rRNA and cytochrome c oxidase subunit the Northwest Pacific. The species status of one I gene sequences. The morphological differences species, Syncarpa composita (Tokioka, 1951) (type between S. composita and S. oviformis may be due locality: Akkeshi, Japan), has been doubted in to intraspecific variation. Syncarpa composita is relation to another, S. oviformis Redikorzev, 1913 possibly a junior synonym of S. oviformis. In our (type locality: Ul’banskij Bay, Russia). To eluci- phylogenetic tree, Syncarpa formed a well-sup- date the taxonomic identity of S. composita, we ported clade together with Dendrodoa. In these examined the morphology of one of the syntypes two genera, one gonad is present on the right and freshly collected topotypes of S. composita, in side of the body wall, a character state unique comparison to a syntype of S. oviformis. In this among other styelids and thus can be regarded paper, we redescribe S. composita and infer its as a synapomorphy for this clade.
100 P16 ANOCTAMIN IS ESSENTIAL FOR NOTOCHORD DEVELOPMENT IN CIONA INTESTINALIS
Zonglai Liang, Marios Chatzigeorgiou
Sars International Centre for Marine Molecular Biology, Thormøhlensgt 55, 5006 Bergen, Norway
Anoctamin/TMEM16 is a recently identified studies of Ci-Ano10 translational fusions show group of proteins, which are highly conserved that the protein is localized mostly in the ER and across the metazoans. They have been show to possibly to the plasma membrane of notochord function either as Ca2+ -activated Cl− channels cells. Combining transient transgenesis with ge- (CaCCs) or phospholipid scramblases[1-5]. Few netic purturbations and confocal imaging, we reports have explored the function of Anoct- demonstrate that the Ci-Ano10 is required for amins in animal development[6]. We examined the proper migration and intercalation of noto- the function of Ci-Ano10 during early develop- chord cells. We are currently characterizing the ment in Ciona intestinalis. WMISH of Ci-Ano10 biophysical properties of Ci-Ano10 using heter- showed that during early phases of develop- ologous expression systems combined with func- ment Ci-Ano10 gene is broadly expressed, while tional imaging and electrophysiology. Our study during the tailbud stages its expression is re- thus reveals the Anoctamin is essential for em- stricted to the notochord, a hallmark structure of bryo notochord development in Ciona intestinalis. the chordate body plan. Subcellular localization
1. Yang, Y.D., et al., TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature, 2008. 455(7217): p. 1210-5. 2. Schroeder, B.C., et al., Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell, 2008. 134(6): p. 1019-29. 3. TMEM16A, A Membrane Protein Associated with Calcium-Dependent Chloride Channel Activity. scinece, 2008 4. Yang, H., et al., TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell, 2012. 151(1): p. 111-22. 5. Suzuki, J., et al., Calcium-dependent phospholipid scrambling by TMEM16F. Nature, 2010. 468(7325): p. 834-8. 6. Rock, J.R., C.R. Futtner, and B.D. Harfe, The transmembrane protein TMEM16A is required for normal development of the murine trachea. Dev Biol, 2008. 321(1): p. 141-9. 101 P17 SINGLE-CELL ANALYSIS OF THE MOLECULAR LINEAGE OF CHORDATE EMBRYOGENESIS
Tengjiao Zhang1,*, Yichi Xu2, Teng Fei3, Kaoru Imai4, Tianwei Yu3, Yutaka Satou5, Zhirong Bao2, Weiyang Shi6
1. School of Life Sciences and Technology, Tongji University, Shanghai 200092, China 2. Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA 3. Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA 4. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan 5. Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan 6. Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean university of China, Qingdao 266003, China
In multicellular organisms, a simple fertilized detected DEGs, we projected that there are about egg develops along divergent embryonic lineag- 400 DEGs for early lineage differentiation in Cio- es to produce distinct cell types. What governs na. To explore the requirement of FGF signaling these processes is central to the understanding for embryonic cell types, we sequenced single of cell fate specification and stem cell engineer- cells from U0106 treated embryo and when ana- ing. Recent advances of single-cell technologies lyzed together with wt cells, we readily detected enabled systematic characterization of cell types all the known FGF-dependent cell type transfor- as well as the molecular programs that define mations. In addition, we also identified several them. The fixed embryonic lineage and low cell novel fate transformation events, such as the number of Ciona early embryo presents a per- TVC to muscle conversion. Finally, we compared fect opportunity to test the utility of single cell the single cell expression profiles of Ciona early transcriptomics in identifying embryonic devel- embryo to those of the mouse. Between these two opmental program. In this study, we generated species, we showed a substantial drift of gene ex- single cell transcriptomes of essentially every pression over chordate evolution, with similarity cell from Ciona savignyi embryos staged from the between homologous cell types largely limited zygote to the 110-cell stage when major tissue to key regulators of cell fates. We also provided types appear using the Smart2-seq approach. In further evidence that rudimentary programs for total, we obtained 745 single cell expression pro- neural crest cells, an innovation in vertebrates, files with two to eight embryos for each stage. exist in the form of overlapping transcriptional We conducted de novo reconstruction of the programs between neural and mesodermal fates trajectories based on the gene expression pro- in Ciona. This study demonstrates a general ap- files and successfully recovered 78% of the pro- proach to study embryonic development and genitor-progeny relationships across stages. 293 gene network regulation across a broad spectrum genes were identified as differentially expressed of non-traditional model organisms at an unprec- genes (DEGs) and the success rate was about edented single cell resolution. 70%. Based on the success rate and the number of
102 P18 EFFECTS OF BISPHENOL A ON PIGMENTED ORGAN DEVELOPMENT IN ASCIDIANS
Silvia Messinetti*, Silvia Mercurio, Alessandro Pennati, and Roberta Pennati
Università degli studi di Milano, Dipartimento di Scienze e Politiche Ambientali, Via Celoria 2, 20133 Milano, Italia
Bisphenol A (BPA) is an organic compound trations caused altered pigment deposition. Ab- used in the manufacturing of polycarbonate sence of one or both pigmented organs or ex- plastic and epoxy resins, employed for food and tranumerary pigmented organs were observed beverage containers, dental materials and water after exposure to high BPA concentrations, either supply pipes. After degradation of these prod- two otoliths or two ocelli can differentiate in ucts, BPA is released into the environment, where treated larvae. it can act both as an endocrine disruptor and as a Moreover, BPA alters the expression of some teratogenic molecule. transcription factors localized in the sensory Alteration of otolith and eye dysplasia induced vesicle. These genes are under the regulation of by exposure to BPA were reported in zebrafish FGF and Wnt signaling, suggesting that BPA acts and amphibian respectively. In zebrafish, the at some point of the cascade activated by these malformations were due to BPA interaction with pathways. Estrogen-Related Receptor (ERR). The co-exposure to BPA and 4-OHT resulted We analyzed the effects of BPA exposure on in a partial rescue of the normal phenotype, sug- the development of pigmented organs in Ciona gesting that BPA could act through binding to robusta and Phallusia mammilata. A co-exposure ERR also in ascidians. with an ERR antagonist (4-OHT) was performed These results highlight that ascidians are valu- to elucidate the role of these receptors in the tera- able invertebrate models for testing pollutants togenic mechanisms. and investigating their mode of action. In both species, exposure to low BPA concen-
103 P19 3D OVARY ORGANOID CULTURE IN CIONA INTESTINALIS
Rose E. Jacobson1, & Steven Q. Irvine1
1. University of Rhode Island, Kingston, RI USA
Tissue culture is an important tool in biochem- riod. Culture medium was optimized using cell istry, cell biology, and physiology research. There counts over time for different combinations of are currently few cell culture methods in marine salinity, antibiotics, and growth additives such as invertebrates for in vitro study. We describe gen- fetal bovine serum and Ciona hemolymph in L-15 eration of 3D primary tissue culture organoids medium. Of note, we found that Ciona hemo- from dissociated ovarian cells of Ciona intestina- lymph was the best growth additive for optimal lis. We used the “3D Petri Dish” system (Microtis- cell proliferation, and protects the cell culture sues, Inc.) which is a device to mold microwells from protist contamination. Histology revealed in agarose in which dissociated cells are seeded. that the organoids develop morphology similar Comparison of cell proliferation of the 3D cul- to that of ovaries. These findings suggest that 3D ture and traditional cell culture on culture treated culture is a viable technique for tissue and organ- plastic showed that the 3D culture system results oid culture in C. intestinalis, opening up new ave- in 185% more cell proliferation over a 22-day pe- nues for in vitro research.
104 P20 EFFECT OF ENVIRONMENTAL STRESSORS ON EMBRYONIC SHELL DEVELOPMENT IN MYTILUS GALLOPROVINCIALIS: A PHENOTYPIC AND MOLECULAR ANALYSIS.
A. Miglioli 2*, L. Besnardeau1, T. Balbi2, M. C. Bitter5, J.-P. Gattuso4, L. Canesi2, L. Kapsenberg3# and R. Dumollard1#
1. Ascidian BioCell Group, Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), UMR7009, 181 chemin du Lazaret, 06230 Villefranche-sur-Mer, France. 2. Dipartimento di Scienze della Terra, dell’Ambiente e della Vita, DISTAV, Universita` di Genova. 3. Institute of Marine Sciences (ICM), Passeig Marítim de la Barceloneta, 37-49. E-08003 Barcelona, Spain. 4. Laboratoire d’Oceanographie de Villefranche-sur-mer (LOV), Sorbonne Universite´, CNRS, 181 chemin du Lazaret, 06230 Villefranche-sur-mer, France. 5. Department of Ecology and Evolution, University of Chicago, Chicago, IL, USA
# contributed equally
Embryonic shell development of Mytilus spp. shape and calcification patterns thus confirming has long been used as a model to test the effect of its pivotal role in shell formation. The effect of stressors and toxicants because of the similar and exposure to BPA or to acidified SW (pH=7.4) on consistent shell phenotypes readily identifiable shell morphogenesis and on the spatio-temporal in the D-Veligers. However, the absence of earlier expression of Mg-TYR was then studied. In tro- time-points of analysis or comparison between chophores, both stressors induced hinge indenta- different stressors prevent the discrimination of tions, delay in matrix expansion and shell depo- specific phenotypes or the identification of com- sition; BPA also caused asymmetric expansion of mon targets. The present study investigates the the organic matrix. In D-Veligers, the stressors effect on shell development of Mytilus gallopro- induced similar shell malformations, although, vincialis of two environmental stressors, Bisphe- their intensity and frequency were clearly dif- nol-A and pH, known to induce shell malfor- ferent. Mg-TYR expression levels were higher in mations. We focused on the time course of shell trochophore than in D-Veligers and the pattern of biogenesis during trochophore stages in physio- expression blueprinted the expansion of the or- logic conditions and after exposure to both stress- ganic shell matrix. Exposure to BPA altered Mg- ors using the double staining Calcofluor and Cal- TYR transcripts level and pattern of expression cein to identify and compare the first occurrence as the signal of Mg-TYR matched shell malfor- of shell phenotypes. As molecular target, we se- mations. We are now investigating the expres- lected an embryonic Tyrosinase gene (Mg-TYR), sion of TYR in low pH exposed embryos. This a phenol oxidase enzyme, key effector gene for work shows that both stressors induced similar the secretion of embryonic organic matrix whose but not equal shell malformations from the tro- expression level in adults of Mytilus edulis is sig- chophore stage and that their occurrence might nificantly altered by exposure to low pH. We first be caused by a modulation of Tyrosinase expres- inhibited TYR enzymatic activity with N-Phen- sion and activity. yltiourea (PTU) and found that it altered matrix
105 P21 RELATIONSHIP BETWEEN TAIL BEATING AND CA2+ OSCILLATION IN THE MOTOR GANGLION OF DEVELOPING ASCIDIAN EMBRYO
Taichi Akahoshi1*, Kohji Hotta 1, and Kotaro Oka1
1. Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Kouhoku-ku, Yokohama 223-8522, Japan,
Ascidian larva shows swimming behavior un- eggs prior to fertilization. We found the Ca2+ os- til metamorphosis. The motor neurons located in cillation is derived from a single pair of posterior the motor ganglion control rhythmic tail muscle motor neurons, A10.64. In the mid-tailbud (stage contractions, and these are only five pairs of cho- 23), the Ca2+ oscillations showed no synchroniza- linergic motor neurons (Horie et al., 2009). These tion in a pair of motor neurons and no correla- motor neurons and their projection to tail muscles tion with the excitation of tail muscles. In the late have been investigated by immunohistochemis- tailbud (stage 24), the Ca2+ oscillations of single try or electron microscopy in larva. However, it neuron were correlated with the excitation of ip- is still unknown how these neurons regulate the silateral tail muscle. After stage 24, the timing of tail beating during development. In the previous the Ca2+ oscillation synchronized with each other study, we identified that the Ca2+ oscillation was motor neuron. Furthermore, the interval of Ca2+ observed in motor ganglion at the mid-tailbud oscillation decreased gradually from approxi- stage, with a duration of 22 ± 4 s (Akahoshi et mately 80 seconds (stage 23) to approximately 15 al., 2017). In this study, we investigated the re- seconds, approached to the interval of tail beat- lationship between the Ca2+ oscillation and tail ing of swimming-larva (stage 27). These results beating until larval stage. To identify the cell lin- allowed us to understand how motor neurons eage and the number of cells showing Ca2+ oscil- wire the network for tail beating. lation, H2B-GCaMP6s mRNA was injected into
References 1. Akahoshi, T., Hotta, K., and Oka, K. (2017). Characterization of calcium transients during early embryogenesis in ascidians Ciona robusta (Ciona intestinalis type A) and Ciona savignyi. Dev. Biol. 431, 205–214. 2. Horie, T., Nakagawa, M., Sasakura, Y., and Kusakabe, T.G. (2009). Cell type and function of neu- rons in the ascidian nervous system. Dev. Growth Differ. 51, 207–220. 106 P22 ZOOID EXPERIMENTAL REMOVAL IN THE COLONIAL ASCIDIAN BOTRYLLUS SCHLOSSERI: HOMEOSTATIC CAPABILITIES AND REGENERATIVE POTENTIAL.
