2nd CONFERENCE OF THE VISEGRÁD GROUP SOCIETY FOR DEVELOPMENTAL BIOLOGY

2 – 5 September, 2021, Szeged, Hungary

IN THE ORGANIZATION OF THE V4 SOCIETY FOR DEVELOPMENTAL BIOLOGY (V4SDB) AND THE HUNGARIAN GENETICS SOCIETY (MAGE)

ABSTRACTS ORAL AND POSTER PRESENTATIONS

ABSTRACTS | ORAL PRESENTATIONS 3

chromatin remodelling. In most animal, a characteristic feature of ABSTRACTS this change is the replacement oocyte/early embryo specific linker ORAL PRESENTATIONS histone by somatic histone H1 variants. To uncover the specific role of this alternative linker histone in early Drosophila embryogenesis, we established fly lines in which domains of embryo specific linker histone, BigH1 have been replaced partially or completely with that T1. KEYNOTE TALK 1 of somatic H1. Analysis of this mutant Drosophila lines revealed that Cytoplasmic forces functionally reorganize nuclear conden- H1 can substitute BigH1 under normal conditions, however at low sates in oocytes temperature, globular and C-terminal domains of BigH1 are nec- Marie-Hélène Verlhac 1 essary for proper development. We showed that in the presence 1 Center for Interdisciplinary Research in Biology, College de France, of BigH1, nucleosome stability increased compared to H1 however Paris, France core histone exchange during replication is more dynamic. Based on our results we propose a model explaining how BigH1 ensures fast Abstract was not received. paced replication cycles before zygotic genome activation.

T1. KEYNOTE TALK 2 T4. Understanding changing architecture of the mammalian p38-MAPK-mediated translation regulation primes blastocyst embryo development and is required for primitive endoderm differen- 1 Berenika Plusa tiation in mice 1 University of Manchester, Division of Developmental Biology and Pablo Bora 1, Lenka Gahurova 1,2, Tomáš Mašek 3, Medicine, United Kingdom Andrea Hauserova 1, David Potěšil 4, Denisa Jansova 2, Andrej Susor 2, During mammalian preimplantation development, cells reduce in Zbyněk Zdráhal 4, Anna Ajduk 5, Martin Pospíšek 3, size and the architecture of the embryo changes significantly. Such Alexander W. Bruce 1 changes are accompanied by dynamic adjustments in gene expres- 1 Laboratory of Early Mammalian Developmental Biology (LEMDB), sion and specification of the first three embryonic lineages. Two of Department of Molecular Biology & Genetics, Faculty of Science, Univer- them, the trophectoderm (TE) and the primitive endoderm (PrE) sity of South Bohemia, Branišovská 31, 37005 České Budějovice, Czech will give rise to extraembryonic tissues such as placenta and part Republic; 2 Laboratory of Biochemistry and Molecular Biology of Germ of the yolk sac respectively. At the same time, a group of cells, the Cells, Institute of Animal Physiology and Genetics, CAS, Rumburská epiblast (Epi), needs to be protected from the differentiation signals 89, 27721 Liběchov, Czech Republic; 3 Laboratory of RNA Biochemistry, to assure development of the foetus. Dynamic crosstalk between Department of Genetics and Microbiology, Faculty of Science, Charles the gene regulatory networks and the evolving architecture of the University, Viničná 5, 12844 Prague 2, Czech Republic; 4 Central Euro- mammalian embryo are notoriously difficult to study and currently, pean Institute of Technology, Masaryk University, 62500 Brno, Czech no easily accessible open-source tool exists that would allow for Republic; 5 Department of Embryology, Institute of Developmental Biology large-scale, quantitative assessment of the changes in the cell and Biomedical Sciences, Faculty of Biology, University of Warsaw, Miec- microenvironment. With this in mind, we developed the quantifi- znikowa 1, 02-096, Warsaw, Poland cation pipeline IVEN (Internal Versus External Neighbourhood), an Keywords: mouse blastocyst, primitive endoderm differentiation, open-source and user-friendly software that can be used in con- regulation of translation junction with existing open-source image analysis software like ImageJ, as well as commercially available programs like IMARIS. Successful specification of the two mouse blastocyst inner cell mass With the use of IVEN, we studied the changes in cell neighbour- (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a pre- hood architecture that accompany self-organisation and lineage requisite for continued development and requires active fibroblast formation in the mammalian embryo and revealed how modulation growth factor 4 (FGF4) signaling. Previously, we identified a role of signalling pathways can alter embryo geometry. for p38-mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained T3. unresolved. Here, we report an early blastocyst window of p38- Is bigger also better? Functional comparison of somatic and MAPK activity that is required to regulate ribosome-related gene larger, embryo specific linker histone during early Drosophila expression, rRNA precursor processing, polysome formation and embryogenesis. protein translation. We show that p38-MAPK inhibition-induced László Henn 1, 2, Anikó Szabó 1,3, Balázs Vedelek 3, Imre M. Boros 1, 3 PrE phenotypes can be partially rescued by activating the transla- 1 Institute of Biochemistry, Biological Research Centre, Szeged, Hungary; tional regulator mTOR. However, similar PrE phenotypes associated 2 Institute of Genetics, Biological Research Centre, Szeged, Hungary; with extracellular signal-regulated kinase (ERK) pathway inhibition 3 Department of Biochemistry and Molecular Biology, University of targeting active FGF4 signalling are not affected by mTOR acti- Szeged, Hungary vation. Moreover, we identify and verify translation related can- didate p38-MAPK effectors/ substrates and confirm their role in Keyworlds: Chromatin, linker histone, embryogenesis blastocyst maturation. These data indicate a specific role for p38- The earliest steps of animal development are driven by maternally MAPKs in providing a permissive translational environment during deposited gene products since transcription program is still inac- mouse blastocyst PrE differentiation that is distinct from classically tive. The zygotic genome activation is accompanied by a general reported FGF4-based mechanisms. 4 ABSTRACTS | ORAL PRESENTATIONS

T5. and cell fate of rabbit isolated ICMs. Our study points to significant The detachment of the blastoderm-vitelline envelope interac- inter-species differences, most notably en extended period of ICM tion and blastoderm chirality potency to differentiate into TE lineage. Giulia Serafini 1, Marina Cuenca 1, Pavel Tomančak 1,2 We first identify GATA3 as an early marker of rabbit TE and CDX2 1 Max Planck Institute of Molecular Cell Biology and Genetics – as a marker of more mature TE, as CDX2 expression is initiated at MPI-CBG, Dresden, Germany; 2 Central European Institute of Technology mid-blastocyst stage. We then analyse developmental potential – CEITEC, Brno, Czech Republic of rabbit ICMs isolated by immunosurgery and subsequently cul- tured in vitro. ICMs originating from early- to mid-blastocyst stage Keywords: gastrulation, attachment, embryonic chirality embryos are able to re-form a blastocyst-like structure, with a func- Gastrulation is a complex and well-coordinated process that, tional TE, and an ICM containing both SOX2-positive epiblast cells through a precise combination of tissue rearrangements and cel- and SOX17-positive primitive endoderm cells. We further observe lular migrations, leads to the segregation of the germ layers in the that rabbit ICMs isolated form later blastocyst stages lose the ability developing embryo. Its principal issue is movement: both single cells for TE specification. Instead, these ICMs form a halo-like cavity with and tissues change dramatically their relative positions over time, an outer layer of SOX17-positive cells, indicating that the potential being strongly influenced by the interaction with their surround- for TE differentiation gives way to formation of a different type of ings. Of particular interest to us is the role played by the vitelline epithelial extraembryonic layer. envelope, the innermost layer of the eggshell. Our group recently described that integrin-mediated attachment of the blastoderm T7. to the vitelline envelope is required for the proper gastrulation of The role of activin A in the regulation of preimplantation both Drosophila melanogaster and Tribolium castaneum. The dis- development of mouse embryo ruption of the attachment dramatically alters gastrulation in both Eliza Winek 1,2, Katarzyna Szczepańska 1, Aneta Suwińska 1 organisms and in Drosophila the defect is scoreable as a twisted 1 Department of Embryology, Institute of Developmental Biology and Bio- gastrulation (TG) phenotype. Exploiting this, we searched for addi- medical Research, Faculty of Biology, University of Warsaw, Warsaw, tional molecular players involved in the attachment besides integ- Poland; 2 Doctoral School of Exact and Natural Sciences, Warsaw, Poland rins. Surprisingly, when quantifying the TG phenotype, we detected Keywords: activin A, mouse, blastocyst, cell lineages a bias in the handedness of the twist. This led us to hypothesize that the attachment could have the role of keeping the embryo Activin A, encoded by InhbA gene, is a protein belonging to the symmetric, preventing the germ band from following the intrinsic transforming growth factor β (TGFβ) family, which is known for its chirality of the tissue during germ band extension. Since the ear- involvement in the patterning of the embryo after implantation. liest developmental process showing chirality is gut formation, our Although the expression of activin A is also detected in the preim- results suggest that the left/right asymmetry could be established plantation stages of mouse (and human) embryo development, its much earlier, during gastrulation. Moreover, the deletion screening role in this period of embryogenesis remains undiscovered. on mutants showing the TG phenotype provided a few interesting We aimed to determine whether activin A pathway is engaged candidate genes, which will allow us to look at left/right asymme- in the commitment of primary cell lineages in the mouse pre- try establishment in Drosophila embryo from a novel and surprising implantation embryo. To this end, we analyzed development of point of view. activin A-deficient mouse embryos InhbA( -/-) from the zygote to the blastocyst stage using time-lapse microscopy and compared them T6. with the stage-matched heterozygotes and wild-type embryos. We Rabbit inner cell mass retains potential to differentiate into the revealed that the absence of zygotic activin A did not affect the rate -/- trophectoderm lineage of development of InhbA embryos (pronuclear envelope break- Katarzyna Filimonow 1, Anna Chołoniewska 1, Jan Chołoniewski 2, down, cleavages, compaction, cavitation, hatching, duration of cell Zofia Eliza Madeja3 , Katarzyna Barłowska 1, Elżbieta Wenta- cycles and synchrony of cleavage rounds). However, we observed Muchalska 1, Berenika Plusa 4, Anna Piliszek 1 that the absence of zygotic activin A disrupts the correct propor- 1 Department of Experimental Embryology, Institute of Genetics and Ani- tion of cells within the inner cell mass (ICM), increases the partici- mal Biotechnology of the Polish Academy of Sciences, Jastrzebiec, Poland; pation of epiblast (EPI) at the expense of primitive endoderm (PE) 2 Center of Excellence for Complex Systems Research, Faculty of Physics, cells. Thus, our preliminary results indicate that activin A may be Warsaw University of Technology, Warsaw, Poland; 3 Faculty of Veter- engaged in establishment of the final EPI:PE ratio during preimplan- inary Medicine and Animal Sciences, Poznan University of Life Sciences, tation period of mouse development. -/- Poznan, Poland; 4 Division of Developmental Biology & Medicine, Univer- Since InhbA embryos were able to reach the blastocyst stage, our sity of Manchester, Manchester, United Kingdom next goal is to verify whether maternal activin A can partially com- pensate for the loss of zygotic gene function. Keywords: lineage specification, trophectoderm, rabbit Acknowledgement: The project is financed by the grant OPUS 17 In the course of mammalian development initial state of totipo- 2019/33/B/NZ3/02906 from the National Science Centre (Poland). tency has to be lost to allow acquisition of a specific cell fate. The first differentiation event results in the formation of trophectoderm (TE) and the inner cell mass (ICM). In the mouse embryo the cell fate of these two compartments is set quickly after formation of a blastocyst. Here we present a detailed analysis of development ABSTRACTS | ORAL PRESENTATIONS 5

T8. KEYNOTE TALK 3 T10. Keynote lecture: Aberrant activation of developmental pro- Unique stem cell subpopulation which ensures mesenchymal grams in adult disease regeneration of continuously growing teeth contributes to Ángela Nieto various tissue organogenesis Instituto de Neurociencias (CSIC-UMH), Alicante, Spain Jan Krivanek 1, Josef Lavicky 1, Petr Taus 2, Marcos Gonzalez Lopez 1, Vladislav Rakultsev 1, Tereza Kurucova 2, Aneta Anna Dunajova 1, Epithelial homeostasis is crucial to maintain tissue architecture, and Marcela Buchtova 3,4, Marie Sulcova 3,4 therefore, it needs to be tightly regulated in the adult. By contrast, 1 Department of Histology and Embryology, Faculty of Medicine, Masaryk embryonic cells show a high degree of epithelial plasticity required University, Brno, Czech Republic; 2 Central European Institute of Technol- for proper morphogenesis and, in particular, for the implementation ogy, Masaryk University, Brno, Czech Republic; 3 Laboratory of Molecular of massive cell movements that occur during gastrulation and neu- Morphogenesis, Institute of Animal Physiology and Genetics, Czech Acad- ral crest delamination among other processes. We have been inter- emy of Science, Brno, Czech Republic; 4 Department of Experimental Biol- ested in the analysis of cell movements, plasticity and epithelial to ogy, Faculty of Science, Masaryk University, Brno, Czech Republic mesenchymal transitions (EMT) for many years, and found that the aberrant activation of developmental EMT-like programs in adult Keywords: Stem cells, development, odontogenesis, regeneration cells leads to several pathologies including tumor progression and In rodents and several other species, a continuously growing teeth organ degeneration. While the epithelial and mesenchymal cells are have evolved. This adaptation on specialized style of living ensures usually considered as extreme phenotypes, intermediate EMT states permanent replenishing of dental tissues worn by constant gnawing also exist. Under those circumstances cells depict a hybrid pheno- and provides an attractive system for studying of stem cell niche, type expressing both epithelial and mesenchymal markers and from cell differentiation or injury-induced regeneration. Recent advances which they can reverse to the original state or move towards a more in single cells RNA sequencing and lineage tracing methods enabled mesenchymal phenotype. Hybrid transitory states can favor coordi- to perform an unbiased and reliable analysis of this organ and to nated cell migration or wound healing but they can also enable the study different stem cell populations responsible for permanent formation of clusters of migratory cancer cells with increased met- growth. astatic potential. However, in contrast to the situation in cancer, the Using this approach, we found a novel, quiescent and long-lasting intermediate phenotype holds promise for new antifibrotic ther- population of mesenchymal stem cells contributing to the perma- apeutic approaches, as inhibiting EMT can attenuate established nent tooth growth. This, up to now unknown, stem cell population fibrosis. I will discuss different scenarios in which this intermediate is spatially restricted and gives rise to all mesenchymal parts of phenotype is observed both in development and in disease. dental pulp, including different types of dental pulp cells and den- tin-producing odontoblasts. Further analyses showed a multipotent T9. characteristic of this unique population and uncovered molecular Pharyngeal component in the premandibular segment of the background responsible for differentiation (bifurcations) into dis- vertebrate head tinct terminally differentiated cell states. Based on our detailed Horackova, A., Stundl, J., Minarik, M., Fabian, P. & Cerny, R. study of this exemplary model system on single cell level we Dept. Zoology, Charles University in Prague uncovered the role of the same type of stem cell population during The segmental formation of the pharynx represents a fundamental embryonic development of several organ systems. part of the metameric organization of the vertebrate head and face. Taken together we discovered a novel, highly specific mesenchymal Pharyngeal pouches maintain populations of the craniofacial mes- stem cells which plays role during permanent adult tissue growth enchyme separate, and signalling from the endodermal pouches is and contribute to formation of different organs during development. essential for induction and pattern of serially arranged cranial skel- etal elements. Segmentation of the vertebrate head is thus orches- T11. trated around pouching of the primitive gut cavity, which forward Developmental anoikis: A novel, protective safeguarding expansion is thought to be limited by the oral (stomodeal) invag- mechanism in the embryonic neocortex ination during the mouth formation. Recently, we ascertained the Zsolt Lele 1*, Zsófia I. László1* , Fruzsina Mógor 1, Dániel Nagy 1, existence of the preoral gut (POG), the rostral-most foregut pouch Dárius Leszkó 1, Susanne Prokop 1, Gabriel M. Simon 2, Ken Mackie 3, located dorsoanterior to the forming mouth, and thus extending to Benjamin F. Cravatt 2 and István Katona 1,3 the pre mandibular segment. POG represents a classical, yet forgot- *=contributed equally ten textbook embryonic domain, and we argue that it presents a 1 Institute of Experimental Medicine, Hungarian Academy of Science, Buda- deep deuterostome heritage of the pan-vertebrate pharynx. Devel- pest, Hungary; 2 Department of Chemical Physiology, The Scripps Research opmental and evolutionary significance of the pharyngeal pouch- Institute, La Jolla, California, USA; 3 Gill Institute and Department of like component in the rostral-most, premandibular head segment Psychological and Brain Sciences, Indiana University, Bloomington, Indi- will be discussed in the context of the segmental nature of the ver- ana, USA tebrate head. Proliferating progenitor cells in higher organisms are generally located in designated stem cell niches where they are immobilized via cell-to-cell and cell-to-ECM connections. In the embryonic cor- tex the ventricular zone (VZ) is such a neurogenic niche occupied mostly by modified neuroepithelial cells called radial glia progen- 6 ABSTRACTS | ORAL PRESENTATIONS

itor cells (RGPCs). Asymmetric division of RGPCs produce a radial T13. glia cell and an intermediate progenitor cell (IPCs) or fate-comit- Shedding light on the details of hemocyte transdifferentia- ted neuroblast which migrate radially to their proper destination. tion in Drosophila melanogaster with an artificial intelligence To escape the VZ, these cells need to loose their cadherin-based based technology adherens junctions to neighbor cells at the apical, and laminin-inte- Erika Gábor 1, Aliz Géczi 1, Ede Migh 2, Attila Beleon 2, grin based cell-ECM connections at the pial surface. This process is Péter Horváth 2, Bence Széplaki 3, Márton Enyedi 3, Lajos Pintér 3, largely reminiscent of the classic epithelial-mesenchymal transition Lajos Haracska 3, Viktor Honti 1 (EMT). EMT is essential during certain steps of development (gas- 1 Drosophila Blood Cell Differentiation Group, Genetic Institute, ELKH, trulation, neural crest) but can be a harmful process in the adult Szeged, Hungary; 2 Laboratory of Mutagenesis and Carcinogenesis organism (fibrosis, metastasis). To prevent unwanted migration Research, Genetic Institute, ELKH, Szeged, Hungary; 3 Laboratory of of cells in adult epithelial tissues, loss of cell to ECM connections Microscopic Image Analysis and Machine Learning, Genetic Institute, usually evokes a specific apoptotic process called anoikis. In this ELKH, Szeged, Hungary talk I’ll present data from a project in which we sought to establish whether there is a similar protective cell death process in the devel- Keywords: Drosophila, hemocyte, differentiation, transdifferentia- oping brain and share some details about the molecular machinery tion, immunity behind this phenomenon. Organisms are highly adaptive to the ever-changing environment. To establish a fast and efficient response, reusing the already active T12. energy sources is a requirement. A good example for this phenom- Primary Cilia and Kidney Cysts Development in Nek8-deficient enon is transdifferentiation, during which a functional cell further Mice differentiates into a functionally distinct one without the involve- Kristekova Daniela 1,2, Buchtova Marcela 1,2 ment of stem cells. Both in vertebrates and invertebrates, blood 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physiol- cells are capable for transdifferentiation under certain conditions. ogy and Genetics, v.v.i., Czech Academy of Sciences, Brno 602 00, Czech For detailed investigation of transdifferentiation, the fruit flyDro ( - Republic; 2 Section of Animal Physiology and Immunology, Department of sophila melanogaster) is a perfect model: the vast majority of its Experimental Biology, Faculty of Science, Masaryk University, Brno 625 signaling pathways, transcription- and epigenetic factors regulating 00, Czech Republic hematopoiesis are evolutionary conserved, and its hemocytes show remarkable similarities in their function and differentiation to the Keywords: primary cilia, kidney, cyst formation vertebrate myeloid cells. Polycystic kidney diseases are a family of genetic disorders man- Previously we discovered that the phagocytic plasmatocytes ifested by renal cystic growth, resulting in kidney failure. Mouse are capable of converting into encapsulating lamellocytes upon line C57BL/6J-Nek8jck/J carries the Nek8 mutation, which in reces- immune induction (e.g. wasp infestation, wounding). The latest sin- sive homozygotes leads to juvenile cystic kidneys-like phenotype. gle-cell transcriptome data proved that plasmatocytes - which were The first small cysts, mostly located in the kidney cortex, can be believed to be homogeneous - form several functional clusters. observed at 4 weeks with typical progressive growth, which can We established a novel method to explore the plasmatocyte-lamel- be observed with the age of animals. The lifespan of the Nek8-mu- locyte transdifferentiation. In anex vivo culture, we identify and tant mice is around 20 – 25 weeks. Nek8-mutation was previously follow hemocytes by their cell-type specific transgenic fluorescent associated with longer cilia and the accumulation of polycystin-1 reporter signal and take time-lapse images, which are analyzed with and polycystin-21 in the cilia. The dysfunctional serine/threonine an artificial intelligence-based machine learning technology. kinase Nek8 leads to disruption of cystogenesis by altering the We would like to shed light on the details of transdifferentiation cilia morphology and function in the distal tubule of nephrons. In upon immune induction, as well as in tumorous mutants. Due to our study, we focus on the changes in the expression of the genes evolutionary conservation, our results can open up new fields in the associated with FGF/FGFR signalling and SHH pathway during the investigation of mammalian hematopoiesis and blood cell originated development of the cysts with the aim to reconstruct the aber- cancer research. rant processes in Nek8-mutants leading to kidney failure and their association to primary cilia function failure. Kidneys from various T14. KEYNOTE TALK 4 stages of both mutant and wild-type mice were collected, analyzed Posttranscriptional control of the germline-soma dichotomy for histopathological changes and alterations in gene expression. Rafal Ciosk Moreover, we examined the possible curative effect of FGFR inhib- Dept. of Biosciences, University of Oslo, Norway itor, Derazantinib (ARQ-087) on cyst formation in Nek8-deficient mice. In summary, our study can provide us with deeper insight into Cell fate commitment and reprogramming are fundamental for molecular regulations leading to the cyst formation caused by the development and tissue homeostasis. A profound cellular repro- disruption of primary cilia and also lead to the design of new treat- gramming occurs during the oocyte-to-embryo transition, when ments, which effect can be easily tested on the postnatal kidney specialized reproductive cells - egg and sperm - give rise to a totipo- cysts development in our model. tent embryo. The majority of reported “roadblocks” controlling this Acknowledgement: This work was supported by the Ministry of reprogramming are transcription factors and chromatin modifiers. Health (NU20-08-00205). However, we demonstrated that RNA-binding proteins (RBPs) fulfill a similar role in the oocyte cytoplasm. Using the C. elegans model, we showed that conserved RBPs function as components of a ABSTRACTS | ORAL PRESENTATIONS 7