Virginia Vanni1,*, Fabio Gasparini1, Chiara Anselmi1 and Lucia Manni1
1. Department of Biology, University of Padova, Italy
In the colonial ascidian Botryllus schlosseri three capacities, this species has been recognized and generations of zooids coexist (adults, buds, and adopted since 1950as model for the study of a va- budlets). These generations develop synchro- riety of biological processes. Here we present the nously in relation to each other in a blastogenetic experiments based on the removal of buds and cycle that lasts one week at 18°C. A radial ves- adult zooids from colonies, which have been per- sel forms in each individual when passing from formed in the last decades to assess the cross talk budlet to bud life phase, and serves for the con- between different blastogenetic generations. In nection to the common colonial vasculature that particular, we show in detail the surgical manipu- synchronize colony development. Budlets, mean- lations to induce a different array of responses by time, appear and develop from the body wall in a removing one or more generations of zooids. We process called palleal budding mediated by stem highlight colony responses in terms of variation cells. A second type of budding, called vascular in blastogenetic potential, bilateral asymmetry, budding, can be induced when all the individ- and growth of the remaining zooids. We discuss uals are removed from a colony, and circulating the results in terms of homeostatic capabilities, stem cells aggregate and organize a new budlet, regenerative potential, methods used, and future able to restore the colony. Thanks to the relatively perspective offered by this tunicate model. fast blastogenetic cycle and its high regenerative
107 P23 NON-NEURONAL CELLS IN THE NERVOUS SYSTEM OF INVERTEBRATE CHORDATES
Riccardo Esposito1,*, Stephanie Bertrand2, Hector Escriva2, Marios Chatzigeorgiou1
1. Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen 5006, Norway 2. Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins, BIOM, F-66650 Banyuls-sur-Mer, France
Neurons are the primary signaling units of the cells, which line the cavity of the sensory vesi- nervous system. However, in order for them to cle (Nishitsuji et al., 2012), while morphologi- function correctly, they rely on the surrounding cal studies in the cephalochordate amphioxus non-neuronal cells. These cells play important demonstrated the presence of supportive cells roles during neurogenesis, synapse formation in the anterior neural tissue of the larvae (Lacal- and pruning in early development. Moreover, li and Kelli 2002). We are performing a more in they supply essential nutrients to neurons and depth characterization of these cell types using participate in neurotransmitter reuptake. Fur- the tunicates Ciona Intestinalis, Oikopleura dioica thermore, it is now clear that non-neuronal cells and the cephalochordate Branchiostoma lanceola- can also actively participate in neuronal signal- tum. We will present the results of a candidate ing. Given their importance in the development gene screen based on vertebrate markers across and function of the nervous system, these cells these three organisms. In addition, through func- represent a very interesting cell type class to tional imaging and behavioural analysis, we will study. Non-neuronal cells have been described provide further insight on their contribution to in several taxa among bilateria, but the presence nervous system development and function. Our of these cells in many groups, including inver- results highlight the importance of studying tebrate chordates remains an open question non-neuronal cells to elucidate their role in the (Hartline, 2011). Tunicates possess ependymal nervous system evolution.
References 1. Hartline, D.K., The evolutionary origins of glia. Glia, 2011. 59(9): p. 1215-36. 2. Lacalli, T.C. and S.J. Kelly, Floor plate, glia and other support cells in the anterior nerve cord of amphioxus larvae. Acta Zoologica, 2002(83): p. 87-98. 3. Koki Nishitsuji, Takeo Horie, Aoi Ichinose, Yasunori Sasakura, Hitoyoshi Yasuo and Takehiro G. Kusakabe. (2012). Cell lineage and cis-regulation for a unique GABAergic glycinergic neuron type in the larval nerve cord of the ascidian Ciona intestinali. Develop. Growth Differ. 54, 177–186 108 P24 DNA BARCODING STRATEGIES FOR FUNCTIONAL GENOMICS BY MULTIPLEXED SINGLE CELL RNA-SEQ ANALYSES
Wei Wang, Andreas Tjärnberg, and Lionel Christiaen*
Center for Developmental Genetics, Department of Biology, New York University, New York, NY, USA
In the past few years, single cell genomic tech- Ciona. Indeed, ascidians, with their fixed lineag- nologies have revolutionized many fields in the es, limited cell numbers and easy to dissociate life sciences, including developmental genetics embryos are particularly well suited for single and systems biology. Providing single cell sus- cell analyses. pensions can be obtained, most biological sys- Comparatively limited efforts have attempted tems are amenable to high-throughput single cell to go beyond systematic descriptions of control genomics, especially RNA-seq. Single cell RNA- animals and the building of catalogs. To harness seq (scRNA-seq) has provided unprecedented the power of single cell methods for function- insights into the cellular diversity of complex al genomics and combine systematic CRISPR/ tissues and organs, as well as a high-resolution Cas9-mediated mutagenesis with scRNA-seq, we view of the dynamics of developing systems, are developing DNA barcoding strategies to mix owing to such emerging concepts as pseudo- samples from different sources in multiplexed time1. Concomitant to the rapid development assays, followed by in silico deconvolution using of technologies, there has been an explosion of both user-defined sample barcodes and random systematic efforts to build comprehensive atlases cell barcodes. We will present progress in har- for various species, stages and tissues of interest2. nessing novel experimental and computational Work in tunicates has not escaped the single cell approaches for functional genomics and analysis frenzy, with reports of single cell transcriptomes of systems dynamics, with a special focus on the in the cardiopharyngeal lineage3, followed by cardiopharyngeal mesoderm and surrounding nervous system4 and whole embryo5 profiling in tissues.
References 1. Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotempo- ral ordering of single cells. Nat. Biotechnol. 32, 381–386 (2014). 2. Regev, A. et al. The Human Cell Atlas. Elife 6, (2017). 3. Wang, W. et al. A single cell transcriptional roadmap for cardiopharyngeal fate diversification. (2017). doi:10.1101/150235 4. Sharma, S., Wang, W. & Stolfi, A. Single-cell transcriptome profiling of the Ciona larval brain.Dev. Biol. (2018). doi:10.1016/j.ydbio.2018.09.023 5. Horie, R. et al. Shared evolutionary origin of vertebrate neural crest and cranial placodes. Nature 560, 228–232 (2018). 109 P25 EXPRESSION AND FUNCTIONAL ANALYSES OF THE FOUR ECTODERMAL TRANSCRIPTION FACTORS IN THE ASCIDIAN, HALOCYNTHIA EMBRYOS
Shih Yu1,*, Wang Kai 1,2, and Nishida Hiroki1
1. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan 2. Present address: Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China
Spatio-temporal expression of zygotic genes most of transcription factor genes expressed in is regulated by transcription factors, which pro- the vegetal hemisphere have been comprehen- motes cell fate decision and morphogenesis. sively analyzed previously. Functional analyses Investigation of the transcriptional regulatory suggested that SP8 doesn’t regulate any devel- relationships would be one of the crucial ways opmental process, although SP8 is expressed to understand embryonic development. In our during blastula stages. Knockdown embryos of previous research, staged RNA-seq of the as- FoxJ-r showed disruption in laterality and the cidian, Halocynthia roretzi, has shown that ten absence of mono-cilia, suggesting its function in transcription factors are transiently expressed cilia formation and left-right asymmetry. Knock- at the blastula stage, which is the stage that cell down of SoxF resulted in two phenotypes, some fates are specified and differentiation starts. Six developed into disorganized cell mass while the of these transcription factors have already been others had protruded cells presented outside at shown to play important roles during early de- the tailbud stage. However, function of PRDM1-r velopment, while the remaining transcription is still unknown due to non-specific effect of the factors, PRDM1-r, SP8, FoxJ-r and SoxF were still morpholino, which caused failure in gastrula- unknown. tion. These findings provide information for fur- ther experiments to investigate the regulatory Investigation of the spatial and temporal ex- functions of these transcription factors, and im- pression patterns showed that all of the four prove our understanding about ascidian embry- genes are expressed in the animal hemisphere onic development. from the 16-cell stage, which is probably because
110 P26 A GENOME-WIDE SURVEY OF MUSCLE STRUCTURAL GENES IN MOLGULA TECTIFORMIS SUGGESTS AN ANCIENT ORIGIN OF ITS ANURAL MODE OF DEVELOPMENT
Fuki Gyoja1,*, Miyuki Kanda2, Takehiro G. Kusakabe1, and Nori Satoh3
1. Department of Biology, Konan University, Japan 2. DNA sequencing Section, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan 3. Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan
Some ascidian species in the families Molgulidae and quenced using Illumina and nanopore sequencers, and Styelidae do not develop into a conventional urodele then assembled and scaffolded. Scaffold N50 values are larva. Instead, they form an anural (tailless) larva. An 864 kbp in M. tectiformis and 1624 kbp in M. manhatten- anural larva does not have tail muscle, notochord or sis. Genes were predicted by Braker/Augustus. BUSCO sensory organs. Previous studies revealed several mo- analysis suggested approximately 85-90% genes were lecular aspects underlying the anural mode of develop- predicted with high accuracy. ment. For example, a) mRNA for a putative Zn finger transcription factor, Manx, is stored in urodele Molgula To characterize larval muscle loss in M. tectiformis, oculata oocytes but not in those of its anural sister spe- we surveyed muscle structural genes in both genomes. cies, M. occulta. Manx seems to have an essential role M. manhattensis has many larval muscle actin copies in in tail formation1. b) Larval muscle actin genes have the genome. M. tectiformis has at least three muscle ac- become pseudogenes in the anural M. occulta2 and M. tin pseudogenes. Those pseudogenes have much more bleizi. c) The expression of muscle structural genes, such substitutions, insertions and deletions compared to as muscle actin, myosin, tropomyosin, and troponin, those in M. occulta, suggesting their ancient origin. We is largely suppressed in anural M. tectiformis embryos speculate that acquisition of anural development in M. and larvae, which was revealed from a large-scale EST tectiformis may have occurred earlier than that in M. oc- analysis3. Recently, genomes of three Molgulid species, culta in the course of Molgulid evolution. namely M. oculata (urodele), M. occulta (anural), and M. We also surveyed several other muscle structural occidentalis (urodele), were decoded. A genomic survey genes including myosin, tropomyosin, and troponin has revealed that tyrosinase and its related genes have 4 genes. For each muscle structural gene, one or more become pseudogenes in the anural species M. occulta . copy for both larval and adult form was obtained in the Because it was shown that anural development occurred M. manhattensis genome. In contrast, no seemingly-func- several times independently in ascidian evolution, it is tional larval muscle structural genes were obtained in desirable to decode genomes of more Molgulid species, M. tectiformis. Furthermore, few pseudogene candidates especially anural ones, to obtain deeper insights into the for those genes were obtained. We speculate that these emergence of anural development. genes have become pseudogenes in anural evolution In this study, we newly decoded two Molgulid ascid- leading to M. tectiformis, but their DNA sequences may ian genomes, the anural species M. tectiformis, and the have accumulated too many mutations to be detected. urodele species M. manhattensis. Genomic DNA was se- References 1. Swalla BJ, Jeffery WR. 1996. Requirement of the Manx gene for expression of chordate features in a tailless ascidian larva. Science 274, 1205-1208. 2. Kusakabe T, Swalla BJ, Satoh N, Jeffery WR. 1996. Mechanism of an evolutionary change in muscle cell differentiation in ascidians with different modes of development.Dev. Biol. 174, 379-392. 3. Gyoja F, Satou Y, Shin-i T, Kohara Y, Swalla BJ, Satoh N. 2007. Analysis of large scale expression sequenced tags (ESTs) from the anural ascidian, Molgula tectiformis. Dev. Biol. 307, 460-482. 4. Racioppi C, Valoroso MC, Coppola U, Lowe EK, Brown CT, Swalla BJ, Christiaen L, Stolfi A, Ris- toratore F. 2017. Evolutionary loss of melanogenesis in the tunicate Molgula occulta. Evodevo 8, 11. 111 P27 GATA IS REQUIRED FOR THE EARLIEST ZYGOTIC GENE TRANSCRIPTION IN THE ASCIDIAN EMBRYO
Kaoru S. Imai 1,*, Kenji Kobayashi 2, Naoki Ookubo1, Izumi Oda-Ishii2 and Yutaka Satou2
1. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan. 2. Department of Zoology, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan
Maternally expressed Gata.a, which is known is required for the expression of not only animal to direct animal hemisphere-specific expression, hemisphere-specific genes, but also vegetal hemi- was required for activating genes in various re- sphere-specific genes. On the basis of this find- gions of early ascidian embryos, with priming ing, we performed a global analysis, and found being the most likely mechanism. that genes expressed in early embryos have sig- nificantly more Gata motifs in their upstream re- In ascidian embryos, the earliest transcription gions. These data suggest that Gata.a function is from the zygotic genome begins between the not limited to the genes expressed specifically in 8-cell and 16-cell stages. Gata.a, a maternally ex- the animal or vegetal hemispheres, and that Ga- pressed Gata transcription factor, activates tar- ta.a plays an important role in the earliest tran- get genes specifically in the animal hemisphere, scription of the zygotic genome. Because Gata.a whereas the complex of β-catenin and Tcf7 an- is present even in the nuclei of 2-cell embryos, tagonizes the activity of Gata.a and activates tar- Gata.a binding may precede and facilitate the get genes specifically in the vegetal hemisphere. binding of Tcf7 or other factors in the upstream Here, we show, through knockdown experi- regions of genes expressed in early embryos. ments for Gata.a and reporter assays, that Gata.a
112 P28 GENOMIC APPROACHES TO STUDY LONG- AND SHORT-TERM CHIMERA FORMATION IN THE GLOBAL INVADER DIDEMNUM VEXILLUM
Marta Pascual1*, Maria Casso1,2, Davide Tagliapietra3, and Xavier Turon2
1. Dept. of Genetics, Microbiology and Statistics, and IRBio, University of Barcelona, Catalonia, Spain 2. Center for Advanced Studies of Blanes (CEAB, CSIC), Catalonia, Spain 3. CNR - National Research Council of Italy, ISMAR - Marine Sciences Institute, Venice, Italy
The formation of chimeric entities through experiment 45 pairs, corresponding to 15 intra- colony fusion has been hypothesized to favour colony and 30 intercolony pairs, were fixed on a colonisation success and resilience to multiple slide using cotton threads, with contacting edges, stressors in modular organisms. We tested the and monitored over 4 weeks. Single zooids per prevalence of chimerism in the ascidian Didem- colony were analysed as mentioned above. In num vexillum at the Ebro Delta population and about 50% of the intra and intercolony pairs one the capacity of chimera formation through col- or both of the fragments regressed and died. In ony fusion experiments in the Venetian lagoon. the remaining pairs all the isogeneic pairs fused, For the prevalence study, we sampled five dif- while only 31% resulted in fusion in allogeneic ferent fragments from 9 large colonies on oyster contacts. We found no significant differences in cultures. For each fragment, we analysed a sin- overall genetic distances between fused and non- gle zooid by whole genome amplification and fused allogeneic pairs. We conclude that colony genotyping-by-sequencing. The percentage of fusion occurs frequently in Mediterranean pop- shared genotypes allowed us to clearly identify ulations of D. vexillum and seems to be unlinked pairs of zooids with the same genotype (96.8± to genetic relatedness among fusing genotypes. 0.14 identical loci) and with different genotypes However, we cannot discard that a few loci are (48.07±0.07). Our results showed that 44% of the responsible for increasing compatibility among colonies were in fact chimeras. For the fusion them.