posttranscriptional network that prevents oocytes from precocious T16. reprogramming into embryonic-like cells. In the absence of this layer Golden hamster piRNAs are necessary for zygote development of regulation, oocytes prematurely undergo embryonic-like differ- and establishment of spermatogonia entiation and form teratomas. We are interested in understanding Zuzana Loubalova 1†, Helena Fulka 1,†‡, Filip Horvat 1,2, the underlying molecular mechanisms, the connection between Josef Pasulka 1, Radek Malik 1, Michiko Hirose 3, Atsuo Ogura 3,4, cytoplasmic and nuclear processes, and the coordination between Petr Svoboda 1 cellular reprogramming and other developmental events. 1 Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic; 2 Bioinformatics Group, T15. Division of Molecular Biology, Department of Biology, Faculty of Science, Cytoplasmic RNA exosome is essential for post-implantation University of Zagreb, 10000, Zagreb, Croatia; 3 Bioresource Engineering mouse embryo development Division, RIKEN BioResource Research Center, 305-0074 Ibaraki, Japan; Michał Brouze 1, Marcin Szpila 1, Tomasz Kuliński 1, Wiktor Antczak 2, 4 Bioresource Engineering Laboratory, RIKEN Cluster for Pioneering Areta Czerwińska 4, Jakub Gruchota 1, Ewa Borsuk 1,3, Research, 351-0198 Saitama, Japan. Andrzej Dziembowski 1,2 † These authors contributed equally 1 Laboratory of RNA Biology, International Institute of Molecular and ‡ Current address: Institute of Experimental Medicine of the Czech Cell Biology, Warsaw, Poland; 2 Laboratory of RNA Biology, Institute of Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic Genetics and Biotechnology, University of Warsaw, Poland; 3 Department Keywords: piRNA, PIWI, small RNAs, Syrian hamster, retrotrans- of Developmental Biology, Institute of Developmental Biology and Bio- posons, oogenesis, spermatogenesis medical Sciences, Faculty of Biology, University of Warsaw; 4 Department of Cytology, Institute of Developmental Biology and Biomedical Sciences, PIWI-associated RNAs (piRNAs) maintain the integrity of the ger- Faculty of Biology, University of Warsaw, Poland mline genome by suppressing retrotransposon activity, and their functions in gene regulation have recently emerged. The under- Keywords: mouse development, RNA, exosome standing of the role of piRNA pathway in mammals was shaped by a RNA exosome is an evolutionary conserved ribonucleolytic complex. mouse model in which piRNAs are essential for male but not female In mammals, cytoplasmic and nuclear exosomes have a non-over- fertility. We report that the role of the piRNA pathway substan- lapping set of catalytic subunits. Nuclear physiological processes tially differs in golden hamsters, whose piRNA pathway setup more such as degradation of pervasive transcription products are medi- closely resembles that of other mammals, including humans. We ated by DIS3 and EXOSC10. The role of the cytoplasmic exosome, show that the loss of Mov10l1 helicase, an essential factor initiating equipped with DIS3L as a catalytic subunit, is less clear. piRNA biogenesis, generates novel phenotypes in hamsters. Unlike As a part of our effort to study this role, we used CRISPR/Cas9 mice, female Mov10l1–/– hamsters are sterile. Mutant fully-grown technology to create a transgenic mouse line with knock-out (KO) oocytes show minor changes in the transcriptome and moderate mutation in Dis3l. This mutation causes embryolethality between to low increase in retrotransposon transcripts. Although Mov10l1–/– day 6,5 and 7,5 of embryo development, with the ratio of KO to hamster oocytes appear largely normal and give rise to zygotes, wild-type embryos being reduced after day 4,5. At blastocyst stage they cannot support normal embryonic development. In another KO embryo growth rate is not impaired, but the development of contrast to mice, Mov10l1–/– male hamsters have impaired estab- particular cell lineages is affected as indicated by changed cell num- lishment of spermatogonia accompanied by transcriptome dysregu- ber of trophectoderm and primitive endoderm as well as the low lation and a surge of expression of a specific young retrotransposon efficiency with which KO blastocyst produce embryonic stem cells subfamily, which is the primary target of retrotransposon-derived (ES cells). Supplementation of 4-cell KO embryos with tetraploid piRNAs in early spermatogenesis. In addition, we examined groups wild-type embryos didn’t produce any offspring, pointing to the of piRNAs during different stages of spermatogenesis and demon- issue with inner cell mass as a source of embryolethality. strate that the regulation of specific retrotransposon subfamilies While sequencing of RNA from KO ES cell lines suggested downreg- is stage-dependent. Our results not only refute the notion that in ulation of 21 transcripts, RNA sequencing of single KO blastocysts mammals the piRNA pathway is important only in males but also revealed the opposite trend of 11 deregulated transcripts out of demonstrate the adaptive nature of the mammalian piRNA path- which 9 were upregulated. way, which allows to confront emerging genomic threats and Taken together, these results allow us to describe the role of DIS3L acquire new critical roles in both sexes. and cytoplasmic exosome in the development of mouse embryo and its specific cell lineages, as well as identify transcripts regulated T17. by DIS3L activity in developing embryo. The snoRNAome of zebrafish Danio( rerio) Renáta Hamar and Máté Varga Department of Genetics, ELTE Eötvös Loránd University, Budapest, Hungary Small nucleolar RNAs (snoRNAs) are one of the most abundant and evolutionary ancient group of functional non-coding RNAs. They were originally described as guides of post-transcriptional rRNA modifications, but snoRNAs fulfill an impressive variety of cellular functions. These range from guiding site-specific chemical modifications in several RNA classes and affecting the nucleolytic 8 ABSTRACTS | ORAL PRESENTATIONS

processing of ribosomal RNA (rRNA) to their involvement in the reg- Biologically, the most meaningful way to register the images is by ulation of chromatin architecture and alternative splicing. Previous identifying cellular correspondences between these two imaging analysis suggests that snoRNAs and the modifications they mediate modalities. In this way, one can bring the two sources of infor- are highly conserved across species. mation into a single domain and combine dynamic information on Based on common sequence motifs and structural features snoRNAs morphogenesis with static gene expression data. Here we propose are classified in two major families, box C/D and box H/ACA snoR- a new computational pipeline for identifying cell-to-cell correspon- NAs. The identification of snoRNAs based on homology search is dences between images from multiple modalities and for using often difficult due to the lack of overall sequence conservation, small these correspondences to register 3D images within and across size (60-300bp) and short sequence motifs. Here, we use size-frac- imaging modalities. We demonstrate this pipeline by combining tionated RNA sequencing data from adult zebrafish tissues to define four-dimensional recording of embryogenesis of Spiralian annelid the snoRNAome for this species. Our approach allowed us to identify ragworm Platynereis dumerilii with three-dimensional scans of fixed several hitherto unannotated snoRNAs in the zebrafish genome. Platynereis dumerilii embryos stained for the expression of a variety We created an analysis pipeline where the raw reads were aligned of important developmental genes. The complete pipeline is avail- to the latest zebrafish genome assembly (GRCz11) usingBowtie2 able for public use through a napari plugin. aligner and we identified putative snoRNA sequences using the blockbuster algorithm. Using several snoRNA predictor methods we T19. were able to confirm the presence of multiple previously predicted How to choose the most suitable increased fidelity SpCas9 snoRNAs in the data and also identified ~80 new snoRNA-like nuclease for high specificity genome editing sequences missing from the current Ensembl database (v99). Based Péter István Kulcsár 1,2, András Tálas 1,3, Sarah L Krausz 1,3, on our preliminary survey our snoRNAome dataset represents the Zoltán Ligeti 1,4, Vanessza L Végi 1, Eszter Tóth 1, Zsófia Rakvács1 , most reliable set of snoRNAs to date in this species. Krisztina Huszár 1, Ervin Welker 1,5 We also compared all putative, novel snoRNAs with ncRNA sets 1 Research Centre for Natural Sciences, Institute of Enzymology, Budapest, from zebrafish and other species viaRNAcentral to identify already Hungary; 2 Department of Biophysics and Radiation Biology, Semmelweis annotated zebrafish snoRNAs. We noted that four of these were University, Budapest, Hungary; 3 Semmelweis University, School of Ph.D. already included in some (non-Ensemble) databases. We also noted Studies, Budapest, Hungary; 4 University of Szeged, Szeged, Hungary; that another six of them were in zebrafish long-non-coding RNA 5 Biological Research Centre, Institute of Biochemistry, Szeged, Hungary genes and yet another six snoRNA sequences could be found in the ncRNA sets of closely related species. The propensity for off-target activity of the Streptococcus pyogenes Finally, we also present our results that show the dynamic expres- Cas9 (SpCas9) has been considerably lowered by increased fidelity 1 2 3 4 5 sion of some snoRNAs during the early stages of zebrafish devel- (high-fidelity) variants, such as e- , -HF1 , Hypa- , evo- , Sniper- , 6 7 opment and tissue-specific expression patterns for others in adults. HeF- and Blackjack variants . Although exhibiting decreased off-target propensity, they still cleave many target sequences with T18. off-targets, which are difficult to predict and have been shown to contain mismatches at even up to 6 positions. Furthermore, they Multi-modal Volumetric Image Registration by Establishing frequently show decreased on-target activities at several target Cell Correspondence sites that are otherwise cleaved by the wild-type SpCas9. Manan Lalit 1,2, Mette Handberg-Thorsager 1,2, Florian Jug 3, A systematic comparison of 20 increased fidelity nucleases revealed Pavel Tomancak 1,2,4 that they can be ranked according to their increasing fidelity and 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, their concomitant decreasing average cleavage efficiency. More Germany; 2 Center for Systems Biology, Dresden, Germany; 3 Fondatione instrestingly, we also found that target sequences can be ranked Human Technopole, Milano, Italy; 4 IT4Innovations, VŠB – Technical Uni- according to their cleavability by these nuclease variants. At one versity of Ostrava, Ostrava-Poruba, Czech Republic end of the target ranking there are easy-to-cleave sequences that Keywords: Platynereis dumerilli, multi-modal registration, instance all variants can cleave, but only the highest fidelity variant cleaves segmentation, tracking them with minimal off-target. At the other end are thehard-to- cleave sequences that are cleaved efficiently (and without off-tar- Early development of an animal from an egg involves a rapid get) only by the ‘less’ increased fidelity variants. increase in cell number and several cell fate specification events This tendency of targets makes efforts to develop a “general” nucle- accompanied by dynamic morphogenetic changes. In order to cor- ase variant that would cleave every target with both high efficiency relate the morphological changes with the genetic events, one and high specificity pointless. Building on the above knowledge, we typically needs to monitor the living system with several imaging show that apparently all targets can be edited without any detect- modalities offering different spatial and temporal resolution. Live able, genome-wide off-target by matching the right increased fidel- imaging allows monitoring the embryo at a high temporal resolu- ity nuclease with the rank of the target sequence. Furhermore we tion and observing the morphological changes. On the other hand, demonstrate a method of choosing the most suitable SpCas9 vari- confocal images of specimens fixed and stained for the expression ant when high precision editing is required. of certain genes enable observing the transcription states of an 1 Slaymaker IM et al.; Science (2016); 2 Kleinstiver BP et al.; Nature embryo at specific time points during development with high spa- (2016); 3Chen JS, Dagdas YS, Kleinstiver BP et al.; Nature (2017); tial resolution. The two imaging modalities cannot, by definition, be 4Casini A et al.; Nature Biotechnology (2018); 5Lee et al. Nature applied to the same specimen and thus, separately obtained images Communications (2018); 6 Kulcsár PI et al.; Genome Biology (2017); ofdifferent specimens need to be registered. 7 Kulcsár PI et al.; Nature Communications (2020) ABSTRACTS | ORAL PRESENTATIONS 9

T20. KEYNOTE TALK 5 T22. Whole-body connectomics and the study of motor control in The paradise fish Macropodus( opercularis) as a complemen- larval Platynereis tary model species of behavioral and developmental genetics Gáspár Jékely Erika Fodor 1, Tony Patelunas 2, Nóra Szabó 1, Zoltán K. Varga Living Systems Institute, University of Exeter, UK 3, Kata Szabó 1, Dávid Czimer 1, Anita Rácz 1, Ádám Miklósi 3, Shawn Burgess 2 and Máté Varga 1,* We are interested in how whole-body neural circuits coordinate 1Department of Genetics, ELTE Eötvös Loránd University, Budapest, Hun- the movement of an animal body. To understand this, we study gary; 2 Translational and Functional Genomics Branch, National Human the experimentally accessible and small larval stages of the marine Genome Research Institute, Bethesda, MD, USA; 3 Laboratory of Transla- annelid Platynereis. The nereid has recently emerged as a powerful tional Behavioral Neuroscience, Institute of Experimental Medicine, Eötvös new experimental system for neural circuits and development. With Loránd Research Network , Budapest Hungary; 4 Department of Ethology, serial electron microscopy, we have reconstructed the entire ner- ELTE Eötvös Loránd University, Budapest, Hungary vous and effector systems of the larva. Through activity imaging, behavioural experiments, transgenes and CRISPR manipulations we In the last half century a small number of model species – each can link genes to neurons and behaviours. In the talk I will discuss representative to a bigger taxa – have been dominating the field the coordination of locomotor cilia, the startle circuit and the pos- of biological research. In regards to fish, zebrafishDanio ( rerio) has tural control system of the larva. The nereid is an ideal system for outshined almost every other species and while it is an extremely connectome mapping and experiments and offers a fresh perspec- useful and convenient model organism, it has its own limitations. tive on how the brain coordinates an entire body. The advent of new transgenic and genome editing techniques has opened up the possibility of using nontraditional model species to T21. address specific research hypotheses and the scientific community Revealing secrets of Proteus anguinus by 3D X-ray imaging also realize the potential in using more unconventional experimental Markéta Tesařová 1, Lucia Mancini 2, Edgardo Mauri 3, organisms. Broadening the field of research would allow us to have Gregor Aljančič 4, Magdalena Năpăruş-Aljančič 4,5, Tomáš Zikmund 1, a more complex view on evolution, learn more about development Markéta Kaucká 6, Federica Papi 3, Igor Adameyko 7, Jozef Kaiser 1 and behavior and to look for better alternatives if a question cannot 1 Central European Institute of Technology, Brno University of Technology, be answered with traditional models. Czech Republic; 2 Elettra-Sincrotrone Trieste S.C.p.A., Basovizza, Trieste, Our aim is to develop a previously popular behavioral genetics Italy; 3 Speleovivarium Erwin Pichl - Adriatic Speleology Society, Trieste, organism, the paradise fish Macropodus( operularis) into a new Italy; 4 Tular Cave Laboratory, Society for Cave Biology, Kranj, Slovenia; complementary model species of developmental and behavioral 5 Research Centre of the Slovenian Academy of Sciences and Arts: Karst genetics. Recently we have established breeding protocols and Research Institute, Postojna, Slovenia; 6 Max Planck Institute for Evolution- adapted molecular methods to work with paradise fish, and assem- ary Biology, Plon, Germany; 7 Medical University of Vienna, Vienna, Austria bled and annotated the draft genome of this obligate air-breather species. We present a brief summary of our results together with Keywords: Proteus, salamander, microCT, microtomography, 3D imaging some intriguing behavioral observations that underscore the poten- The darkness of caves harbour an astonishing biodiversity. Cave tial of paradise fish for behavioral research. animals show diverse morphological and physiological adaptations which enabled them to survive in lightless and low food environ- T23. ment. In our work, we look at the endangered Proteus anguinus Blastomeres Derived from the Vegetal Pole are the Extraem- - the only European amphibian that lives exclusively in caves. Pro- bryonic Nutrition in Sturgeon Embryo: Transition from Holo- teus has been under the attention of scientists for centuries. It is blastic to Meroblastic Cleavage a top predator in the subterranean food chain, hence an excellent Mujahid Ali Shah 1, Viktoriia Iegorova 2, Roman Franěk 1, indicator of groundwater pollution. The use of contrast-enhanced Martin Pšenička 1, Taiju Saito 1,3 X-ray computed microtomographic imaging allowed the 3D seg- 1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries mentation of soft tissues in the head of both larval and adult spec- and Protection of Waters, South Bohemian Research Center of Aquacul- imens to help understanding how an amphibian adapted to harsh ture and Biodiversity of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany, conditions of caves. The synergy of rare material and life sciences 2 Czech Republic; Laboratory of Gene Expression, Institute of Biotechnol- has impacted research by proposing new tools for investigation. ogy of the Czech Academy of Sciences, Vestec, Czech Republic; Multi-disciplinary team composed of clinicians, biologists, engineers 3 South Ehime Fisheries Research Center, Ehime University, Ainan, Ehime, and imaging experts are currently pushing forward the understand- 798-4206, Japan. ing of biological questions using 3D approaches. We show cartilage of chondrocranium, the position and shape of the brain, remnant Keywords: Evolution, Fate-mapping, Vertebrates, Vegetal blasto- eyes and olfactory epithelium with possible analysis that can be mere, Yolk cells, Extraembryonic conducted on such 3D models. Above that, we compare Proteus Generally, amphibian (holoblastic) embryos in which all blastomeres anguinus with Ambystoma mexicanum as an exemplary comparison contribute to one of the germ layers are preserved as a stem lineage between cave- and surface-dwelling salamanders. Proteus angui- of vertebrates, and meroblastic cleavage has evolved independently nus has shrouded its ecology, evolution and physiology in mysteries. in each vertebrate lineage. The increasing egg size / yolk volume is Our high-resolution images and accurate segmentation processing the key factor for transition from holoblastic to meroblastic cleav- could help to reveal some of these secrets and further the research age pattern. Sturgeon (Acipenser) eggs are two times larger than on very accurate 3D microtomography data. 10 ABSTRACTS | ORAL PRESENTATIONS

Xenopus laevis (amphibian); despite varying size, sturgeon embryos T25. retain nearly same developmental characteristics as Xenopus lae- Insight into the maternal gene complement of Priapulus cau- vis. Interestingly, due to increased yolk volume, sturgeon’s vegetal datus embryos blastomeres are bigger and divide slower than animal blastomeres– Ferenc Tibor Kagan, Andreas Hejnol mimic the meroblastic cleavage pattern. Therefore, detailed under- University of Bergen standing of sturgeon cleavage pattern might reveal whether the transition of cleavage pattern (holoblastic-meroblastic) in the acti- Within Metazoa the early development of different species is partly nopterygian lineage is correlated with egg size / yolk volume. In orchestrated by maternally provided factors. The impact of their this study, we found that sturgeon vegetal blastomeres formed only regulation can be highly variable (maternal factors set up body axes primordial germ cells, and the rest of the descendants were larger in several invertebrates, but not in mammals). Their crucial role in vegetal macromeres „yolk cells (YCs).” Morphological and pheno- development has been demonstrated in several model systems. typic characteristics revealed that after the 1 k-cell / mid-blastula Ecdysozoan models such as Drosophila melanogaster and Caenor- transition, YCs became transcriptionally inactive and served only habditis elegans have been rich sources of information, but their for nutrition as larvae developed. Furthermore, inhibition of vegetal development is highly derived and therefore make it difficult to build blastomeres showed embryogenesis without producing YCs instead hypotheses of the evolutionary ancient maternal gene repertoire. formed acellular yolk mass similar to teleosts, which could suggest Priapulids are marine worms forming likely the sister group to that meroblastic cleavage in the actinopterygians, teleosts, might all remaining Ecdysozoa. Their fossil records indicate their abun- evolved by the fusion of vegetal blastomeres. dant presence in the Early Cambrian and also show a conserved body plan. Combined with a slow nuclear and mitochondrial gene T24. sequence evolution indicate that some priapulids represent a slowly evolving branch. Allele distribution modelling predicts climate change adapta- Using RNA sequencing we sampled the early development of Pri- tion in the bank vole apulus caudatus. This dataset has led to an insight into the maternal Marco Alejandro Escalante 1, Silvia Marková 1, Jeremy Searle 2 and factors in this species. We were able to identify maternal genes that Petr Kotlík 1 are also required for Drosophila melanogaster and Caenorhabditis 1 Laboratory of Molecular Ecology, Institute of Animal Physiology elegans development. Furthermore, from the PAR system used by and Genetics of the Czech Academy of Sciences, Rumburská 89, 277 21 the nematode to establish anterior-posterior axis we found only the Liběchov, Czech Republic.; 2 Department of Ecology and Evolutionary par3-par6-cdc42-apkc axis and not its antagonist axis par1-par2- Biology, Cornell University, Ithaca, NY 14853, USA. lgl1. Our analysis of the maternal gene complement shows that the Evolutionary adaptation is an important mechanism of species’ overall conservative evolution in priapulids, is also reflected on the response to climate variation whereby populations develop physio- level of maternal genes during the development. logical alterations to adjust to new conditions. Therefore, evolution- ary adaptation will likely be important for the ability of organisms T26. to cope with future climate change. This is especially relevant to the X-ray Micro computed tomography imaging based collection of species occurring on islands, with limited opportunities to respond reptile skull models for morphological analysis of tooth attach- to changing environmental conditions by changing their distribu- ment tions. Bank voles are widespread in Britain and carry two func- Michaela Kavková 1, Marie Šulcová 2, Tomáš Zikmund 1, Martin tionally distinct haemoglobin variants HbS and HbF with a striking Pyszko 4, Jozef Kaiser 1 and Marcela Buchtová 3 north-south geographic pattern thought to be driven by environ- 1 Central European Institute of Technology, Brno University of Technol- mental selection. The HbF variant confers increased erythrocyte ogy, Brno, Czech Republic; 2 Department of Experimental Biology, Faculty resistance to a free-radical attack compared to HbS and is thus of Science, Masaryk University, Brno, Czech Republic; 3 Laboratory of likely adaptive under environmental conditions favouring oxida- Molecular Morphogenesis, Institute of Animal Physiology and Genetics, tive stress. This is important, for example, because exposure dur- v.v.i., Czech Academy of Sciences, Brno, Czech Republic; 4 Department of ing early development to environmentally induced oxidative stress Anatomy, Faculty of Veterinary Medicine, University of Veterinary Sci- can constrain the offspring phenotype. We therefore modelled the ences Brno, Brno, Czech Republic current distributions of the alleles encoding HbS and HbF based on their response to environmental factors and forecasted their Keywords: microCT, 3D imaging, reptiles, tooth-bone attachment distributions under different future climate change scenarios. The The superorder of Lepidosaurian reptiles presents a large hetero- areas with the currently most favourable environmental conditions genity in the teeth morphology. The main variability is based on for HbS were predicted to be in northern Britain, while for HbF in the different tooth shape and on the type of tooth attachment the south of the island, in agreement with their present ranges. to the underlaying bone. There are at least four different types of However, under the future climate warming scenarios, it was fore- tooth-bone attachment ranging from mammalian-like thecodont casted that HbF will replace HbS throughout northern Britain, which attachment found in crocodilian to the simple acrodont implanta- indicates the species’ capacity for evolutionary adaptation to future tion occurring in some lizards. The type of tooth attachment to the global warming. jawbone is also associated with the number of tooth generations, which is another interesting feature varying in reptilian species. Here, we introduce a collection of micro CT based scans of skulls of various reptilian species. We selected model species, which possess ABSTRACTS | ORAL PRESENTATIONS 11