References Stefaniak, L., Lambert, G., Gittenberger, A., Zhang, H., Lin, S., & Whitlatch, R. B. (2009). Genetic con- specificity of the worldwide populations of Didemnum vexillum Kott, 2002. Aquat. Invasions 4, 29–44. 113 P29 RIMBP2/3, A NEW MARKER FOR PIGMENT CELL PRECURSORS AND PERIPHERAL NERVOUS SYSTEM IN CIONA ROBUSTA
Ugo Coppola1,3, Paola Olivo1, Enrico D’Aniello1, Christopher Johnson2, Alberto Stolfi2, Filomena Ristoratore1.
1. Biology and Evolution of Marine Organisms Department, Stazione Zoologica Anton Dorhn, Villa Comunale, 80121 Napoli, Italy 2. Georgia Institute of Technology, School of Biological Sciences, Atlanta, GA, USA. 3. Now at: Molecular Cardiovascular Biology Division, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.
Rimbp (RIM-binding proteins) is a poorly stud- lation of this gene we identified a cis-regulatory ied gene family involved in the proper function- region of 0.3 kb conserved with sibling species ing of presynaptic machinery calcium-depend- C. savignyi harboured inside its seventh intron ing. Starting from an ascidian Rimbp gene, we (intR7), which guides a strong GFP expression in shed light on the evolution of Rimbp family in BTNs and Ascending Motor Ganglion neurons metazoans, defining how duplications modelled (AMGNs) at larva stage. Moreover, we unrav- its evolutionary scenario, mainly in vertebrates. elled the intR7>eGFP presence also in two glu- The unique Rimbp2/3 of tunicates, is orthologous tamatergic neurons of palps (papillae)belonging to vertebrate vertebrate Rimbp2 and Rimbp3. In to PNS, documenting, by double electroporation sea squirt Ciona robusta, Rimbp2/3 is expressed in experiments, that these neurons originated from the pigment cell precursors (that will give rise to FoxC+ territory of palps. otolith and ocellus) and in cells belonging to both In sum, we produced first data regarding the central and peripheral nervous system (CNS and expression of unique family member Rimbp2/3 in PNS). In situ hybridizations indicated expres- tunicates. Our findings speak in favour of a con- sion, at early tailbud stage, in bipolar tail neurons served involvement in the functions of distinct (BTNs), prior of the expression in otolith and cells belonging to the nervous system. ocellus precursors. In the aim to study the regu-
114 P30 LABORATORY CULTIVATION OF CIONA AND OTHER TUNICATES
Schires Gaëtan1, Henry Sébastien1 , Garnier Ronan1, Lasbleiz Régis2
1. Centre de Ressources Biologiques Marines, FR2424 Sorbonne Université / CNRS, Station Biologique de Roscoff, 29680 Roscoff, France 2. Centre de Ressources Biologiques Marines, FR3761 Sorbonne Université / CNRS, Institut de la Mer de Villefranche, 06230 Villefranche-sur-Mer, France
Tunicates have long been the subject of scien- for the laboratory cultivation of Ciona intestinalis tific research due to their close phylogenetic re- have been defined and the full life cycle complet- lationship with vertebrates. Interest increased ed ex situ, allowing the establishment of laborato- in the 2000s with publication of the full genome ry strains (wild-type/inbred), and, since 2016, of sequence of Ciona robusta. In order to accompany a semi-intensive “farming” mode of production. the development of model species by the scien- These techniques have been fully or partially tific community, the Marine Biological Ressource transferred to the cultivation of other species of Center of the Roscoff Biological Station started a tunicate, including Ciona robusta, Phallusia mam- breeding program on several ascidian species in millata, Ascidiella aspersa, and Styela clava. 2008 in the context of the EMBRC infrastructure and associated projects. Zootechnical parameters
115 P31 EVOLUTION OF THE NOTOCHORD AND CARDIO- PARAXIAL MUSCLE IN CHORDATES INFERRED FROM THE APPENDICULARIAN ACTINS.
Alba Almazán, Alfonso Ferrández-Roldán, Ricard Albalat and Cristian Cañestro*
Department of Genetics, Microbiology and Statistics, & Biodiversity Research Institute (IRBio), University of Barcelona, Spain
The evolution of the notochord and paraxi- reveal differences in the temporal-regulation and al muscle linked to locomotion powered by tail tissue-specificity of different actin paralogs, sug- beating is likely one of the key innovations that gesting complex processes of subfunctionaliza- facilitated the origin and radiation of chordates. tion during the evolution of urochordates. Our This innovation was accompanied by gene du- results suggest the presence of a “cardio-parax- plications that gave rise to muscular actins from ial” muscular actin in the last common ancestor cytoplasmic ancestral forms, which acquired of vertebrates and urochordates. Cytoplasmic contractile capability thanks to the recruitment actins show highly dynamic tissue-specific ex- of the myosin motor-machinery. In our work, we pression domains, which include the notochord, have characterized the complete actin catalogue ciliated cells and neurons with axonal projec- of the appendicularian Oikopleura dioica, an uro- tions, which challenge the classic housekeeping chordate that maintains a chordate body plan notion ascribed to these genes. Considering that throughout their life, including the notochord in previous work had demonstrated the existence a muscled tail that confers an active free-living of notochord-specific actins in cephalochordates, pelagic style. Our genomic survey, phylogenet- the tissue-specific expression of two cytoplasmic ic analyses and Diagnostic-Actin-Values (DAVs) actins in the notochord of O. dioica suggests that reveal that O. dioica has four muscular actins this pattern plausibly reflects the ancestral condi- (ActnM1-4) and three cytoplasmic actins (Act- tion of chordates, and provides new insights to nC1-3), most of which originated by appendic- better understand the evolutionary origin of the ularian-specific gene duplications. Our results notochord.
Reference Almazán A, Ferrández-Roldán A, Albalat R, Cañestro C. 2018 Developmental atlas of appendicular- ian Oikopleura dioica actins provides new insights into the evolution of the notochord and the car- dio-paraxial muscle in chordates. Dev Biol. pii: S0012-1606(17)30888-6. PMID:30217598 116 P32 DIVING INTO THE UNKNOWN OF ASCIDIAN DIVERSITY IN ECUADOR
M. Gabriela Agurto1,2,4, David Santos-Soares4,5, Rosana M. Rocha2, Jenny Rodriguez1, Federico D. Brown1,3,4,5 *
1. Escuela Superior Politécnica del Litoral, ESPOL, (Centro Nacional de Acuicultura e Investigaciones Marinas, CENAIM), Campus Gustavo Galindo Km. 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador 2. Departamento de Zoologia - Setor de Ciências Biologicas - Universidade Federal do Paraná, Brazil 3. Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia 4. Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Brazil 5. Centro de Biologia Marinha, Universidade de São Paulo, SP, Brazil
To date, only few species of sessile tunicates ing nine new species: Aplidium lambertae sp. nov. (i.e. ascidians) have been recorded for the Trop- Ascidia valdiviensis sp. nov., Ascidia vulgaris sp. ical Eastern Pacific. To fill this gap, we collected nov., Ascidia huancavilca sp. nov., Eudistoma caras specimens along a 600 km stretch of coastline sp. nov., Polyandrocarpa teticas sp. nov., Polyandro- in central and southern Ecuador (3°11’40.28” S carpa santaclarensis sp. nov., Pyura machallila sp. - 0°34’54.6” W). In several expeditions that last- nov., and Trididemnum azureus sp. nov. We report ed over three years, we collected by scuba-div- four new geographic records in the Pacific Ocean ing (5 – 20 meters in depth) at sites suggested for Eudistoma platense, Eudistoma clarum, Eudi- by local divers or fishermen to contain a high stoma obscuratum and Perophora carpenteria. The diversity of marine sessile invertebrates on hard highest number of ascidian species was collected substrata, i.e. rocks and corals. We also collected in Isla Santa Clara close to the border with Peru. in the intertidal zones during low tides, as well The cold Humboldt Current converges with the as from artificial substrates and docks. Samples warm Equatorial Current at this site allowing were relaxed in menthol, fixed in formaldehyde both cold and warm water species to co-inhabit (4%), preserved in ethanol (80%), and species here, which could explain the high diversity of were identified. We collected approximately 393 ascidians reported for this area. Species that were specimens in total, and in this manuscript we re- found across distant sites and presumably with port the identification of 155 samples, which rep- broad distribution include Aplidium lambertae sp. resents about 40% of the total collection. We re- nov., Ascidia vulgaris, Ascidia huancavilca sp. nov., port 27 species of three ascidian orders (6 species Ascidia sydneiensis, Cystodytes dellechiajei, and Eu- of Phlebobranchia, 11 species of Aplousobran- distoma clarum. chia, and 10 species of Stolidobranchia), includ-
117 P33 PROVISION OF COMMUNITY TOOLS FOR THE TUNICATE COMMUNITY
Delphine Dauga,*
Bioself Communication, Marseille, France
Biocurators are involved in the development neers, PhD candidates, postdocs, and students. of tools that allow the community to share and The multitude of platforms allows for choosing comment data. Resources such as repositories, the optimum networking tool: Twitter to main- portals, and websites that specify to a commu- tain a professional network, Facebook to reach nity which data are available and where, as well a mainstream audience whereas scientific net- as shared documents that facilitate interaction works like ResearchGate or MyScienceWork of- between researchers, developers, annotators, fer the possibility of developing a more special- and biocurators in a specific community are nec- ized and more professional network. essary. Biocurators may also be involved in the management of social networks. Social networks I will present here some tools I set up for the and media are improving the connectivity be- tunicate community. tween biocurators, journals, researchers, engi-
Reference Dauga D. Biocuration: A New Challenge for the Tunicate Community (2015). doi: 10.1002/dvg.22842 118 P34 EXPLORING THE ROLE OF CDK5 IN ASCIDIAN DEVELOPMENT
Mercurio S.1*, Messinetti S. 1, Venturin M.2, Manni L.3 and Pennati R.1
1. Department of Environmental Science and Policy, University of Milan 2. Department of Medical Biotechnology and Translational Medicine, University of Milan 3. Department of Biology, University of Padova
Cyclin dependent kinase 5 (Cdk5) is a ser- ent, and, based on our analysis, their expression ine-threonine kinase predominantly expressed patterns are comparable with those reported in in neural tissues and involved in both brain de- vertebrates. Thus, we started exploring Cdk5 in- velopment and degeneration. During vertebrate volvement in larval neural development, specifi- neurogenesis, Cdk5 is implicated in cell cycle cally inhibiting Cdk5 activity by drug treatments. regulation, neural migration as well as synapse Larval central nervous system was highly affect- formation (1, 2). Cdk5 gets activated by its neu- ed by Cdk5/p35 inhibition: sensory vesicle ap- ron-specific activator p35 (3). Stressed conditions peared reduced in size and fused with the motor induce cleavage of p35 to p25 by calpain which ganglion and neurites outgrowth was impaired forms a more stable yet hyperactive Cdk5/p25 along the tail in a dose dependent manner. complex (4), leading to neural disruption. Al- Overall, our results suggested that Cdk5/p35 though Cdk5/p35 is a key player of neural ho- functions are highly conserved between ascidi- meostasis, no study on its involvement in ascidi- ans and vertebrates, setting the stage for further ans neural development has been performed yet. research related to its involvement in ascidian In Ciona intestinalis and C. robusta genomes ho- development. mologs of Cdk5 and its main regulators are pres-
References 1. T. Kawauchi, 2014. Cdk5 regulates multiple cellular events in neural development, function and disease. Development, Growth & Differentiation 56, 335-348. 2. Y. Sheng, L. Zhang, S. C. Su, L.-H. Tsai, J. J. Zhu, 2016. Cdk5 is a New Rapid Synaptic Homeostasis Regulator Capable of Initiating the Early Alzheimer-Like Pathology. Cerebral Cortex 26, 2937-2951. 3. K. Shah, D. K. Lahiri, 2014. Cdk5 activity in the brain – multiple paths of regulation. Journal of Cell Science 127, 2391-2400. 4. G. N. Patrick, L. Zukerberg, M. Nikolic, S. de la Monte, P. Dikkes, L.-H. Tsai, 1999. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615-622. 119 P35 INGESTED MICROPLASTICS TRANSLOCATE FROM THE GUT CAVITY OF JUVENILES OF CIONA INTESTINALIS