unique, dental features among reptiles with aim to evaluate differ- MMP inhibitors. We revealed a negative effect of MMPs inhibition ent types of tooth to bone attachment across the lepidosaurian on formation of actin ring and laminin layer resulting in wound clo- reptiles. We introduce the analysis of tooth-bone attachment in 3D sure defects. In addition, we performed detailed expression ana- view, which brings the new insights into evaluation of this area. The lyzes of mmps and healing markers at the whole embryo and single obtained collection of data will be also valuable for the research of cell levels. skull morphology in lepidosaurians and it will be suitable for the 3D print and follow up use of the 3D printed models in education. T29. This work was supported by CzechNanoLab Research Infrastruc- hiPSC derived cardiomyocytes: struggles and hopes ture supported by MEYS CR (LM2018110) and Grant Agency of the Radaszkiewicz Katarzyna, Streďanská Katarína, Kohoutková Eliška, Czech Republic grant 21-05146S. Pacherník Jiří Department of Experimental Biology, Faculty of Science, Masaryk Univer- T27. KEYNOTE TALK 6 sity, Brno, Czech Republic Newt regeneration – evolution and regulation Keywords: hiPSC, cardiomyocytes, maturation, regeneration, car- András Simon diovascular disease Karolinska Institutet, Department of Cell and Molecular Biology, Stock- holm, Sweden Cardiovascular diseases, specifically ischemic disease caused by coronary obstruction, are the leading cause of mortality and mor- The ability to regenerate lost body structures is present in diverse bidity worldwide. The inadequate blood supply to heart leads to the animal species ranging from simple organisms, such as the hydra, to loss of cardiomyocytes and subsequently to severe structural and complex vertebrates, such as salamanders. We aim to understand functional defects in the myocardium, which in turn, induces heart how animals with outstanding regenerative capabilities sense what failure. Current therapies usually can slow down the progression of and how much is missing in relation to the normal homeostatic state, the disease, however standard approaches do not solve the major and how they translate that information to the appropriate regen- issue: depletion of cardiomyocytes and its replacement by a fibrotic erative responses. We primarily study an aquatic salamander, the scar. Cardiac regeneration requires new cardiomyocytes, but heart newt, which possesses exceptional regenerative capacities among has an extremely low regenerative ability. Therefore, cardiomyo- adult vertebrates. Recent technical advance from our lab, such as cytes derived from human induced pluripotent stem cells (hiPSC) genome sequencing and in vivo editing, allow now to understand have emerged as a potential source of therapeutic cells as well as newt regeneration at the molecular level. In my presentation I will a platform for disease modelling and drug discovery. Despite the focus on mechanisms of limb regeneration with particular focus on great promise of hiPSC-derived cardiomyocytes in medicine, many the role of skeletal muscle. challenges remain to be solved. Current methods of differentiation usually generate a heterogenous mix of cardiac subtypes and non- T28. cardiac cells. Moreover, in vitro derived cardiomyocytes have an The role of matrix metalloproteinases during embryonic wound immature phenotype corresponding to foetal cardiomyocytes with healing dramatically different structural and functional properties. Due to Paulina Kikinderova, Ravindra Naraine, Pavel Abaffy, Mikael Kubista, these major differences in hiPSC-derived and native cardiomyo- Radek Sindelka cytes, the development of novel maturation protocols remains a Institute of Biotechnology, Czech Academy of Sciences, Vestec, Czech major research goal. Here we show how different approaches such Republic as long-term culture, metabolic restriction, or 3D culture affect hiP- Keywords: wound healing, gene expression, Xenopus, matrix SC-derived cardiomyocyte phenotype. metalloproteinases T30. Two types of cutaneous wound healing could be distinguished: Sertoli cell progenitors as a promising tool for muscle regen- embryonic and adult. Embryonic healing in contrast to adult is eration faster and scar-less and consists of early, middle, and late phases. Vladimír Krylov 1, Aneta Wróblová 2, Jakub Onhajzer 3, Actin ring is formed during the early phase and de novo expression Tereza Tlapáková 1 of healing specific genes is initiated also during middle phase. Our 1 Charles University, Faculty of Science, Department of Cell Biology, understanding about late phase remains elusive. Essential role in Viničná 7, Prague 2, Czech Republic; 2 Clinic for Reproductive Medicine the wound healing is given to matrix metalloproteinases (MMPs). IVF Cube, Evropská 423, Prague 6, Czech Republic; 3 Institute of Molec- These remodeling enzymes are important for releasing the cyto- ular Genetics of the Czech Academy of Sciences, Laboratory of Cell and kines, inflammatory cells, apoptosis, and degradation of extracel- Developmental Biology, Vídeňská 1083, Prague 4, Czech Republic lular matrix. Our laboratory performed temporal RNA sequencing of the healing tissue using tailbud and swimming tadpole embryos. Keywords: XtiSCs, Xenopus tropicalis, muscle regeneration Results showed predominant expression of four mmps: 1, 7, 8 and 9. Muscle regeneration is a limited process in higher vertebrates Injury or amputation caused the upregulation and their expression including human. Striated musculature is renewed from satellite level peaked at 3-6 hours post injury, which corresponds to late Pax7+ cells localized beneath the basal lamina surrounding individ- phase of healing. We used in situ hybridization and scRNA-Seq to ual myofibers. Heart healing after ischemia or infarct is restricted visualize cells expressing mmps. We also studied functional relation- to formation of fibrotic scar without substantial proliferation of car- ships between each selected mmps by using specific and general diomyocytes. We succeeded in the establishment of culture of Ser- 12 ABSTRACTS | ORAL PRESENTATIONS

toli cell progenitors (XtiSCs) derived from testicles of adult Xenopus T32. tropicalis males. XtiSCs possess common features with mesenchy- Are LGR5-positive cells labeling stem cell niche in molars? mal stem cells regarding gene expression profile and differentiation Olbertova K 1,2, Hrckulak D 3, Kriz V 3, Hruba E 1, Korinek V 3, into osteocytes, chondrocytes, and adipocytes. We showed that Buchtova M 1,2 XtiSCs can trigger switch of M1 to M2 macrophage population when 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology injected into the mouse tail vein indicating their pro-regenerative and Genetics, Brno, Czech Republic; 2 Department of Experimental Biology, and anti-inflammatory function. Using X. tropicalis tadpoles as a Masaryk University, Brno, Czech Republic; 3 Laboratory of Cell and Devel- model for the striated muscle regeneration after the tail amputa- opmental Biology, Institute of Molecular Genetics, Praha, Czech Republic tion revealed active migration of microinjected XtiSCs to the wound area and higher proliferation of endogenous Pax7+ satellite cells 3 Keywords: stem cells, odontogenesis, single cell sequencing days after injury if compared with control tadpoles without XtiSCs Functional tooth germs in vertebrates are initiated from the den- injection. Concerning heart muscle regeneration, we amputated tal lamina. The longevity of the lamina plays a role in governing heart apex in adult X. tropicalis individuals and analyze its healing the number of tooth generations. Monophyodont species (such as 7 days later. The experimental group was injected with XtiSCs into mouse) have only rudimental replacement dental lamina. Here, we hind limb muscle bed where cells can produce paracrine factors into analyzed if stem cell niche is also located in the dental lamina of the circulatory system, but they are not able to directly contribute monophyodont species. We uncovered LGR5-positive cells located to heart muscle regeneration (remote control). Area measurement in distinct area of the dental lamina and asymmetrically arranged of cryotome sections at the resection site confirmed significantly cluster of positive cells in the surrounding mesenchyme. Co-expres- lower levels of fibronectin deposition and higher levels of cardiomy- sion analysis of LGR5 with other progenitor markers revealed over- ocytes after XtiSCs injection into skeletal muscle bed compared to lap with SOX2 and SOX9 in the dental stalk. Next, we focused on PBS injection 7 days after surgery. the fate of these epithelial cells and their migration routes through the tooth germ while using lineage tracing to analyze the fate of T31. Lgr5-positive cells and their contribution to different parts of tooth Exploring the Role of TMEM107 in Retinal Development Using germ. Usage of transgenic mice lines such as Lgr5-EGFP-CreERT2 Human Retinal Organoid Model mice and Rosa26-lacZ reporter mice allowed us to precisely deter- Tomáš Bárta 1,2, Kamila Weissová 1,2, Canan Celiker 1,2, Lucie Pešková mine the fate of LGR5-positive cells during different stages of 2, Jana Šebestíková 1,2 odontogenesis. Moreover, single cell sequencing was performed to further characterize differences between individual populations of 1 Institute of Animal Physiology and Genetics CAS, Veveří 97, 602 00 LGR5-positive cells and to uncover signaling regulating side-specific Brno, Czech Republic; 2 Department of Histology and Embryology, Faculty differences in the dental stalk area. In conclusion, based on overlap of Medicine, Masaryk University, Kamenice 3, 62500 Brno, Czech Republic of progenitor markers in the dental lamina, we propose lingual side Keywords: retinal organoids, primary cilium, retinal development of the dental stalk in molars as a new stem cell niche important for replacement tooth formation. Transmembrane protein 107 (TMEM107) is localized in the primary This work was supported by the Czech Science Foundation (19- cilium and is enriched at the transition zone acting as a key regu- 01205S) and by the MEYS CR (CZ.02.1.01/0.0/0.0/15_003/0000460) lator of protein content and composition of the cilium. Mutations in TMEM107 are associated with human syndromes exhibiting a T33. wide range of ciliopathic defects including retinal degeneration. Additionally, recently it has been demonstrated that the absence Setting up the model of Alzheimer’s disease using cerebral of TMEM107 leads to disruption of primary cilia and SHH signalling organoids 1,2 1 1 3 associated with numerous distinct developmental defects including Tereza Vanova , Jan Raska , Jiri Sedmik , Petr Taus , Hana Hrib- 1 1 1 microphthalmia and/or anophthalmia in mice suggesting a critical kova , Veronika Pospisilova , Veronika Fedorova , Karla Plevova 3,4 1,2 role of TMEM107 in eye development. , Dasa Bohaciakova 1 Here we used retinal organoids derived from human pluripotent Department of Histology and Embryology, Faculty of Medicine, Masaryk 2 stem cells as a model to closely investigate the role of TMEM107 University, Brno, Czech Republic; International Clinical Research Center 3 in retinal development. We used shRNA and CRISPR/Cas9 approach (ICRC), St. Anne’s University Hospital, Brno, Czech Republic; Central to perform TMEM107 loss-of-function studies. TMEM107 deficiency European Institute of Technology, Masaryk University, Brno, Czechia; 4 results to: I) impaired differentiation towards retinal structures, II) Department of Internal Medicine - Hematology and Oncology, University profound structural changes to retinal organoids, III) absence of Hospital Brno, Brno, Czechia primary cilia on retinal cell types, and IV) impaired SHH signalling. Keywords: Induced pluripotent stem cells, Cerebral organoids, Alz- Our results indicate that TMEM107 is crucial for retinal development heimer’s disease, Single-cell sequencing and SHH signalling. We envisage to use retinal organoids to further explore the molecular mechanisms underlying the role of TMEM107. Alzheimer’s disease (AD) is a chronic neurodegenerative disease Acknowledgement: This work was supported by the Czech Science with brain tissue undergoing pathological changes such as accu- Foundation (21-05146S) and the European Regional Development mulation of β-amyloid plaques (Aβ) and hyperphosphorylation of Fund - Project INBIO (No.CZ.02.1.01/0.0/0.0/16_026/0008451). Tau protein (p-Tau). However, despite a large number of studies, We acknowledge the core facility CELLIM supported by MEYS CR the mechanism of pathogenesis, and thus the potential treatment (LM2018129 Czech-BioImaging) of Alzheimer’s disease, remains unclear. Importantly, the advance- ments in stem cell biology and the possibility of obtaining stem cells ABSTRACTS | ORAL PRESENTATIONS 13