Pennati A.1*, Messinetti S.1, Mercurio S.1, Scarì G.1 and Pennati R.1
1. Department of Environmental Science and Policy, University of Milan
Microscopic plastics, 1 µm-1 mm diameter, are particles on the larval and juvenile development widespread in marine environment and can be in the ascidian Ciona intestinalis. At the highest ingested by a variety of marine organisms1,2,3. concentration tested, they caused a delay in the They derive from the fragmentation of larger juvenile growth, probably due to inadequate plastic debris, such as plastic bottles and bags4,5. food intake. Instead, larval development was not The impact of these particles can depend on their affected by the presence of microplastics. concentration and size6. Filter-feeders like ascidi- ans are more sensitive to microplastics pollution A histological analysis of juveniles revealed then predators due to their less selective strategy that 1 µm particles can translocate from the gut of feeding7,8. Once ingested microplastics can ex- to the internal extracellular compartment in just 4 days and they can be phagocytized by specific ert different effects, from damaging and blocking 11 the feeding appendages and digestive system9, to blood cells identified as granular amoebocytes limiting the food intake. Only few studies have with phagocyte activity. investigated the translocation of microplastics Therefore, small microplastics can exert also an from the gut cavity to the circulatory system and immune response in ascidians with consequenc- 10 body tissues . es that are still to be clarified thus raising the con- We analyzed the effects of 1 µm polystyrene cern regarding the presence of these pollutants in marine environment. References 1. Boerger CM, Lattin GL, Moore SL, Moore CJ. 2010. Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre. Marine pollution bulletin 60: 2275-78 2. Davison P, Asch RG. 2011. Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre. Marine Ecology Progress Series 432: 173-80 3. Wright SL, Rowe D, Thompson RC, Galloway TS. 2013. Microplastic ingestion decreases energy reserves in ma- rine worms. Current Biology 23: R1031-R33 4. O’Brine T, Thompson RC. 2010. Degradation of plastic carrier bags in the marine environment. Marine pollution bulletin 60: 2279-83 5. Browne MA, Crump P, Niven SJ, Teuten E, Tonkin A, Galloway T, Thompson R. 2011. Accumulation of microplas- tic on shorelines woldwide: sources and sinks. Environmental science & technology 45: 9175-79 6. Bigelow LK, Lasenby DC. 1991. Particle size selection in cadmium uptake by the opossum shrimp, Mysis relicta. Bulletin of environmental contamination and toxicology 47: 790-96 7. Gallo A, Tosti E. 2015. The Ascidian Ciona Intestinalis as model organism for ecotoxicological bioassays. Journal of Marine Science: Research & Development 5: e138 8. Rummel CD, Löder MGJ, Fricke NF, Lang T, Griebeler E-M, Janke M, Gerdts G. 2016. Plastic ingestion by pelagic and demersal fish from the North Sea and Baltic Sea. Marine pollution bulletin 102: 134-41 9. Derraik JGB. 2002. The pollution of the marine environment by plastic debris: a review. Marine pollution bulletin 44: 842-52 10. Browne MA, Dissanayake A, Galloway TS, Lowe DM, Thompson RC. 2008. Ingested microscopic plastic translo- cates to the circulatory system of the mussel, Mytilus edulis (L.). Environmental science & technology 42: 5026-31 11. Rowley AF. 1981. The blood cells of the sea squirt, Ciona intestinalis: morphology, differential counts, and in vitro phagocytic activity. Journal of Invertebrate Pathology 37: 91-100 120 P36 ELUCIDATING THE MECHANISMS DRIVING SECONDARY NOTOCHORD-ENRICHED GENE EXPRESSION
Matthew Harder*, Wendy Reeves, Chase Byers, Mercedes Santiago, and Michael Veeman
Division of Biology, Kansas State University
The primary (A-line) and secondary (B-line) minimal cis-regulatory module revealed evi- Ciona notochord lineages have distinct cell be- dence for distinct positive elements that activate haviors, but most notochord-enriched genes are notochord expression and an important silencer thought to be uniformly expressed across the two element that shuts expression off in the primary lineages. We recently identified three notochord notochord. This minimal CRM contains predict- genes, C11.331, C12.115, and C8.891, with ex- ed binding sites for ETS, GATA, LMX, and Myb pression that is specifically enriched in only the in or near the silencer region. Mutation of each secondary notochord. The tail tip expresses both of these sites individually reduces expression of Wnt and FGF ligands, so we tested pharmaco- the minimal reporter in the secondary notochord, logical modulators of these pathways and found but these mutations neither eliminate expression that both C11.331 and C12.115 are positively reg- altogether nor release the primary notochord ulated by FGF and Wnt signaling. We developed from silencing. These results suggest multiple a dual reporter strategy to quantify regional- inputs into the regulation of the C11.331 second- ized expression while accounting for transgene ary notochord-enriched expression pattern, in- mosaicism. Extensive analysis of the C11.331 cluding Wnt and FGF ligands from the tail tip, upstream regions revealed multiple non-over- notochord-specific regulators, and a yet to be lapping regions that drive enriched expression identified silencer in the primary notochord. in the secondary notochord. Dissection of one
121 P37 INSIGHTS ON THE MITOCHONDRIAL GENOME OF APPENDICULARIANS
Dorothee Huchon1,2,*, Maria Novosolov1, Rade Garic3, Noa Shenkar1,2, Thomas Stach4 and Carmela Gissi5
1. School of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 2. The Steinhardt Museum of Natural History and National Research Center, Tel Aviv University, Tel Aviv, Israel 3. Institute for Marine and Coastal Research, University of Dubrovnik, Dubrovnik, Croatia 4. Institut für Biologie, Molekulare Parasitologie, Humboldt-Universität zu Berlin, Berlin, Germany 5. Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari “Aldo Moro”, Italy.
Oikopleura dioica (Appendicularia) is a model sites were observed between Mediterranean and organism to understand the evolution of chor- Japanese populations of O. dioica suggesting a dates. Although its nuclear genome has been rapid evolution of these sites. sequenced, its mitochondrial (mt) genome has While tRNAs usually punctuate the pro- not been determined, probably because of their tein-coding genes in chordate mt genome, only fast evolutionary rate and extensive RNA edit- one tRNA, tRNAmet, was identified among O. ing. We sequenced cDNA of the cox1 gene and dioica mt contigs assembled from published nuclear genomes (using Illumina) from O. dioi- cDNA reads. To test if mt tRNAs could have ca and Fritillaria pellucida. Mapping the resulting been lost, we searched the genomic assembly for DNA reads onto the cox1 cDNA sequences, we the presence of mt aminoacyl tRNA synthetase confirmed the previously-published observation (aaRS) genes, which encode the proteins respon- that, at DNA level, long poly-T stretches inter- sible for the aminoacylation of its cognate tRNA. rupt the mt ORFs, hampering their identification. No specific mt aaRS genes were found in the nu- In addition, we noticed that mitogenomic poly-T clear genome assembly of O. dioica, except for mt- correspond at RNA level to TTTTTT (6T) regions MetRS, which catalyzes the mt-tRNAMet amino- in O. dioica but to shorter TTTT (4T) regions in acylation. This suggests that the mt genome of O. F. pellucida, suggesting species-specific RNA ed- dioica encodes only the single tRNAMet. itings by deletion. Remarkably, different editing
122 P38 A LIBRARY OF SINGLE GUIDE RNA CONSTRUCTS FOR SYSTEMATIC CRISPR/CAS9-MEDIATED MUTAGENESIS IN CIONA ROBUSTA
Margaux Failla1, Yelena Bernadskaya1, and Lionel Christiaen1
1. Department of Biology, New York University, New York, NY, United States of America
The use of model organisms in research has ed mutagenesis4, 5. been paramount in understanding animal devel- Here, we report the development of a library opment, as well as evolution of developmental of single guide RNA constructs. We describe diversity. Many powerful developmental models optimized strategies for sgRNA design and val- have benefited from genetic screens to identify idation, as well as a high-throughput cloning regulators of different developmental processes. method. Further, we identified an optimal Cas9 Our lab studies the specification and devel- variant for gene knockout. We also report the opment of the cardiopharyngeal cell lineage1, 2 development of imaging chambers designed to using the ascidian Ciona robusta, a model organ- accommodate Ciona embryonic development. ism highly amenable to Cell and Developmental We are now developing a pipeline for high-con- Systems Biology. Our lab has extensively pro- tent microscopy and image analysis to screen for filed the cardiopharyngeal transcriptomes3, and cell migration and division phenotypes in the we are thus poised to systematically interrogate cardiopharyngeal lineage. This approach will the function of zygotically expressed and lin- permit the reconstruction of biomolecular net- eage-specific genes in cardiopharyngeal develop- work models for cardiopharyngeal cell behavior ment. To this aim, we have adapted the CRISPR/ in Ciona robusta, by analogy with previous work Cas9 system to be used in Ciona and have de- in C. elegans6, and empower the community with scribed a protocol to design efficient single guide a resource for functional genomics analyses of a RNAs for lineage-specific CRISPR/Cas9-mediat- variety of developmental systems.
References 1. Razy-Krajka, F. et al. Collier/OLF/EBF-dependent transcriptional dynamics control pharyngeal muscle specification from primed cardiopharyngeal progenitors. Developmental cell 29, 263-276 (2014). 2. Wang, W., Razy-Krajka, F., Siu, E., Ketcham, A. & Christiaen, L. NK4 antagonizes Tbx1/10 to pro- mote cardiac versus pharyngeal muscle fate in the ascidian second heart field. PLoS biology 11, e1001725 (2013). 3. Wang, W. et al. A single cell transcriptional roadmap for cardiopharyngeal fate diversification. Nature Cell Biology, In Press (2019) 4. Stolfi, A., Gandhi, S., Salek, F. & Christiaen, L. Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141, 4115-4120 (2014). 5. Gandhi, S., Haeussler, M., Razy-Krajka, F., Christiaen, L. & Stolfi, A. Evaluation and rational de- sign of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona. Developmental bi- ology 425, 8-20 (2017). 6. Green, R.A. et al. A high-resolution C. elegans essential gene network based on phenotypic profil- ing of a complex tissue. Cell 145, 470-482 (2011). 123 P39 IS BRACHYURY A UNITARY MASTER REGULATOR OF CIONA NOTOCHORD FATE?
Wendy M. Reeves*, Kotaro Shimai, Yuye Wu, Matthew J. Harder and Michael T. Veeman
Division of Biology, Kansas State University, Manhattan KS 66506, USA
We previously used FACS-RNAseq and in situ also continued to express many of their own hybridization to quantify and validate an exten- unique markers. This suggests that Brachyury is sive set of genes enriched in the Ciona notochord. not a true master regulator, as it can only partial- This newly comprehensive notochord-enriched ly transform other cell types to notochord fate. gene set allows us to test the long-held model To test whether Brachyury is necessary for the that Brachyury is a master regulator of the no- expression of all notochord-enriched genes, we tochord gene regulatory network. Specifically, is disrupted it by somatic CRISPR ribonucleopro- Brachyury function necessary for expression of tein injection followed by RNAseq transcription- the full notochord transcriptome, and is ectopic al profiling. Surprisingly, we found that many expression of Brachyury sufficient to induce no- notochord enriched genes were not strongly af- tochord fate in other tissues? fected by loss of Brachyury function. Enhancer To test Brachyury’s ability to induce the noto- TFBS analysis and perturbation of notochord fate chord-specific transcriptional program in other induction identified other transcription factors cell types, we ectopically expressed Brachyury that may be acting in parallel to Brachyury. We using three different tissue-specific enhancers. are currently testing the function of these candi- Whole embryo RNAseq showed that the ma- date transcription factors in the notochord GRN. jority of notochord-enriched genes were neither Instead of being a straightforward regulatory significantly nor strongly induced by ectopic cascade entirely downstream of Brachyury, the Brachyury expression and that targeted tissues notochord GRN appears to be more complex.
124 P40 ITERATIVE AND COMPLEX ASYMMETRIC DIVISIONS CONTROL CELL VOLUME DIFFERENCES IN CIONA NOTOCHORD TAPERING
Konner Winkley*, Spencer Ward, Wendy Reeves, and Michael Veeman
Division of Biology, Kansas State University, USA
Consisting of only 40 cells in a small embryo divide asymmetrically to give daughter cells of well suited for quantitative in toto microscopy, different size and fate. Here, by contrast, a series the Ciona notochord is a tractable system for of subtle but iterative and finely patterned asym- studying the cellular behaviors giving rise to or- metric divisions controls the shape of an entire gan shape. It forms a single-file column at tailbud organ. We have quantified how these asymme- stages, which has a tapered shape that involves tries are driven by distinct cellular mechanisms differences in cell volume along the anterior-pos- including mitotic spindle displacement, mother terior axis. We have quantified sibling cell vol- cell shape, and effects occurring post-anaphase ume asymmetry throughout the developing that potentially involve unequal cortical con- notochord and find there are distinctive, stereo- tractility. We find that different combinations of typed patterns of unequal cleavage in all 4 bilat- these mechanisms are used in each blastomere. eral pairs of A-line primary notochord founder Inhibition of Nodal signaling reverses the di- cells and their descendants, and also in the B-line rection of asymmetric division in the A8.5 and derived secondary notochord lineage. A quanti- A8.6 blastomeres, largely via changes in spindle tative model confirms that the observed patterns displacement. These results demonstrate a new of unequal cleavage are sufficient to explain all role for asymmetric division in directly shaping the anterior-posterior variation in notochord cell a developing organ and point towards complex volume. Many examples are known of cells that underlying mechanisms.
125 P41 INSIGHTS INTO THE CELLULAR AND MOLECULAR MECHANISMS OF POLYCARPA MYTILIGERA’S EXTRAORDINARY REGENERATIVE ABILITIES
Tal Gordon1*, Mark Kowarsky2 Ayelet Voskoboynik3, Lucia Manni4, Noa Shenkar1, 5
1. School of Zoology, George S. Wise Faculty of Life Sciences, Tel-Aviv University 6997801, Tel-Aviv, Israel 2. Department of Physics, Stanford University, Stanford, United States 3. Institute for Stem Cell Biology and Regenerative Medicine and Hopkins Marine Station, Stanford University, United States 4. Department of Biology, University of Padova 35121, Padova, Italy 5. The Steinhardt Museum of Natural History, National Research Center, Tel-Aviv University 6997801, Tel-Aviv, Israel
Regeneration is widespread in the animal terize the cellular process and time-course of its kingdom, and a variety of model systems are regeneration using immuno- and histochemical employed to better understand the principles methods. One week following neural complex and genetic programs underlying this process. removal, we observed wound healing process Ascidians are remarkable for their regenerative and 21 days following its amputation the neural abilities; and while the majority of regenerative complex was completely regenerated. studies focused on well-known models such as P. mytiligera transcriptomic profiling, performed Ciona intestinalis, our previous work suggested a at five different time points of CNS regeneration, new model system: the solitary ascidian Polycar- revealed many genes with dynamic expression pa mytiligera (order Stolidobranchia). over time. This includes genes involved in the In vivo experimental observations revealed this regulation of cell cycle and in Wnt and Hedge- species extraordinary ability to regenerate all ma- hog signaling pathways. jor body parts following their removal, including Our new findings provide an in-depth charac- the digestive system, nervous system, and heart. terization of P. mytiligera’s regeneration, present- Our current study further describes P. mytilig- ing insights into the cellular and molecular as- era’s impressive regenerative potential and pro- pects of CNS regeneration, further emphasizing vides a cellular description of its regeneration this ascidian’s potential to serve as a new model processes over time. We focus on the central system for studying regeneration and its evolu- nervous system (CNS) regeneration and charac- tion within the tunicates.
126 P42 DEVELOPMENT OF A GENETIC ABLATION SYSTEM TO STUDY THE MOSAIC TO REGULATIVE TRANSITION AND ORGAN REGENERATION IN CIONA
Keaton J. Schuster1,*, and Lionel Christiaen1
1. Center for Developmental Genetics, Department of Biology, New York University, New York, NY, USA
Ascidians such as Ciona robusta are classic devel- system to ablate specific lineages in a temporally opmental models for deterministic or “mosaic” controlled manner to identify the transition from development. Mosaic/determinative develop- determinant development to a more regulative ment is typified by the embryo’s strict adherence mode of development that allows for the ability to invariant cleavage patterns and the inability to to amount a regenerative response. As a comple- recover lost lineages after ablation. Despite be- ment, we have designed an experimental strate- ing unable to replace lost cells as embryos, adult gy to harness the power of single cell transcrip- ascidians have remarkable regenerative abilities tome profiling and characterize this “regulative ranging from the replacement of specific organs transition” in whole metamorphosing larvae. In in solitary ascidians, to the remarkable whole- addition to identifying the mosaic to regulative body regeneration found in colonial ascidians. transition, a genetic ablation system will empow- An open question is how they can acquire such er studies of the potential regenerative ability of regenerative capacity as adults after having a internal organs that are traditionally difficult to highly deterministic embryonic development. To access surgically, such as the heart and body wall answer this question, we are developing a genetic muscles.
127 P43 MOLECULAR SIGNATURES OF CHORDATE DEVELOPMENT: TWO DISPARATE PATHWAYS, ONE TUNICATE
Mark Kowarsky1*, Chiara Anselmi2, Kohji Hotta3, Paolo Burighel2, Giovanna Zaniolo2, Federico Caicci2, Benyamin Rosental4,5, Norma F Neff6, Katherine J Ishizuka4,5, Karla J Palmeri4,5, Jennifer Okamoto6, Tal Gordon7, Irving L Weissman4,5,6, Stephen R Quake6,8, Lucia Manni2, Ayelet Voskoboynik4,5,6
1. Department of Physics, Stanford University, CA 94305, USA 2. Dipartimento di Biologia, Università degli Studi di Padova, Padova, Italy 3. Department of Biosciences and Informatics, Keio University, Yokohama, 223-8522, Japan 4. Institute for Stem Cell Biology and Regenerative Medicine, and Ludwig Center, Stanford University School of Medicine, Stanford, CA 94305, USA 5. Department of Biology, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950, USA 6. Chan Zuckerberg Biohub, San Francisco CA 94158, USA 7. Zoology Department, Tel Aviv University, Tel-Aviv Israel 8. Department of Applied Physics and Bioengineering, Stanford University, CA 94305, USA
Sexual development in chordates is well-de- Using the model organism Botryllus schlosseri scribed by embryogenesis. Other developmental we have combined transcriptome sequencing pathways including asexual reproduction and of major embryonic and blastogenic stages with whole body or tissue regeneration share some confocal, two-photon and electron microscopy essential processes such as establishment of the to characterize the molecular and morphological body axes, morphogenetic patterning and organ signatures along both developmental pathways. formation but differ in origin. Although studies We identify de novo, periods of transcriptional have identified conserved aspects of embryo- transition and shared molecular characteristics genesis across and within phyla, this pathway including stem cell associated transcription fac- has not been linked to the other developmental tors. Phylotypic conservation is investigated by pathways. In particular, it is unknown if conver- comparing developmental gene signatures from gent morphology implies convergent molecular other chordates. This study generated a complete mechanisms. Colonial tunicates provide a key gene profile database on the entire embryogene- to answering these questions, they are unique sis process and is the first to similarly describe amongst chordates in possessing two disparate asexual development. By combining microscopy developmental pathways that produce the adult with transcriptome sequencing, it demonstrates body, either sexually through embryogenesis, or the extent to which convergent morphology im- through a stem cell mediated asexual renewal plies different molecular mechanisms and reveals termed blastogenesis. the basic principles and evolutionary conserved elements of chordate development.
128 P44 ASYMMETRIC DIVISIONS AND THE CARDIOPHARYNGEAL NICHE
Nicole Kaplan1,*, Alberto Stolfi 2, and Lionel Christiaen1
1. New York University 2. Georgia Institute of Technology
During development, multipotent progenitors contact with an extrinsic source of Fgf ligand(s). divide and generate a diverse range of cell types. Towards this aim, we implicate the RhoGAP This often requires precise spatiotemporal coor- protein Depdc1.a in the regulation of oriented, dination between cell-intrinsic properties and asymmetric TVC divisions. We propose a model extracellular cues. Here we focus on mechanisms whereby TVCs divide stereotypically along the of fate specification in the cardiopharyngeal me- medio-lateral axis of the embryo to position pha- soderm of the simple chordate, Ciona robusta. Ci- ryngeal muscle precursors directly adjacent to a ona has two pairs of cardiopharyngeal precursors mesenchyme-derived source of Fgf ligand, thus called trunk ventral cells (TVCs). They divide restricting pharyngeal muscle fate to laterally asymmetrically and are specified as either - car positioned cells. These observations provide the diac or pharyngeal muscle precursors. As mul- foundation for an ongoing project aimed at un- tipotent progenitors, TVCs are transcriptionally derstanding the mechanisms by which oriented primed for both developmental programs. We cell divisions instruct fate specification in cardio- focus on the cell biology, with a specific empha- pharyngeal progenitors. sis on cell behaviors that orchestrate asymmetric
129 P45 IDENTIFICATION OF ENHANCER ELEMENTS RESPONSIBLE FOR TRANSCRIPTIONAL ACTIVATION OF CDX IN THE CIONA ROBUSTA NEURAL PLATE
Yoshie Nishimura, Shihori Yoshitake, Sylvia Lai, Ryoma Kondo, Shigeki Fujiwara
Department of Chemistry and Biotechnology, Kochi University, Kochi 780-8520, Japan.
The central nervous system of ascidian larvae is these cells is unclear. We cloned a 6.1-kb 5’ flank- formed through vertebrate-like neurulation pro- ing region of the Cdx locus and carried out re- cesses in the dorsal midline. The transcription porter analyses. Deletion analyses revealed that factor Cdx is important for neural tube formation, the region between -4469 and -2250 contained since suppression of its function leads to incom- enhancer elements activated in A9.15, A9.29, and plete closure of the neural tube. Cdx is expressed A9.31 cells at the late gastrula stage. Although in A9.15, A9.29, and A9.31 cells in the neural plate further deletion diminished the enhancer activi- of the Ciona robusta gastrula. The expression of ty, the region between -3319 and -2249 appeared Cdx in these cells requires the growth factor Nod- to contain a minimal essential element. We are al. A9.29 and A9.31 receive Nodal, while A9.15 now trying to identify transcription factors and does not. How Nodal activates Cdx in both of their binding sites within this region.
130 P46 COMPARATIVE ANATOMY OF ASCIDIAN MINIATURE TAILBUD
Kaoru Matsumura1*, Mitsuru J. Nakamura2, Wataru Koizumi1, Kohji Hotta1 and Kotaro Oka1
1. Department of Bioscience and Informatics, Faculty of Science and Technology, Keio University 2. Research Center for Complex Systems Biology, University of Tokyo
The tailbud stage belongs to the evolutionary cell number of each tissue, cell volume between conserved period (organogenesis period) among dwarf tailbuds and normal one, by constructing chordates and is essential for deciding the charac- dwarf 3D virtual mid-tailbud embryo (3DVMTE teristics of chordate body plan. Although the ra- [2]). As a result, the ratio of volume per tissue is tio of egg volume between Halocynthia roretzi and maintained in the dwarf tailbud embryo. To com- Ciona sp. is about 8 times, their shape of tailbud pare each cell volume quantitatively, receiver op- embryos are similar and basic structure is main- erating characteristic (ROC) curve are drew and tained. Halocynthia can produce normal-looking calculated area under curve (AUC). Epidermis, miniature (dwarf) tailbud embryos when eggs are nervous system and mesenchyme reduced the artificially cut in a plane. Although cell lineages number of cells but maintained cell volumes in during ascidian embryogenesis are known to be the dwarf. On the other hand, notochord, mus- invariant, it was reported that the number of cell cle and endoderm maintained the number of divisions in the dwarf embryo is changed by a cells but relatively reduced their cell volumes in distinct mechanism in each tissue [1]. In this study, the dwarf. Germline precursor doesn’t belong to to elucidate the size regulation of each tissue in both groups. These results showed there are the the dwarf tailbud embryo, we compared ana- distinct strategies of size regulation in each tissue tomical structure, the developmental speed, the of the dwarf tailbud embryo.
References: 1. Atsuko Yamada and Hiroki Nishida (1999). Distinct parameters are involved in controlling the number of rounds of cell division in each tissue during ascidian embryogenesis. J. of Exp. Zool, 284:378-391. 2. Mitsuru J Nakamura, Jun Terai, Reiko Okubo, Kohji Hotta, Kotaro Oka (2012). Three-Dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution. Developmental biology, 372(2):274-84. 131 P47 NANOPLASTICS AFFECT EMBRYOGENESIS OF CIONA ROBUSTA
Eliso M.C.1,2,*, Bergami E. 2, Murano C. 1,2, Corsi I. 2, Spagnuolo A.1
1. Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn Naples, Italy 2. Department of Physical, Earth and Environmental Sciences, University of Siena, Italy
Nanoplastics (<100 nm) represent an emerg- PS NPs were characterized in natural seawater ing threat for marine ecosystem and can have a (NSW) exposure media by DLS, while embrio- significant impact on aquatic organisms due to toxicity was evaluated looking at % of larval de- dimensions and higher surface area. The aim of velopment and morphology. PS-COOH formed this study is to evaluate the effect of polystyrene micro-aggregates (Z-Average >1 µm) and no sign nanoparticles (PS NPs) on the embryogenesis of of embryotoxicity were recorded up to 100 µg/ the tunicate Ciona robusta. Ascidians, are recog- mL. On the contrary amino-modified PS NP (PS-
nized as valuable biological models for ecotoxic- NH2) still in nano-scale form (Z-Average in NSW ity studies thanks to their rapid embryonic and ≈60 nm) compromise hatching and the normal larval development and resemblance to verte- development of the trunk of the larvae even at brates. Based on recent findings on embryo and the lowest concentration tested with an EC50 of larval disruption upon nanoplastics exposure 6.81 µg/mL. Our results suggest that differenc- in marine invertebrates species, ascidians also es in PS NP surface charges and aggregation in might represent a target which deserves further seawater affect their behavior and embriotoxici- investigation. PS NPs, owing negative and pos- ty in ascidians therefore further investigation on itive surface charges, respectively as carboxyl- mechanism of toxicity of amino-modified PS NPs
ated (PS-COOH) and amino-modified (PS-NH2) (PS-NH2) are strongly required due to their eco- were tested for 22 h in the range of 0-100 µg/mL. logical role in marine coastal environments.