directly from patients represent a great promise of modeling com- of NTs in vivo, the important aspects of their growth and function plex neurodegenerative diseases, including AD. need to be elucidated. Thus, our study aimed to create an in vitro model of Alzheimer’s In our study we want to visualize and characterize membrane NTs disease directly from patients’ cells and subsequently to study the between the epiblast cells of zebrafish embryos. For visualization development of AD pathogenesis. Using cell reprogramming, we unstained and DiI labelled pre-gastrulation phase embryos were successfully created induced pluripotent stem cells from cells of used. Laser-scanning confocal microscope and transmission elec- patients with familial AD as well as from healthy controls. We sub- tron microscope (TEM) were applied to examine the NTs between sequently created 3D cerebral organoids (miniature organs resem- the epiblast cells of 4-8 hours-old embryos. bling the human brain) and 2D neuronal cultures from these cells The characterization of membrane nanotubes shows that their length with detectable AD pathology, i.e., accumulation of Aβ and p-Tau. is approximately identical with the individual cell diameters, and their On these cell systems, we are currently testing several hypotheses occurrence is pronounced in the animal pole of the embryo. on the primary cause of Aβ accumulation. We have also recently Our electronmicroscopic experiments confirmed the in vivo exis- performed single-cell RNA sequencing and are currently analyzing tence of NTs, but further experiments are needed to reveal their the results. Altogether, our data will not only help to elucidate the possible function in zebrafish embryo. development of AD pathology but will also allow the testing of sub- References: stances that may prevent its development under in vitro conditions. [1] Davis D.M., Sowinski S. (2008) Membrane nano- This study was supported by the Czech Science Foundation (21- tubes: dynamic long-distance connections between ani- 21510S), AZV (NV19-08-00472 and NU21-08-00373), and by the mal cells. Nature Reviews Molecular Cell Biology 9. 431–436. project „SORLA-FIX“ no. 8F20009 funded by JPND/EU-MSMT. [2] Gerdes, H.-H. (2008) Intercellular transfer mediated by tunneling nanotubes. Curr Opin Cell Biol 20(4):470–475. [3] Caneparo L., Pantazis P., Dempsey W., Fraser S. E. (2011) Inter- T34. KEYNOTE TALK 7 cellular Bridges in Vertebrate Gastrulation. PLoS One 6(5):e20230. Towards understanding the dynamics of cell fate decisions and organization in the early mammalian embryo T36. Eszter Pósfai DUSPing mouse embryonic stem cells – role of dual specificity Princeton University, Department of Molecular Biology, Princeton, USA phosphatases in differentiation Setting apart embryonic and extraembryonic cell types are a series Stanislava Sladeček, Katarzyna Anna Radaszkiewicz, Martina Bőhmová, of crucial events needed for the successful outcome of mammalian Tomáš Gybeľ, Tomasz Witold Radaszkiewicz, Jiří Pacherník pregnancy and are the earliest fate decisions cells encounter following Department of Experimental Biology, Faculty of Science, Masaryk Univer- fertilization. Using the mouse as a model system, we are interested sity, Brno, Czech Republic in understanding the rules of blastocyst development by addressing Keywords: DUSP, MAPK, mouse embryonic stem cells the dynamics of molecular and cellular mechanisms underlying cell fate choice and morphogenesis. I will discuss the recent technologi- Dual specificity phosphatases (DUSP) are a broad family of enzymes cal advances enabling such studies: our highly efficient genome engi- that can dephosphorylate phosphoserine/phosphothreonine as well neering method, enabling us to generate reporter mouse models, and as phosphotyrosine residues within the same substrate. Although our long-term multi-color live imaging approach coupled with auto- the substrate specificity or substrate preference of DUSPs can vary, mated image analysis tools, allowing us to track cells through differ- they generally dephosphorylate and inactivate members of mitogen entiation and development in an unprecedented manner. activated protein kinases (MAPK). DUSP6, DUSP7 and DUSP9 are cytosolic enzymes which are closely related based on their struc- T35. ture and substrate specificity. In our study we focus on their impor- In vivo examination of actin based membrane nanotubes in tance during the in vitro differentiation of mouse embryonic stem developing zebrafish embryos cells. Knock-out cell lines of these phosphatases do retain some of Katalin Türmer 1, Miklos Nyitrai 1,2, Edina Szabo-Meleg 1,2 their basic characteristics, but the lack of individual DUSP, or their 1 Department of Biophysics, Medical School, University of Pés, Pés, Hun- combination, affects their in vitro differentiation. We have observed gary; 2 Szentagothai Research Centre, University of Pés, Pés, Hungary that cells which do not express these DUSPs form less of the cardiac mesoderm, which in longer differentiation results in the formation Keywords: membrane nanotubes, actin, zebrafish embryos of a lower number of cardiomyocytes, while the formation of neu- Membrane nanotubes (NT) are transient long-distance connections roectoderm and neurons is increased. However, the severity of this between cells and can facilitate intercellular communications [1]. shift in differentiation varies depending on which DUSP is depleted. NTs are actin-based cell protrusions which were first reported in Absence of DUSP6 has the lowest impact on cell differentiation, 2004 between rat pheochromocytoma (PC12) cells [2]. Membrane while in the absence of DUSP7, the number of formed cardiomy- nanotubes were described both in vitro (in cell cultures) and in vivo ocytes is the lowest. The importance of DUSP7 for mouse embry- (eg. between dendritic cells in mouse cornea [1] or within zebrafish onic stem cells and their differentiation is further supported by the embryos [3]). The mechanism of NT formation is not completely fact, that we were not able to create double knock-out cell lines understood yet, nevertheless based on in vitro studies conducted with Dusp7 in the combination of knock-out genes. Taken together, on cell cultures, these structures can be developed as actin-driven this data suggests that DUSP6, DUSP7 and DUSP9 play and impor- membrane protrusions from directed, filopodium-like structures or tant role in in vitro differentiation and that out of the three stud- they can be formed when migratory cells separate after close con- ied phosphatases, DUSP7 is pivotal in the maintenance of mouse tact. Although convincing results are available about the presence embryonic stem cells and their early differentiation. 14 ABSTRACTS | ORAL PRESENTATIONS

T37. The epithelium of all three different ChP types arises from the pro- Avian ceca are required for hindgut enteric nervous system genitor domains termed as cortical hem and Wnt1+ rhombic lip. development by promoting enteric neural crest cell prolifera- Besides the non-neuronal cell types, these domains produce also tion and inhibiting neuronal differentiation via non-canonical the neuronal cells within the telencephalon as well as the hindbrain. Wnt signaling Interestingly, the avian graft experiments of these progenitors Nandor Nagy 1, Tamas Kovacs 1, Rhian Stavely 2, Viktoria Halasy 1, showed their hidden potential as the transplantation of rhombic lips Adam Soos 1, Emoke Szocs 1, Ryo Hotta 2, Hannah Graham 2, cells into the diverse part of the neural tube led to their migration Allan M. Goldstein 2 into the branchial arches via routes of originally Wnt1+ neural crest 1 Department of Anatomy, Histology and Embryology, Faculty of Medi- cells (NCC). cine, Semmelweis University, Budapest, Hungary; 2 Department of Pediatric Here, we explored the idea of the shared origin of all ChPs in the Surgery, Pediatric Surgery Research Laboratories, Massachusetts General „NCC-like” cellular domains. Using the Confetti lineage system, we Hospital, Harvard Medical School, Boston, MA 02114 proved the common origin of ChP epithelial layers in the Wnt1+ cellular population. To distinguish these cells from the original NCC Keywords: neural crest, enteric nervous system, ceca, avian population, we examined the Sox10 Confetti mouse model. Here, embryo, Wnt11 ChP tissue remains unmarked indicating a non-NCC origin of ChPs. The enteric nervous system (ENS), which is derived from enteric Additionally, we identified to-date undescribed Wnt1+ cellular neural crest cells (ENCCs) during gut development, represents the domain within the cortical hem in which possibly origins the epithe- neuronal innervation of the gastrointestinal tract and is critical for lium of telencephalic ChP. regulating normal intestinal function. Compromised ENCC migra- Taken together, our results indicated a converse Wnt1 dependent tion can lead to Hirschsprung Disease, which is characterized by an program of multipotent progenitors cells within the neural tube. aganglionic distal bowel. We find that removal of the ceca, a paired Moreover, this program seems to be repetitively utilized at different structure present at the midgut-hindgut junction in avian intestine, timepoint during embryogenesis. leads to severe hindgut aganglionosis, suggesting that the ceca are required for ENS development. To test this, we replaced the ceca of T39. embryonic day 6 (E6) wild-type chicks with ceca from transgenic The role of CXCR4-CXCL12 signaling in extrinsic innervation of GFP chicks. Interestingly, the entire hindgut ENS arises from the enteric nervous system development GFP+ ceca-derived ENCC population. Comparative transcriptome Viktória Halasy, Tamás Kovács, Emőke Szőcs, Ádám Soós, Dalma profiling of the cecal buds compared to the interceca region shows Jancsovics, Nándor Nagy that the non-canonical Wnt signaling pathway is preferentially Laboratory of Stem Cells and Experimental Embryology, Department of expressed within the ceca. Specifically, Wnt11 is highly expressed Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis in the ceca, as confirmed by RNA in situ hybridization, leading us to University, Budapest, Hungary hypothesize that cecal expression of Wnt11 is important for ENCC colonization of the hindgut. Organ cultures were prepared using E6 Keywords: enteric nervous system, hindgut development, embry- avian intestine, when ENCCs are migrating through the ceca, and onic manipulation techniques showed that Wnt11 inhibits enteric neuronal differentiation. These The intestine is innervated by intrinsic neurons of the enteric nervous results reveal an essential role for the ceca during hindgut ENS for- system (ENS) and by the afferent and efferent axons of peripheral mation and highlight an important function for non-canonical Wnt ganglia. The nerve of Remak (NoR) is a sacral neural crest-de- signaling in regulating ENCC differentiation and thereby promoting rived (NCC) structure, an avian-specific ganglionated structure their migration into the colon. that extends from the cloaca to the proximal midgut and has role OTKA grant: 124740 in extrinsic innervation of the gut. Development of NoR starts at early embryonic day 5 (E5) and axons projecting from the NoR reach T38. the gut wall at E7. The molecular mechanism of NoR-derived axon Wnt1 to rule them all: A story about the shared origin of all growth is unknown. In mammals the presence of CXCR4, a cell surface types of choroid plexi receptor for the chemokine stromal cell-derived factor-1 (SDF1, also 1 1 2 Petra Kompanikova , Karol Kaiser , Jan Prochazka , named CXCL12) is responsible for several embryonic developmental 2 2 2 Michaela Prochazkova , Ivana Bukova , Radislav Sedlacek , processes in response to CXCL12 including migration of neural crest 1 Vitezslav Bryja cells, neuronal survival and axon pathfinding. We have employed 1 Department of Experimental Biology, Faculty of Science, Masaryk Uni- chimeric tissue recombination, in situ hybridization, combined with 2 versity, Brno 61137, Czech Republic; Czech Centre for Phenogenomics immunofluorescence labeling to follow the precise spatiotempo- and Laboratory of Transgenic Models of Diseases, Institute of Molecular ral expression of CXCR4 molecule during migration of sacral NCC Genetics of the CAS, Prague 142 20, Czech Republic within the ENS. We have observed specific CXCR4 expression in NoR Keywords: choroid plexus, Wnt1, progenitor domains and CXCL12 in the mesenchyme around of NoR and pelvic plexus. Embryonic hindgut+NoR cultured in presence of CXCL12 results in The choroid plexus (ChP) is a vascularized tissue protruding into all significant increase of NCC migration and axon projection from the mammalial brain ventricles. Even though ChP does not draw the NoR, while inhibition of CXCR4 signaling with AMD3100 disrupt their attention of many scientists, it is essential for the proper develop- migration out of explant, suggesting a novel role for CXCR4/CXCL12 ment of the organism as it stands behind the production and com- signaling in the extrinsic innervation of the colorectum. position regulation of cerebrospinal fluid. Grant: NKFI 124740 ABSTRACTS | ORAL PRESENTATIONS 15

T40. players in sperm-egg interaction. In particular, we discovered the Diverse roles of ERK and AKT signaling pathways in epithelial small protein Bouncer, which is bound to the egg membrane and morphogenesis is required for sperm entry into the egg. Remarkably, by replac- Jakub Sumbal 1, Vaclav Sadilek 1,2, Jan Elias 1,2, ing zebrafish Bouncer with medaka Bouncer in the zebrafish egg, Miroslav Vorechovsky 1,2, Zuzana Koledova 1 we found that changing this single factor allowed cross-fertilization 1 Department of Histology and Embryology, Faculty of Medicine, Masaryk between these two evolutionarily distant fish species. While fish University, Brno, Czechia; 2 Institute of Structural Mechanics, Faculty of express Bouncer exclusively in the egg, the mammalian homolog Civil Engineering, University of Technology, Brno, Czechia of Bouncer, Spaca4, is required in mammalian sperm for efficient binding to and penetration of the zona pellucida. Keywords: Branching morphogenesis, Organoid, Cell signaling Overall, our studies uncover important mechanisms that regulate Branching morphogenesis is an evolutinarily conserved mechanism the earliest steps at the beginning of an organism’s life. that increases the functional area of numerous organs, including mammary gland. Branching morphogenesis of mammary gland is T42. tightly regulated by local paracrine signals, however, how numer- Patterning and morphogenesis of the head-trunk boundary ous microenvironmental signals are integrated in the epithelium to Bruno Cossermelli Vellutini, Marina Belen Cuenca, Pavel Tomancak result in specific patterns of the gland is poorly understood. Here, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, we have used primary mammary organoid system, coupled with Germany quantitative time-lapse imaging and pharmacological inhibitors, Keywords: cephalic furrow, genetic interactions, tissue mechanics confocal imaging and integrative computational modeling coupling 3D vertex model with activator-inhibitor system to untangle the The head–trunk boundary is a fundamental landmark of animal roles of two major intracellular signaling pathways of developing morphology. However, the developmental mechanisms that estab- epithelium – ERK and AKT. We found two distinct roles for ERK and lish, refine and shape the interface between head and trunk remain AKT. While ERK is a branching promoting pathway that operates poorly understood. Here we integrate genetics, live-imaging, and in defined spatial self-driving domains, AKT induces overall growth in vivo mechanical perturbations to reveal the developmental pro- and epithelial stratification and is more dependent on stable extrin- cesses underlying the formation of the cephalic furrow—a deep sic signals. Moreover, our findings from organoids are corrobo- invagination that demarcates the head–trunk boundary of Dro- rated by in vivo pharmacological inhibition of ERK and AKT, that sophila. We first extend the known cascade of genetic interactions demonstrate the importance of those two pathways for mammary patterning the cephalic furrow by describing the role of a novel branching morphogenesis in mouse. upstream factor that controls the position of the invagination along Taken together, we have identified ERK and AKT as key pathways the anteroposterior axis. We then analyze how defects in cephalic for mammary branching morphogenesis, driving epihtelial branch- furrow formation impact tissue interactions during gastrulation by ing and stratification, respectively, two processes indispensable for imaging mutant embryos under lightsheet microscopy at high-tem- proper mammary gland development. Furthermore, our data sug- poral resolution. Surprisingly, mutant embryos which fail to initi- gest, that disharmony of these pathways may cause dismorphic ate the cephalic furrow form a series of late ectopic folds at the growth of mammary epithelium during breast cancer. head–trunk interface. We show these ectopic folds form due to Acknowledgements: MUNI/G/1446/2018 and MUNI/G/1775/2020. mechanical forces generated by the germ band extension, revealing an unexpected long-range tissue interaction between the head and T41. KEYNOTE TALK 8 the trunk domains. Our findings suggest the cephalic furrow act The egg-to-embryo transition – from fertilization to transla- as a buffer for these conflicting tissue interactions by priming the tional regulation position of folding at the head–trunk boundary early in Drosoph- Andrea Pauli ila gastrulation. Altogether, our work reveals the interplay between IMP (Research Institute of Molecular Pathology), Vienna, Austria molecular patterning and tissue morphogenesis shaping a key trait of bilaterian animals. Life of any sexually reproducing organism starts with the fusion of two specialized cells, the egg and the sperm, to form a single cell, T43. the zygote. This totipotent cell gives rise to all cells of the future Mechanical forces generated by fibroblasts regulate morpho- organism. Despite the importance of sperm-egg fusion in initiat- genesis of mammary epithelium ing embryogenesis and decades of research devoted to its under- Jakub Sumbal, Denisa Belisova, Anas Rabata, Zuzana Koledova standing, the mechanisms that keep the egg dormant for prolonged Department of Histology and Embryology, Faculty of Medicine, Masaryk periods of time and ready to be fertilized have remained largely University, Brno, Czech Republic unknown. Moreover, the underlying molecular mechanisms that mediate sperm-egg binding and fusion in vertebrates are still mys- Keywords: branching morphogenesis, mechanobiology, mammary terious. gland, organoid In my talk, I will share new insights into the egg-to-embryo tran- Branching morphogenesis of mammary epithelium entails coordi- sition, using zebrafish as main model organism. Structural, bio- nated epithelial proliferation, differentiation, and collective migra- chemical and in vitro and in vivo studies revealed a new mechanism tion, through which the elaborate mammary epithelial tree-like that keeps ribosomes repressed and preserved in the egg. Genetic structure of adult female mammary gland is formed. While system- studies in zebrafish, medaka and mice uncovered new essential ically orchestrated by hormones, the complex processes of mam- 16 ABSTRACTS | ORAL PRESENTATIONS

mary morphogenesis are fine-regulated locally by epithelial-stromal of the different MTOCs during the late stages of spermatogenesis. interactions, which remain incompletely understood. In this study, Funding: NKFIH_OTKA 132155, GINOP 2.3.2-15-2016-00032, using genetic mouse models, advanced mammary organoid-fibro- GINOP-2.3.2-15-2016-00035 blast co-cultures, time-lapse imaging and high-end confocal imag- ing with quantification, we demonstrate that mammary stromal T45. fibroblasts induce epithelial branching using their mechanical force. The biological significance of nuclear actin We show that physical contact between mammary epithelium and Péter Borkúti, Ildikó Kristó, Zoltán Kovács, Anikó Szabó and fibroblasts is required to induce folding of mammary epithelium Péter Vilmos to branches in ex vivo organoid-fibroblast co-cultures, and that Biological Research Centre, Szeged, Hungary genetic ablation of fibroblasts or fibroblast-specific knockout of myosin II gene Myh9 retards mammary epithelial outgrowth and Keywords: Nuclear actin, Nuclear transport, Importin reduces mammary epithelial branching in vivo. Finally, we identify Actin is present in the cell nucleus, where it participates in multiple spatial anisotropy of YAP and ERK signaling activity in the epithe- tasks. However, the biological relevance of its nuclear activity is still lium as downstream effectors of fibroblast-induced mammary epi- unclear. We decided to investigate this question with the help of thelial branching. Together, our study unveils a novel mechanism of Drosophila melanogaster, by using combinations of various trans- mammary epithelial morphogenesis via mechanical forces exerted genes and mutations, and monitoring the development and viability by fibroblasts and a new mechanically active fibroblast subpopula- of the animals. tion in developing mammary gland, which might represent a normal The labelling of actin with a Nuclear Export Signal (NES) results in counterpart of cancer-associated fibroblasts. constant removal of the protein from the nucleus. Our first set of Acknowledgements: MUNI/G/1446/2018 and MUNI/11/ experiments showed no change in the number of surviving Act5C SUP/20/2020. null mutant males upon rescuing with NES-Act5C expressing trans- gene, suggesting that tagging of actin with NES tag alone cannot T44. lower significantly the amount of the protein in the nucleus. There- Microtubule organizing centers contain testis-specific γ-TuRC fore, we severed the putative nuclear retention motif of actin, and proteins in spermatids of Drosophila melanogaster deleted the RanBP9, which encodes the only known importin for Elham Alzyoud 1,2, Viktor Vedelek 1, Zsuzsanna Réthi-Nagy 2,3, actin, and Act42A genes. The combined effect of NES tagging of Zoltán Lipinszki 3, Rita Sinka 1 actin and the homozygous null mutation of RanBP9 significantly 1 Department of Genetics, University of Szeged, Szeged, Hungary; lowered the number of mutant male progenies. The experiments 2 Doctoral School of Biology, Faculty of Science and Informatics, Univer- also provided evidence that RanBP9 is not the exclusive nuclear sity of Szeged,Szeged, Hungary; 3 Biological Research Centre, Institute of importin of actin, therefore we screened for other nuclear import Biochemistry, MTA SZBK Lendület Laboratory of Cell Cycle Regulation, factors of actin and identified new importins that interact with ELKH, Szeged, Hungary actin. Our results unambiguously demonstrate the robustness of the Keywords: Drosophila, MTOC, spermatogenesis nuclear localization of actin, and show that multiple parallel path- Gamma Tubulin Ring Complex (γ-TuRC) is known as the main player ways ensure this localization. At the same time, this observation for microtubules nucleation in MTOC. Proteins involved in the strongly argues for the great significance of actin in the nucleus. formation and the development of MTOC in Drosophila are con- served among eukaryotes. Mutations of many of MTOC proteins are T46. known to cause diseases in human. Centrosomes undergo massive Effect of mir-302B microRNA inhibition on chicken PCG prolif- changes during spermatogenesis, as they turn into basal bodies, eration and apoptosis rate which nucleate and stabilize microtubules. We aim to study the Nikolett Tokodyné Szabadi 1, Mahek Anand1, Ganna Stepanova 1, γ-TuRC distribution and function during late stages of Drosophila Bence Lázár 1,2, Roland Tóth 1, András Ecker 1, Martin Urbán 1, spermatogenesis. γ-tubulin exists in two complexes in Drosophila: Krisztina Sima 3, Tamara Kepler 3, Zoltán Hegyi 4, László Homolya 4, γ-Tubulin Small Complex (γ-TuSC) and γ-TuRC. We conducted phylo- Eszter Patakiné Várkonyi 2, Bertrand Pain 5, Elen Gócza 1 genetic analysis and identified three testis-specific γ-TuRC proteins. 1 Hungarian University of Agriculture and Life Sciences, Gödöllő, Hun- γ-TuSC represented by t-Grip84 and t-Grip91 a paralogue of Grip84 gary; 2 National Centre for Biodiversity and Gene Conservation, Gödöllő, and Grip91 respectively, the third one is t-Grip128 a paralogue of Hungary; 3 Pázmány Péter Catholic University, Budapest, Hungary; 4 Grip128. This suggests the existence of testis-specific γ-TuRC (t-γ- Research Centre for Natural Sciences, Eötvös Loránd Research Network, TuRC). Analyzing the phenotype of t-γ-TuRC mutants we found that Budapest, Hungary; 5 U1208 INSERM, Bron, France t-Grip84 mutant is male sterile, t-Grip91 mutant is male semi-ster- ile and t-Grip128 mutant fertility is normal. We made transgenic Keywords: chicken embryo, PGCs, microRNA lines and checked the localization of the t-γ-TuRC proteins. They Primordial germ cells (PGCs) are the first germ cell population localize to the centriole adjunct after meiosis, and the nuclear tip established during development and are precursors for both the during nuclear elongation, then to the surface of the mitochondria oocytes and spermatogonia. PGCs have an essential function in during cyst elongation. We proved the binding of the t-γ-TuRC to stem cell biology. Investigation of the factors affecting cell prolifer- γ-Tubulin, Mzt1 and to each other biochemically. Our results pro- ation is essential in stem cell biology. vided the first proof of the existence and essentiality of t-γ-TuRC, The cell number in two male (M1, M2) and two female (F1, F2) PGC which can lead us to understand better the molecular composition lines in a 96-well plate was measured every 4 hours (H) for three ABSTRACTS | ORAL PRESENTATIONS 17

days using the XLS Imaging system with a built-in incubator. We T48. inhibited the gga-miR-302b-5P and gga-miR-302b-3P miRNAs Embryonic cells can act as non-professional phagocytes and using miRNA inhibitors: miR-302b-5p (5P), miR-302b-3P (3P) and undertake the clearance of dying cells in mouse blastocysts using mixture of the miR-302b-5P and the miR-302b-3P inhibitors Dušan Fabian 1, Jozef Pisko 1, Alexandra Špirková 1, Štefan Čikoš 1, (5P/3P) at treatments. Comparing the control and treated lines on Lucia Olexiková 2, Veronika Kovaříková 1, Zuzana Šefčíková 1 the first day (28H/4H) and second day (52H/28H), male lines had 1 Institute of Animal Physiology, Centre of Biosciences, Slovak Academy of a higher proliferation rate independently from the treatments. Not Sciences, Košice, Slovak Republic; 2 Research Institute for Animal Pro- treated (control) and 5P/3P inhibited PGC lines show significantly duction Nitra, National Agricultural and Food Centre, Lužianky, Slovak higher proliferation rate than 5P and 3P inhibited lines at all time Republic points in all PGC lines. Apoptotic rates, most cases showed a posi- tive correlation with the cell number, but the apoptotic rate of the Keywords: preimplantation embryo, apoptosis, non-professional cells inhibited with miR-302b-5P showed a negative correlation phagocytes with cell number. Cell membranes of the cells inhibited with miR- The study provides the first insight into the machinery of embry- 302b-5P showed some additional “blebbing” on their membrane onic phagocytosis of apoptotic cells (efferocytosis) and quantitative surfaces compared to the untreated ones, as was discovered during analysis of its efficiency. immunostaining assays. Results show that embryonic cells in mouse blastocysts possess all the mechanisms necessary for the recognition, engulfment and T47. digestion of damaged blastomeres. The process of embryonic effero- Deregulation of mitochondrial activity and redox status in cytosis seems to follow the standard pathway: It begins with the rec- oocytes from aged mouse females: is it maternal or postovula- ognition of the apoptotic cell via the binding of various phagocytic tory ageing to blame? receptors to externalized phosphatidylserine or other ligands such Katarzyna Czajkowska and Anna Ajduk as modified lipoproteins. Gene transcript analysis showed that the Reproductive Biology Group, Department of Embryology, Institute of embryonic cells expressed 12 receptors / immunoregulatory proteins Developmental Biology and Biomedical Sciences, Faculty of Biology, Uni- likely involved in phagocytic process. Signaling leads to the recruit- versity of Warsaw , Warsaw, Poland ment of Rho family GTPase RAC1, the actin-dependent formation of the phagocytic cup and targeted internalization, i.e., taking the Keywords: oocyte, mouse, aging, mitochondria, redox status, apoptotic cell into a vacuole (the phagosome) which then undergoes developmental potential maturation (progressive acidification) and gradual degradation. Maternal aging has been reported to affect quality of mammalian Microscopic analysis of mouse blastocysts revealed phosphati- oocytes, including mitochondria and redox status, which results in dylserine externalization to be 10 times less common than inci- their decreased developmental potential. Here we show that ovu- dence of apoptotic cells (as detected by TUNEL). In spite of the lated oocytes from aged mouse females fall into two distinctive low frequency of phosphatidylserine-flipping (in inner cell mass, cathegories: morphologically normal and abnormal (shrunk). We no annexin V staining was recorded), fluorescence staining of the also show that those two cathegories differ in terms of activity and plasma membrane showed more than 20% of apoptotic cells to 2+ distribution of mitochondria, FAD /FADH2 redox status and reactive have been engulfed by neighbouring blastomeres. The mean fre- oxygen species (ROS) amount), abnormal oocytes displaying much quency of apoptotic cells escaping phagocytosis by their extrusion more severe signs of deregulation of mitochondrial activity and into blastocyst cavities did not exceed 10%. It would thus appear redox status than normal oocytes. Importantly, when maternally that intact early embryonic cells can undertake the clearance of the aged oocytes mature in vitro, they display mitchondrial activity and majority of dying cells in blastocysts. distribution the same as the young oocytes, although they contain The study was funded by the project APVV-18-0389. higher amount of ROS. It suggests that the abnormal phynotype frequent in maternally aged ovulated oocytes may be predomi- T49. nantly caused by postovulatory, not maternal, aging. Indeed, our Lysosome Related Organelles promote stress and immune results indicate that young oocytes aged for 49-57 hours resemble defenses in C. elegans abnormal aged oocytes. As abnormal aged oocytes are resistant to Gábor Hajdú 1, Csaba Sőti 1 fertilization, we followed developmental potential only for normal 1 Semmelweis University, Department of Biochemistry and Molecular Biol- maternally aged oocytes. Their calcium response to fertilization dif- ogy, Budapest, Hungary feres from the response of young oocytes, although the alterations are relatively mild. Maternal aging decreases oocytes’ fertilizability, Keywords: LROs, Hormetic stress PC, autofluorescence, cytopro- but it affects neither their cavitation rate after successful fertiliza- tection, immune defense tion, nor their ability to differentiate into the first embryonic cell lin- Lysosome-Related Organelles (LROs) of multicellular organisms play eages and develop to term. In summary, our research indicates that divergent roles through storage and controlled secretion. Human most of the severe mitochondrial phenotypes associated previously LRO dysfunction causes severe diseases with hypopigmentation, with maternal aging may be due to increased representation of pos- bleeding diathesis and immunodeficiency. LROs in Caenorhabditis tovulatory aged oocytes in the pool of ovulated oocytes. elegans enterocytes share glo (gut granule loss) human LRO bio- genesis gene orthologs. Although the age-dependent accumulation of autofluorescent material is a marker of healthspan, their biolog- ical function is unclear. 18 ABSTRACTS | ORAL PRESENTATIONS