132 P48 PROBABLE RECORD OF PHALLUSIA DEPRESSICULA AND P. PHILIPPINENSIS ALONG WITH MORPHOMETRIC COMPARISON WITH P. NIGRA FROM ANDAMAN AND NICOBAR ISLANDS, INDIA
Jhimli Mondal1,* and C. Raghunathan2
1. Centre for Marine Living Resources and Ecology, Ministry of Earth Sciences, Kendriya Bhavan, 6th Floor, Kakkanad, Kochi - 682037, India 2. Zoological Survey of India, Prani Vigyan Bhawan, M-Block, New Alipore, Kolkata, West Bengal- 700053, India
Altogether 70 species of ascidians under 25 in anatomical characteristics. Morphological- genera, 11 families and 3 orders including 2 new ly P. nigra is with jet black colour along with its species were documented during the study peri- non-papillated pre-pharyngeal area which is eas- od (September 2013- November, 2016) from An- ily distinguishable from other two species. How- daman and Nicobar Islands. Among the three or- ever, other two species is quite similar in external ders of Class Ascidiacea most of the species were morphology as well as in anatomy i.e. in both identified under the order Stolidobranchia i.e. the species 12 lobes found on branchial aperture 28 species (40%) followed by 25 species (35.71%) and 8 on atrial aperture along with massive cau- from Aplousobranchia and 17 species (24.29%) liflower shaped ovary. Besides, in P. depressicula under Phlebobranchia. Genus Phallusia is belong- has about 45 branchial tentacles in three different ing to the family Ascidiidae currently comprise orders whereas it is 60 branchial tentacles in four of 20 valid extant species world-wide (Ascidiacea different orders forP. philippinensis. The anal bor- World Database). Till date seven species under der of P. depressicula is plicated without definite the genus Phallusia are recorded from Andaman roll and it is bi-lobed anus with rolled rim for P. and Nicobar Islands. However, Phallusia depres- philippinensis. The morphological characteristics sicula (Heller, 1878), P. philippinensis Millar, 1975 features of these three species are compared. and P. nigra Savigny, 1816 are closely resembled
Key words: Phallusia, Taxonomy, Pre-pharyngeal band, India. References Ascidiacea World Database: http://www.marinespecies.org/ascidiacea/aphia.php?p=taxdetails&id=103485 as viewed on 30/03/2019 133 P49 PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENE AND IDENTIFICATION OF AN INDEPENDENT GLYCOSYL HYDROLASE FAMILY 6-DOMAIN GENE OF CIONA
Jun Inoue, Kun-Lung Li, Keisuke Nakashima, and Noriyuki Satoh
Okinawa Institute of Science and Technology Graduate University 1919-1 Tancha, Onna, Okinawa 904-0412, Japan
Horizontal gene transfer is one of the import- the spatial and temporal expression of the GH6-1 ant force that shapes animal genomic architec- gene in Ciona by in situ hybridization and quan- tures and contributes to biological diversity. The titative PCR. The GH6-1 gene was expressed at tunicate cellulose synthase gene CesA [1], a gene epidermis of tailbud embryos, and its expression transferred from bacteria, potentiates the struc- level was increased around ten hours after fertil- tural innovation of the tunic and the house. The ization (mid-tailbud stage). Although a mutant, tunicate CesA gene contains not only a CesA do- swimming juvenile, had been generated, in which main of the glycosyl transferase family 2 (GT2) the CesA expression was disrupted [3], the mech- but also a glycosyl hydrolase family 6 (GH6) do- anisms underlying its phenotypic changes were main, which is similar to bacterial cellulases [1]. still enigmatic. To investigate the function of the During our phylogenetic analysis on CesA using CesA and GH6-1 genes further, we are perform- the ORTHOSCOPE tool [2], we identified a sep- ing functional studies to clarify how these genes arate GH6-containing gene (tentatively called are incorporated into the host genetic networks. GH6-1) in Ciona intestinalis type A. We found that Our results support the uniqueness of tunicate CesA and GH6-1 homologs were present in seven CesA genes in the animal kingdom and provide tunicate genomes but not in any other non-tuni- knowledge of horizontally transferred genes ac- cate metazoans. In this study, we also examined quiring expression control in the animal host.
References 1. Nakashima, K., Yamada, L., Satou, Y., Azuma, J., and Satoh, N. (2004). The evolutionary origin of animal cellulose synthase. Dev Genes Evol 214, 81-88. 2. Inoue, J., and Satoh, N. (2019). ORTHOSCOPE: an automatic web tool for phylogenetically infer- ring bilaterian orthogroups with user-selected taxa. Mol Biol Evol 36, 621-631. 3. Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., and Satoh, N. (2005). Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc Natl Acad Sci U S A 102, 15134-15139. 134 P50 BOTRYLLUS SCHLOSSERI SECONDARY BUD SURVIVAL AFTER ZOOIDECTOMY MIMICS WHOLE BODY REGENERATION STUDIES
Shane Nourizadeh*, Susannah Kassmer, Delany Rodriguez, and Anthony W. De Tomaso
Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara
Understanding the regenerative mechanisms eration is stimulated by injury, and occurs ecto- of tissues in model species helps advance our pically compared to blastogenesis. Blastogenesis knowledge for how to treat limb, tissue, and or- and WBR have been studied in several related gan damage in humans. The present study fo- botryllid ascidians, and both processes show cuses on regeneration in the colonial ascidian Bot- species-specific characteristics. Here we utilized ryllus schlosseri, which under normal conditions time-lapse microscopy to characterize WBR in grows via a regenerative process called blasto- B. schlosseri. Our data suggests that, unlike other genesis where entire bodies, including all somatic botryllid species, WBR in B. schlosseri is not due and germline tissues, are generated de novo on to a novel ectopic budding process, but that suc- a weekly basis. In addition, B. schlosseri has also cessful regeneration always was initiated in spe- been reported to undergo a process called whole cific niches, which we found were remnants of body regeneration (WBR), during which regen- the blastogenic process itself.
135 P51 FUNCTIONAL ANALYSIS OF HOX 1 IN THE APPENDICULARIAN OIKOPLEURA DIOICA
LE GOFF Carine1,*, DENG Wei1, CHOURROUT Daniel1
1. SARS International Center for Marine Molecular Biology
The appendicularian Oikopleura dioica pos- some Hox genes have acquired lineage specific sesses a simplified chordate body plan, which function. is retained after the metamorphosis. In order to To address this issue, we studied the temporal understand how this simplification proceeded and spatial expression of Hox1, the most anterior at the molecular level, we study conserved key gene. The results showed a high expression level developmental genes, such as Hox genes. Hox at the tailshift stage, and a strong expression at genes are homeobox-containing transcription the trunk-tail transition and in the central ner- factors known to pattern the antero-posterior vous system. To go further, we have generated (AP) axis in many bilaterians. Previously, O. dioica a mutant line using CRISPR/Cas9 system (Deng Hox genes complement and cluster organisation et al., 2018) to unravel the biological function of have been described (Seo et al., 2004), showing Hox1. The Hox1 knock out was confirmed by in the presence of nine Hox genes, and highlighting situ hybridization and immunostaining, and the the disintegration of the cluster and alteration of mutant phenotype appeared to be lethal. We will the spatial collinearity. These features lead us to present morphological and physiological char- comprehend the function of Hox genes in O. dio- acterizations of the homozygotes, focusing more ica development, and to elucidate if the function particularly on the neuroanatomy. in AP axis patterning has been conserved or if
References: 1. Seo, H. C., Edvardsen, R. B., Maeland, A. D., Bjordal, M., Jensen, M. F., Hansen, A., ... & Reinhardt, R. (2004). Hox cluster disintegration with persistent anteroposterior order of expression in Oiko- pleura dioica. Nature, 431(7004), 67. 2. Deng, W., Henriet, S., & Chourrout, D. (2018). Prevalence of Mutation-Prone Microhomology-Me- diated End Joining in a Chordate Lacking the c-NHEJ DNA Repair Pathway. Current Biology, 28(20), 3337-3341. 136 P52 CRYPTIC SPECIES E OF THE BOTRYLLUS SCHLOSSERI SPECIES COMPLEX IS A NEW SPECIES
Carmela Gissi1,2,*, Marika Salonna1, Francesca Griggio3, Federica Montesanto4, Francesco Mastrototaro4, Fabio Gasparini5 and Riccardo Brunetti6
1. Dept of Biosciences, Biotechnologies and Biopharmaceutic, University of Bari “Aldo Moro, Via Orabona, 4 – 70125 Bari, Italy 2. IBIOM, CNR, Via G Amendola 165/A - 70126 Bari Bari, Italy 3. Dept of Biosciences, University of Milan, Via Celoria, 26 – 20133 Milano, Italy 4. Dept of Biology and CoNISMa LRU, University of Bari “Aldo Moro”, Via Orabona, 4 – 70125 Bari, Italy 5. Dept of Biology, University of Padova, Via U. Bassi, 58/B – 35131, Padova, Italy 6. Natural History Museum of Venice, Santa Croce, 1730 - 30135 Venezia, Italy
Based on nuclear and mitochondrial genes, in localities, always in sympatry with clade A. Se- 2012 Bock et al. demonstrated the existence of quencing of the whole mitochondrial genome of five highly divergent clades (A-E), correspond- clade E showed no gene order differences com- ing to morphologically-indistinguishable cryp- pared to clade A. Remarkably, intra-species and tic species, in the cosmopolitan model ascidian congeneric comparisons between public ascidian Botryllus schlosseri (Pallas, 1766). While clade A is mitogenomes revealed that the clade A-clade E globally widespread and its nuclear genome was divergence is incompatible with intra-species already sequenced, clade E was so far identified divergences but very similar to congeneric dis- only in European waters, both Atlantic and Med- tances. Moreover, the clade A-clade E divergence iterranean. Here we present combined molecular is almost identical to that observed in the con- and morphological data definitely demonstrat- generic pairs Ciona intestinalis-Ciona robusta, and ing that clade E is a new species, with unique Botrylloides niger-Botrylloides leachii i.e., in species characters distinguishing it from B. schlosseri sen- considered for long time morphologically indis- su Brunetti et al. 2017 (belonging to clade A) and tinguishable and recently reconsidered as true other botryllids. Samplings in the Venice Lagoon species thanks to genetic data. These molecular and in Southern Italy were carried out so that results were supported by the identification of molecular and morphological analyses were fea- morphological discriminant traits. The descrip- sible on the same specimens. Based on cox1 anal- tion of a new species is therefore presented and yses, clade E was identified in all investigated discussed.
References 1. Bock DG, MacIsaac HJ, Cristescu ME (2012) Multilocus genetic analyses differentiate between widespread and spatially restricted cryptic species in a model ascidian. Proc Royal Soc B 279:2377- 85 2. Brunetti R, Manni L, Mastrototaro F, Gissi C, Gasparini F (2017) Fixation, description and DNA barcode of a neotype for Botryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea). Zootaxa. 4353: 29-50 137 P53 VENTRAL PERIPHERAL NERVOUS SYSTEM FORMATION IN ASCIDIANS AND AMPHIOXUS: INSIGHTS INTO ITS ORIGIN AND EVOLUTION IN CHORDATES
Yann Le Petillon1*, Rafath Chowdhury*, and Sébastien Darras
1. Sorbonne Universités/CNRS, UMR 7232, BIOM, Observatoire Océanologique de Banyuls/Mer, France
Invertebrate chordates, such as ascidians and oxus Branchiostoma lanceolatum and the ascidian amphioxus, possess a peripheral nervous system Phallusia mammillata. To do so, we have done a (PNS) while they are lacking neurogenic plac- differential gene expression analysis by RNA- odes and neural crest. These dorsal structures seq, on both species, after disruption of two are known to be vertebrate-specific and lead to signaling pathways known to be implicated in the formation of vertebrates PNS. In ascidians vPNS formation: BMP and Delta/Notch1,2. Anal- and amphioxus, the PNS is mostly ventral1,2. This ysis of these large datasets allowed us to identify ventral peripheral nervous system (vPNS) likely vPNS candidate genes, for whom expression pro- correspond to an ancient feature present in the files will be determined by in situ hybridization, ancestral chordate that may have been lost in throughout development, in order to validate vertebrates or shifted dorsally to form placodes them as vPNS markers. Alternatively, we have and neural crest. look at the conservation of the regulatory logic controlling vPNS formation between ascidians To test these hypotheses and better understand and amphioxus. Altogether, these are first steps the evolution of PNS formation in chordates, our to reconstruct a vPNS gene regulatory network project aims at performing a side-by-side func- (GRN) for each organism but also for the ances- tional comparative analysis of the vPNS forma- tral chordate. tion in invertebrate chordates, using the amphi-
References 1. Waki, K., Imai, K. S. & Satou, Y. Genetic pathways for differentiation of the peripheral nervous system in ascidians. Nature Communications 6, 8719 (2015). 2. Lu, T.-M., Luo, Y.-J. & Yu, J.-K. BMP and Delta/Notch signaling control the development of am- phioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system. Development 139, 2020–2030 (2012). 138 P54 PATTERNS OF DISTRIBUTION AND DIVERSITY OF ASCIDIANS (TUNICATA: ASCIDIACEA) IN THE CORAL REEFS OF THE SOUTH OF THE GULF OF MEXICO.
*Lilian A. Palomino-Alvarez*, Edlin Guerra Castro2, Rosana Rocha Moreira3 and Nuno Simoes4,
1. PhD Student, Posgrado de Ciencias el Mar y Limnología .UNAM, Unidad Académica Sisal(UAS), Faculty of Sciences, National Autonomous University of Mexico, Puerto de abrigo s/n, Sisal, CP 97356 Yucatán, Mexico. 2. Professor full time, Department of Ecology and Conservation, National School of Higher Studies, Merida Unit, National Autonomous University of Mexico, Merida, Mexico. 3. Profesor full time. Zoology Department, Universidade Federal do Parana – UFPR, CP 19020, CEP 81531-980, Curitiba, PR, Brazil, cLaboratorio Nacional de Resiliencia Costera Laboratorios Nacionales, CONACYT, Mexico City, Mexico. 4. Professor full time Multidisciplinary Unit of Teaching and Sisal Research (UMDI-SISAL), Faculty of Sciences, National Autonomous University of Mexico, Puerto de abrigo s/n, Sisal, CP 97356 Yucatán, Mexico., National Laboratory of Coastal Resilience National Laboratories, CONACYT, Mexico City, Mexico and International Chair for Coastal and Marine Studies, Harte Research Institute for Gulf of Mexico Studies, Texas A&M University–Corpus Christi, Texas, U. S. A.
The ascidians are one of the most diverse taxo- protocol and a hierarchical spatial sampling that nomic groups in the reefs of the Gulf of Mexico allows the identification of spatial scales of vari- and the Caribbean Sea. Actually, 76 species have ation. A total of 930 quadrants have been made, been registered in 5 studies only in Gulf of Méxi- 28 sampling sites in 5 reefs of two reef systems of co. However, the patterns of distribution and di- the Gulf of Mexico. 52 species of ascidians corre- versity of species in these regions have not been sponding to 12 families and 21 genera have been described, using standardized methodologies identified. In the Gulf of Mexico, the diversity of that allow postulating theories about the possi- ascidians decreases in the East-West direction. ble processes that sustain such a level of diver- The greatest number of species was recorded in sity. The objective of this study is to describe the the Reefs Bajos de Campeche (S = 44 n reefs = 2), diversity components of ascidian species in cor- decreasing progressively towards the coral reefs al reefs in the Gulf of Mexico and the Mexican of the Veracruz Reef System, 13 species were re- Caribbean Sea through a standardized sampling corded (n reefs = 3).
References 1. Fraschetti S, Terlizzi A, Benedetti-Cecchi L. 2005. Patterns of distribution of marine assemblages from rocky shores: evidence of relevant scales of variation. Mar Ecol Prog Ser 296:13 2. Guerra-Castro EJ, Conde JE, Cruz-Motta JJ. 2016. Scales of spatial variation in tropical benthic assemblages and their ecological relevance: epibionts on Caribbean mangrove roots as a model system. Mar Ecol Prog Ser 548:97-110 doi:https://doi.org/10.3354/meps11693 3. Legendre, P., Borcard, D. & Peres-Neto, P.R., 2005. Analysing beta diversity: partitioning the spa- tial variation of community composition data. Ecological Monographs, 75(4), pp.435–450. 4. Levin SA. (1992). The problem of pattern and scale in ecology. Ecology 73(6): 1943- 1967. 5. Wulff J. 2017. Bottom-up and top-down controls on coral reef sponges: disentangling within-hab- itat and between-habitat processes. Ecology, 98, 1130-1139. 139 P55 ENHANCER ACTIVITIES OF AMPHIOXUS BRACHYURY GENES IN THE ASCIDIAN CIONA EMBRYOS
Hitoshi Tominaga1, 2, 3, Noriyuki Satoh3, Naoto Ueno1, 2 and ,Hiroki Takahashi1, 2*
1. National Institute for Basic Biology, Japan 2. Graduate University for Advanced Studies, Japan 3. Okinawa Institutes of Science and Technology Graduate University, Japan
The notochord is a prominent organ of and introns of BfBra. The experiment showed chordates. A T-box transcription factor gene that (1) 5’-upstream sequences of both genes pro- Brachyury (Bra) is expressed there and play piv- mote lacZ reporter expression in muscle cells, (2) otal role in the formation. In the cephalochordate 3’-downstream sequences have enhancer activi- Branchiostoma floridae, Bra is duplicated into two ty to promote lacZ expression in notochord cells, genes (BfBra1 and BfBra2), which are expressed and (3) introns of BfBra2 and BfBra1 exhibit lacZ in somite-formation region as well. As a series of expression primarily in muscle and notochord experiments to elucidate chordate Bra enhancer cells, respectively. These results suggest shared activity, we here carried out lacZ reporter assay Bra enhancer machinery between cephalochor- of BfBra in embryos of the ascidian Ciona intesti- dates and urochordates, which was discussed nalis. Vista analyses suggest the presence of con- in relation to the mode of formation of chor- served non-coding sequences (CNSs) not only in date-specific organs in the two lineages. the 5’-upstream but also in the 3’-downstream
References 1. Tominaga H, Satoh N, Ueno N, Takahashi H. (2018). genesis. 56: e23240. 140 P56 QUANTITATIVE IN VIVO IMAGING TO EXPLORE ASCIDIAN EMBRYONIC DEVELOPMENT VARIABILITY.
Laussu Julien,* 1, Leggio Bruno2, Michelin Gaël3, Guignard Léo4, Fiuza Ulla-Maj5, Faure Emmanuel6, Godin Christophe2, Malandain Gregoire3, Lemaire Patrick1.
1. CRBM, Université de Montpellier, CNRS, France 2. Mosaic Inria team, Inria Rhône-Alpes and RDP Research Unit Lyon, France 3. Université Côte d’Azur, Inria, CNRS, I3S, France 4. Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix drive, Ashburn, VA, USA 5. EMBL, Cell Biology and Biophysics Unit, Meyerhofstrasse 1, 69117, Heidelberg, Germany 6. LIRMM, Université de Montpellier, CNRS, France.
First described more than a century ago, as- light-sheet datasets and for the extraction of geo- cidian embryonic cleavage has a stereotypical metric and cell division parameters. pattern. Development however allows for some In this presentation, we will introduce a recent variability, which we need to quantify in space overhaul and extension of ASTEC, focusing on and time as a prerequisite to the study of the two new modules that decrease imaging quality mechanisms constraining ascidian development. requirements, and a refactoring effort to reduce With less than 3000 cells in the hatched larva, the the time of the segmentation. We will also pres- solitary ascidian embryo is an ideal system to sys- ent how Morphonet, has been adapted to manu- tematically investigate individual cell properties ally curate the few remaining segmentation and such as cell shape, cell position, cell division and tracking errors made by ASTEC. This enhanced cell/cell contacts throughout embryogenesis. We set to tools allowed us to carry out a deeper sta- have started to paint a picture of the intra- and tistical analysis of embryonic variability based inter-species variability of embryogenesis using on a set of ten high-quality fully-segmented and live single-cell high-throughput 4D embryo im- tracked WT Phalllusia embryos, which also ne- aging (MuViSPIM technology) (1). cessitated the development of methods to spa- A central part of this research program was the tially and temporally align independently im- development of ASTEC (Adaptative Segmen- aged digital embryos .They also open the way to tation and Tracking of Embryonic Cells) (1), a the comparison of the embryogenesis of the em- high-throughput computational pipeline for the bryos of different ascidian species, in particular digitalization (systematic whole-cell segmenta- Phallusia mammillata and Ascidiella aspersa. tion and tracking) of massive high-throughput
References 1. Guignard, L. et al. Contact-dependent cell communications drive morphological invariance during ascidian embryogenesis. (2017). doi:10.1101/238741 141 P57 ROLE OF PUMILIO DURING GERM CELL FORMATION IN COLONIAL ASCIDIAN, BOTRYLLUS PRIMIGENUS
Takeshi Sunanaga
Laboratory of Cellular and Molecular Biotechnology, Faculty of Science and Technology, Kochi University, Japan
Colonial ascidians generate new individuals venile oocytes in the ovary. Oogonia and primor- from somatic tissues asexually. In breeding sea- dial testis expressed it weakly. No signals were son, each of the asexual individuals produces emitted from germline stem cells and well-de- hermaphrodite gonads. In order to examine how veloped gonad. On the other hand, some of the germ cells differentiate in those animals, we pro- somatic tissues in developing buds expressed Bp- ceed with the isolation of the genes involved in Pum. To identify the target mRNA of PUM-HD the germ cell differentiation. In this study, we in BpPum, we screened the 3’-UTR sequences of focused on PUF protein Pumilio. We examined the expected germline genes. The 3’-UTR of Vasa the structure, expression, and possible function (BpVas) mRNA contained NRE-like sequence. In of a Botryllus primigenus Pumilio gene (BpPum) vitro analyses demonstrated that recombinant during gametogenesis. The deduced amino acid PUM-HD protein directly bound to the NRE-like sequence of BpPum contained PUM Homology sequence and that recombinant BpPum protein Domain (PUM-HD) in the C-terminal region. It bound to recombinant Nanos (BpNos) protein. has been established that PUM-HD functions as When BpPum was knocked down using siRNAs, RNA-binding domain. In B. primigenus, the gam- the amount of BpVas protein was increased in the etes and gonadal primordia originate from cell oocytes. These results suggest that BpPum fine- aggregates of germline precursor. BpPum was tunes the protein expression level of BpVas by expressed strongly in the germline cells such ju- inactivating translation during gametogenesis.
142 P58 TRANSCRIPTIONAL PROFILING OF NEUROGENIN TARGETS IN MIGRATING BIPOLAR TAIL NEURON PRECURSORS
Susanne Gibboney*1, Kwantae Kim1, Sara Shoushtarian1, and Alberto Stolfi1
1. School of Biological Sciences, Georgia Institute of Technology, Atlanta, USA * Presenting author: [email protected]
The Bipolar Tail Neurons (BTNs) are two bilat- and differentiate as an expanded chain of cells. erally symmetric pairs of neurons that delaminate Thus, Neurogenin is a key regulator of BTN mor- from the dorsal midline of the tail tip ectoderm phogenesis, including processes that specifically and migrate anteriorly along the developing tail interest us like delamination, migration, polarity muscle cells until they reach a position in the inversion, and axon extension. We have used flu- middle of the tail. During this process, they first orescence-activated cell sorting (FACS) to isolate extend an anterior, or proximal process, then in- BTN precursors from wildtype embryos as well vert their anterior-posterior polarity and extend as embryos subjected to Neurogenin overexpres- a posterior, or distal process. These processes are sion or expression of a dominant repressor form subdivisions of a single axon, and this bipolar of Neurogenin (Neurogenin::WRPW). RNAseq subdivision allows the BTNs to form a synap- of sorted cell populations from these three con- tic relay between epidermal neurons of the tail ditions was used to identify putative targets of and the central nervous system (Motor Ganglion Neurogenin in the BTNs. Top candidates have and Brain). We have previously shown that the been validated by in situ hybridization and we bHLH factor Neurogenin is necessary and suffi- have begun to test the localization and function cient to specify BTNs from the tail tip ectodermal of potentially interesting effectors of BTN devel- midline, and ectopic BTN precursors all migrate opment.
143 P59 QUANTITATIVE ANALYSIS OF THE ERK PATHWAY DURING EMBRYOGENESIS
Kilian Biasuz1, Julien Laussu1, Bruno Leggio1,2,3, Panajot Kristofori1, Emmanuel Faure1,2,3,4, Leo Guignard1,2,5, Patrick Lemaire1,3
1. CRBM, Université de Montpellier, France 2. Inria project-team Virtual Plants, CIRAD, INRA, Université de Montpellier, France 3. Institut de Biologie Computationnelle, IBC, Université de Montpellier, France 4. IRIT, CNRS, INPT, ENSEEIHT, Universités de Toulouse I et III, France 5. Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix drive, Ashburn, VA, USA
Successful embryogenesis requires the dif- Studies of the vertebrate Ras/ERK pathway are ferentiation of the correct cell types, in defined complexified by the presence of multiple paral- numbers and in appropriate positions. In most ogues for each component, with slightly diver- cases, decisions taken by individual cells are in- gent activities. During my PhD project, I study it structed by signals emitted by their neighbors. in ascidians that have single FGFR, SOS, Ras, Raf, A surprisingly small number of signaling path- MEK and ERK genes and make extensive use of ways act repeatedly in widely different cellular the FGF/Ras/ERK pathway to pattern their ear- contexts to produce diverse output. The Ras/ ly embryos. The embryos of the ascidian Phal- ERK (Extracellular Regulated Kinase) pathway is lusia mammillata have two additional favorable one of these important embryonic signaling av- properties: they are fully transparent and easy enues for which the cascade of activations from to image and they develop with invariant cell the transmembrane receptor to the ultimately lineages, providing a rigid framework allowing phosphorylated ERK nuclear targets has been single cell analysis. well described over the years. Thus, to address this issue, I have started to However, our current knowledge of this path- quantify in real time the dynamics of activation way is mostly static and we lack an integrated of ERK, the most downstream component of the understanding of its spatio-temporal dynamics. cascade, using a live sensor. To study the dy- Indeed, recent studies reveal additional levels of namics and robustness of the pathway output, I complexity showing that, although the core com- now want to combine these measurements with ponents of this pathway are the same, depending spatio-temporal optogenetic modulations of on the cellular context they can specify different upstream components of the cascade including cell fates in function of their dynamic features the FGF receptor (opto-FGFR1) and SOS protein such as amplitude, duration or frequency of acti- (opto-SOS) and ultimately computational model- vation. The emergence of such qualitatively and ling. My project benefits of our MuViSPIM light- quantitatively diverse outputs from a relatively sheet microscope and our unique ability devel- simple pathway is a fascinating basic biology is- oped over the past years to identify/segment/ sue. track individual cells during development.
144 P60 ORAL CONTRIBUTIONS FROM BOTH NEURAL AND ANTERIOR PLACODAL ECTODERM IN CIONA ROBUSTA.
Florian Razy-Krajka, Susanne Gibboney and Alberto Stolfi
1. School of Biological Sciences, Georgia Institute of Technology, Atlanta, USA
In the tunicate model Ciona robusta, a neural OSP-derived cells and the endoderm derived pe- plate consisting of six rows of cells gives rise to ripharyngeal band. We name these peri-oral cells the central nervous system (CNS), the oral si- the “Kano cells”, after Shungo Kano who first phon placode (OSP), and the adhesive papillae. described a folded structure protruding from the Despite the interest fueled by their comparisons CNS in the Ciona larva. Even though the func- with the head development in vertebrates, cur- tion(s) and cell type(s) of the Kano cells remain to rent cell fate descriptions of the most anterior be identified, their morphology evokes connec- rows (IV-VI) of the neural plate remain partial tive tissue rather than a neural fate, hinting at an and contradictory. ectomesenchyme-like cell population. Here, we document the fate of neural (row IV) Using cell fate conversion induced by misex- and placodal ectoderm (row V and VI) from the pression of transcriptions factors and signaling early embryo through metamorphosis to the ju- molecules, we are now investigating the roles of venile stage using several fluorescent reporters the Kano cells and other cell populations origi- expressed in distinct cell populations. nating from the anterior neural plate in Ciona. We show that a subpopulation of cells de- Altogether, our results lead us to compare the scending from row IV separates from the CNS interactions between the Kano cells and the OSP and surrounds the OSP, which derives from a in tunicates to those between the extreme anteri- combination of cells from rows IV and V. During or domain and adjacent neural crest cells during metamorphosis, these cells continue proliferat- primary mouth morphogenesis in vertebrates. ing to form a layer of tissue fully detached from the CNS and positioned between the more rostral
145 P61 ANISEED 2019 : A REBUILT VERSION WITH NEW SPECIES, UPDATED ANNOTATIONS, AND NEW DATA
Justine Dardaillon1, Delphine Dauga2, Alexandra Louis3, Matija Brozovic1, Paul Simion4, Céline Scornavacca4, Frédéric Delsuc4, Emmanuel J. P. Douzery4, Hugues Roest Crollius3, Christelle Dantec1#, Patrick Lemaire1#
1. Centre de Recherche en Biologie Cellulaire, CNRS-U. Montpellier, Montpellier, France 2. Bioself Communication, Marseille, France 3. DYOGEN, IBENS, Ecole Normale Supérieure, CNRS, Inserm, PSL Research University Paris, France 4. ISEM, CNRS-IRD-EPHE-U. Montpellier, Montpellier, France
ANISEED (Brozovic et al. 2017) (Ascidian Net- Moreover, new annotations have been added work for In Situ Expression and Embryological such as GOSlim annotation for each gene and Data) is a major database system for the genetic gene models versioning. Genome annotations control and anatomy of ascidian embryos. Since have also been added for 2 new species : Botryl- 2017, the system has been enriched in several loides leachii (styelid stolidobranchians) and Mol- aspects and its robustness improved. The ANI- gula occulta (molgulid stolidobranchians), raising SEED database, the genomic browser WashU the number of covered species to eleven. (Zhou et al. 2011) and the synteny genomicus For these eleven species and vertebrates spe- browser (Nguyen et al. 2018) were entirely re- cies, improved orthology relationships have been built, the gene model set for Ciona robusta was computed. improved by the inclusion of about 1000 NCBI models of previously missed genes. In addition, We will present the novelties of ANISEED and all gene annotations data and files were updated our strategy for the future. using the latest version of InterPro, blast similar- ity ontologies (Gene Ontology, …).
References 1. Brozovic M. et al. ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets. Nucleic Acids Res.46(D1), D718–D725 (2017). 2. Zhou X. et al. The Human Epigenome Browser at Washington University. Nature Meth. 8, 989–990 (2011). 3. Nguyen NTT. et al. Genomicus 2018: karyotype evolutionary trees and on-the-fly synteny comput- ing. Nucleic Acids Res.46(D1), D816–D822 (2017). 146 P62 COMPLETE TRACING OF SINGLE CELL TRANSCRIPTOME AND MORPHOLOGICAL DYNAMICS FROM ZYGOTE TO GASTRULA IN PHALLUSIA MAMMILLATA
Hanna Sladitschek1,2, Ulla-Maj Fiúza1, Dinko Pavlinic1, Vladimir Benes1, Lars Hufnagel1# and Pierre Neveu1#
1. European Molecular Biology Laboratory, Meyerhofstrße 1, 69117 Heidelberg, Germany 2. Department of Molecular Medicine, University of Padua School of Medicine, viale Colombo 3, 35126 Padua, Italy
In ascidians most cell fates are restricted by the cell types without prior knowledge. The analysis onset of gastrulation. Advances in microscopy, identifies 18 different cell types at 64-cell stage. image analysis and sequencing technologies are We also developed methods identifying moth- allowing to quantitatively measure cell morpho- er-daughter cell relationships for every cell di- logical and transcriptional dynamics paving the rectly from the transcriptome data and allowing ground for rendering ascidians into an in silico to infer some degree of spatial information from modelling system for developmental studies. transcriptional data. We additionally generated In this work we capture the individual cellular digital representations of developing early as- developmental trajectories during ascidian early cidian embryos allowing to extract the tempo- embryonic development (zygote to gastrula) us- rally resolved lineage trees and cell and embryo ing a combination of single-cell RNA sequencing morphological properties. We created an atlas for (scRNAseq) and digital representations of devel- whole embryo cellular gene expression profiles oping embryos derived from light-sheet micros- from zygote to gastrula (64-cells stage) where copy imaging. Using high coverage scRNAseq, gene expression pattern formation and cellular we devised a computational framework that morphology and organisation can be explored stratified single cells of individual embryos into during early ascidian development.
147 P63 SEARCHING FOR STEM CELLS BY COMPARISON OF HAEMOCYTE POPULATIONS BETWEEN TWO COLONIAL ASCIDIAN SPECIES DURING WHOLE-BODY REGENERATION
LA Matas*1, Simon Blanchoud1
1. Department of Biology, University of Fribourg, Fribourg, CH.
Colonial ascidians are the only known class of we have set out to isolate them. Developing a chordates capable of undergoing whole-body re- new intravascular micro-transfusion technique generation (WBR) throughout their adult life. In enabled us to collect pure and healthy haemo- Botryllus schlosseri and Botrylloides leachii, WBR cytes. We thus sampled the early onset of WBR starts after the removal of all adult zooids, and when the stem-like cells have been reported to develops exclusively within the remaining vas- appear in both species. culature. In ten days, fully functional adult zo- By comparing haemolymph composition in oids are regenerated from as little as 200 cells. each species, we searched for cell populations Stem cells in tunicates appear to play a role in a with common stem-like features using flow cy- myriad of biological scenarios, including regen- tometry and immunolabeling. Using FACS, eration. In B. schlosseri, cells with stem-like mor- candidate cell populations could be isolated for phology gather at the site of regeneration. In B. characterization. Cell labeling and re-injection leachii, cells expressing the conserved stemness will assess their contribution during regenera- marker Piwi have been observed in the vascula- tion and transcriptome sequencing will establish ture during the first 48h of WBR. Overall, popu- whether they express known stemness markers. lations of circulating stem-like cells are believed This novel approach is a first step towards char- to be required for WBR in colonial ascidians. acterizing the stem-like cells during regeneration To assess whether these are actual stem cells, in colonial ascidians.
148 P64 PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENE AND IDENTIFICATION OF A GLYCOSYL HYDROLASE FAMILY 6-DOMAIN GENE OF CIONA
Kun-Lung Li, Jun Inoue, Keisuke Nakashima, and Noriyuki Satoh
Okinawa Institute of Science and Technology Graduate University 1919-1 Tancha, Onna, Okinawa 904-0412, Japan
Horizontal gene transfer is one of the import- and temporal expression of the GH6-1 gene in ant force that shapes animal genomic architec- Ciona by in situ hybridization and quantitative tures and contributes to biological diversity. The PCR. The GH6-1 gene was expressed at epider- tunicate cellulose synthase gene CesA [1], a gene mis of tailbud embryos, and its expression level likely transferred from bacteria, potentiates the was increased around ten hours after fertilization structural innovation of the tunic. The tunicate (mid-tailbud stage). CesA gene contains not only a CesA domain of We are trying to use morpholino anitisense the glycosyl transferase family 2 (GT2) but also oligos or TALEN-mediated genome editing to a glycosyl hydrolase family 6 (GH6) domain [1]. knock down or knock out the GH6-1 gene. Also, During our phylogenetic analysis on CesA using we will prepare reporter constructs using the the ORTHOSCOPE tool [2], we identified a sep- genomic DNA neighboring to GH6-1 to find its arate GH6-containing gene (tentatively called driving enhancers. We may show how this likely GH6-1) in Ciona intestinalis type A. We found that horizontally transferred GH6-1 gene is retained CesA and GH6-1 homologs were present in seven in tunicate genomes. tunicate genomes but not in any other non-tuni- cate metazoans. We then examined the spatial
References 1. Nakashima, K., Yamada, L., Satou, Y., Azuma, J., and Satoh, N. (2004). The evolutionary origin of animal cellulose synthase. Dev Genes Evol 214, 81-88. 2. Inoue, J., and Satoh, N. (2019). ORTHOSCOPE: an automatic web tool for phylogenetically infer- ring bilaterian orthogroups with user-selected taxa. Mol Biol Evol 36, 621-631. 149 P65 LEATHERY SEA SQUIRT GENOME PROVIDES INSIGHTS INTO MECHANISMS OF ENVIRONMENTAL ADAPTATION AND LARVAL METAMORPHOSIS IN INVASIVE TUNICATES
Jiankai Wei, Jin Zhang, Ping Ren, Qiongxuan Lu, Xin Guo, Jing Wang, Xiang Li, Yaoguang Chang, Shuai Duan, and Bo Dong
Laboratory of Morphogenesis & Evolution, Ocean University of China, Qingdao 266003, Ch
The invertebrate sea squirt, which undergoes nome, heat shock protein 70 family and the com- a unique vertebrate-like larval stage, occupies plement system genes expanded significantly, a unique evolutionary position between inver- and cold shock protein genes were horizontally tebrates and vertebrates. It exhibits adaptation transferred into the Styela genome from bacte- to broad environmental conditions and is dis- ria. The expanded gene families potentially play tributed globally. Despite hundreds of years of roles in the adaptation of S. clava to its environ- embryogenesis studies, the genetic basis of as- ments. While the gene loss may affect the galac- cidian invasive habits remains largely unknown. tan synthesis pathway and lead to different tunic We utilized a whole genome and transcriptome structure and hardness compared to Ciona. We of leathery sea squirt, Styela clava, the first fos- demonstrated further that the integrated thyroid sil tunicate species identified from the Early hormone synthesis pathway regulates sea squirt Cambrian, to explore genomic and molecular larval metamorphosis. A thyroglobulin-like pre- network-based mechanisms of adaptation to cursor gene, the core of the thyroid hormone syn- environments. Compared to other tunicate ge- thesis system, was identified for the first time in a nomes, the size of S. clava genome expanded urochordate. The results indicated that Styela has two-fold although the gene number was com- genetic mechanisms of regulating physiological parable. Increase in transposon numbers and and developmental processes to facilitate adap- transition types were identified as potential ex- tation to the marine environment. pansion mechanisms. In the expanded Styela ge-
150 LIST OF PARTICIPANTS
Akahoshi Taichi [email protected] Keio University Villefranche sur Mer Developmental Biology Laboratory - Alie Alexandre [email protected] Sorbonne University Anselmi Chiara [email protected] University of Padova
Arnone Ina [email protected] Stazione Zoologica Anton Dohrn
Ballarin Loriano [email protected] University of Padova Villefranche sur Mer Developmental Biology Laboratory - Bekkouche Faisal [email protected] Sorbonne University Bertrand Stephanie [email protected] Observatoire Oceanologique de Banyuls sur Mer
Biasuz Kilian [email protected] CRBM UMR5237
Blanchoud Simon [email protected] University of Fribourg
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Braun Katrin [email protected] Humboldt-Universität zu Berlin Villefranche sur Mer Developmental Biology Laboratory - Brunet Manon [email protected] Sorbonne University Caballero-Mancebo Silvia [email protected] Institute of Science and Technology Austria
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