Here we show that undiluted, toxic benzaldehyde (BA) exposure through promoting the degradation of the mutant α-synuclein cause a rapid induction of autofluorescence and hormesis in a (A53T) protein. These data suggest that Rab2 may serve as a novel glo-1/Rab32, and glo-3/GEF dependent manner. These mutants target where lysosomal degradation can be specifically increased to were more sensitive to BA toxicity, heat and oxidative stress after protect neurons from undergoing demise. preconditioning. Methyl salicylate, chemically related to BA also ele- vated autofluorescence and physiological stress tolerance. We carried out systematic qRT-PCRs to investigate induction of selected LRO biogenesis, stress- and detoxification, as well as immune defense genes upon hormetic BA exposure. mRNA level of LRO biogenesis and LRO-specific transmembrane transporter genes showed glo-1 dependent induction, suggesting an adap- tive stress-response. BA exposure induced cytoplasmic heat shock response (hsp-16.2 and hsp-70), as well as xenobiotic stress response (gst-4 and cyp-35B) mRNA-s in wildtype, but not in glo-1 mutants. Intriguingly, we found glo-1 dependent induction of irg-1, irg-5 and clec-60 gene expressions suggesting a robust activation of the innate immune system. Our results identify C. elegans LROs as an organizer of systemic stress defenses. The importance of LROs in human immunity sug- gests an evolutionary conserved and therapeutically relevant role of LROs in organismal self-protection.

T50. A novel target for autophagy activation in Drosophila neurode- generative and ageing models Eszter A. Kiss 1, Fanni Keresztes 1, Janka Szinyákovics 1, Tibor Vellai 1,2,, Tibor Kovács 1 1 Department of Genetics, Institute of Biology, ELTE Eötvös Loránd Uni- versity, Pázmány Péter sétány 1/C, Budapest, H-1117, Hungary; 2 ELKH- ELTE Genetics Research Group, Pázmány Péter sétány 1/C, Budapest, H-1117, Hungary Keywords: autophagy 1, Rab2 2, small G-protein 3, neurodegener- ation 4, ageing 5, Parkinson’s disease 6, Drosophila 7 Autophagy is a lysosome-mediated degradation process in eukar- yotic cells which plays an essential role in the breakdown of cyto- toxic proteins and damaged organelles. Dysfunction of autophagy has been associated with various age-associated diseases, such as cancer, neurodegenerative diseases, immunodeficiency and tis- sue atrophy. Thus, pharmacological activation of autophagy has a great medical potential. Increasing the formation of new auto- phagic structures alone, without lysosomal degradation, however could be detrimental to neurons: for example, in Alzheimer’s dis- ease (AD) autophagosomes provide an isolated environment in which the maturation of intracellular toxic protein forms can be favoured. This implies that a rapid degradation of autophagosomal content is essential for treating AD. Autophagy proteins regulating lysosomal degradation and acidic hydrolysis may serve as new tar- gets for autophagy stimulation. The fusion of autophagy-related membranes is regulated by small-G proteins including Rab2. The absence of Rab2 function inhibits autophagy. The main goal of our research is to study whether activation of Rab2 can lead to enhanced autophagy, and interfere with ageing and neurodegen- eration in Drosophila models. We show that a constitutively active form of Rab2 (Rab2-CA) can effectively activate autophagy in neu- rons, thereby improving cognitive abilities and extending lifespan in the affected animals. Furthermore, Rab2-CA also improves move- ment and increases life expectancy in a Parkinson’s disease model, ABSTRACTS | POSTER PRESENTATIONS 19

or postnatal lethality due to developmental defects. Using site-spe- ABSTRACTS cific endonucleases, transcription activator-like effector nucleases POSTER PRESENTATIONS (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9), knocking out of genes became much easier than before. Unfortunately, heterozygous mutant mice did not always P1. develop the symptoms of a human disease. Several KO mouse lines Extracellular vesicles microRNA cargo in porcine follicular flu- were generated for modelling certain diseases, but homozygous ids: the potential association with oocyte quality lethality is still an unwanted side effect in case of many genes. The Ahmed Gad 1,2, Matej Murin 1, Alexandra Bartkova 1,3, literature search was conducted using PubMed and Web of Science Kateřina Marcollová 1, Jozef Laurincik 1,3, Radek Prochazka 1 databases until April 30th, 2021. The following terms were com- 1 Institute of Animal Physiology and Genetics, Czech Academy of Sciences, bined to find relevant studies: “lethality”, “mice”, “knock-out”, “defi- Liběchov, Czech Republic; 2 Department of Animal Production, Faculty of cient”, “embryonic”, “perinatal”, “rescue”. Additional manual search Agriculture, Cairo University, Giza, Egypt; 3 Constantine the Philosopher was also performed to find the related human diseases in the Online University in Nitra, Nitra, Slovakia Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. According Keywords: Extracellular vesicles, miRNA, follicular fluids to the literature, the most promising methods to rescue a lethal Extracellular vesicles (EVs) have been detected in the ovarian folli- phenotype are the creation of mosaic mice by TALEN or CRISPR/ cular fluids (FFs) and reported to play essential roles in regulating Cas9 system and the disruption of other genes which are involved oocyte development through their cargo molecules including microR- in the affected pathway. NAs (miRNAs). The objective of this study was to identify miRNA expression profiles of small EVs from porcine FFs in association with P3. oocyte quality. Antral follicles were aspirated individually and oocytes p38-MAPK-mediated translation regulation primes blastocyst were stained with 0.5% Lissamine Green B stain (LB), a vital stain development and is required for primitive endoderm differen- for oocyte quality. Each oocyte was classified separately according to tiation in mice the stain into high-quality (unstained; HQ) and low-quality (stained; Pablo Bora 1,*, Lenka Gahurova 1,2, Tomáš Mašek 3, LQ). Oocyte corresponding FFs were pooled together into HQ and LQ Andrea Hauserova 1, David Potěšil 4, Denisa Jansova 2, groups and their EV-miRNAs were isolated and sequenced. Andrej Susor 2, Zbyněk Zdráhal 4, Anna Ajduk 5, Martin Pospíšek 3, Sequencing analysis revealed that a total of 295 known miRNAs Alexander W. Bruce 1,* were commonly detected in both groups. MiR-27b-3p, miR-140-3p, 1 Laboratory of Early Mammalian Developmental Biology (LEMDB), miR-29a-3p, miR-202-5p, and miR-16 were the top highly abun- Department of Molecular Biology & Genetics, Faculty of Science, Univer- dant miRNAs in both groups. Differentially expression (DE) analysis sity of South Bohemia, Branišovská 31, 37005 České Budějovice, Czech exhibited that 19 miRNAs (including miR-193a-5p, miR-125b, and Republic; 2 Laboratory of Biochemistry and Molecular Biology of Germ miR-320) were up- while 22 (including miR-9, miR-6516, and miR- Cells, Institute of Animal Physiology and Genetics, CAS, Rumburská 206) were down-regulated in the HQ compared to the LQ group. 89, 27721 Liběchov, Czech Republic; 3 Laboratory of RNA Biochemistry, DE-miRNAs target gene analysis uncovered pathways associated Department of Genetics and Microbiology, Faculty of Science, Charles with oocyte development including oocyte meiosis, ubiquitin-me- University, Viničná 5, 12844 Prague 2, Czech Republic; 4 Central Euro- diated proteolysis, and signaling pathways (MAPK, PI3K-Akt, and pean Institute of Technology, Masaryk University, 62500 Brno, Czech FoxO). Our findings indicated that FF-EVs contain different miRNA 5 cargo associated with oocyte quality. These miRNAs could be used Republic; Department of Embryology, Institute of Developmental Biology as potential non-invasive biomarkers for oocyte selection. Further and Biomedical Sciences, Faculty of Biology, University of Warsaw, Miec- investigations of EV-miRNA functions during oocyte development znikowa 1, 02-096, Warsaw, Poland. will give more insights into oocyte developmental competence. Keywords: mouse blastocyst, primitive endoderm differentiation, Supported by IAPG-Matoušek Award-2020 and Ministry of Educa- regulation of translation. tion, Youth and Sports of the Czech Republic (CZ.02.1.01/0.0/0.0/1 5_003/0000460). Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a pre- P2. requisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role Practical methods for rescuing KO lethal phenotypes in mice: for p38-mitogen-activated protein kinases (p38-MAPKs) during a literature review PrE differentiation, but the underlying mechanisms have remained Nándor Lipták 1, Zoltán Gál 1, Bálint Biró, László Hiripi and unresolved. Here, we report an early blastocyst window of p38- Orsolya Ivett Hoffmann MAPK activity that is required to regulate ribosome-related gene 1 These authors contributed equally to this work expression, rRNA precursor processing, polysome formation and Hungarian Agriculture and Life Sciences-Institute of Genetics and Bio- protein translation. We show that p38-MAPK inhibition-induced technology, Animal Biotechnology Department, Gödöllő, Hungary PrE phenotypes can be partially rescued by activating the transla- Keywords: knock-out mice, CRISPR/Cas9, lethality, mosaicism. tional regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition Approximately 35% of the mouse genes are essential for viability, targeting active FGF4 signalling are not affected by mTOR acti- thus, global knock-out (KO) of those genes may result in embryonic 20 ABSTRACTS | POSTER PRESENTATIONS

vation. Moreover, we identify and verify translation related can- P5. didate p38-MAPK effectors/ substrates and confirm their role in Determination the time of the “window” period and confirming blastocyst maturation. These data indicate a specific role for p38- the presence of primordial germ cells for elaboration a com- MAPKs in providing a permissive translational environment during bined method for production germline chimaera in the Hun- mouse blastocyst PrE differentiation that is distinct from classically garian goose (Anser anser domestica) species reported FGF4-based mechanisms. Nikoletta Sztán, Bence Lázár, Mariann Molnár, Krisztina Liptói, Eszter Patakiné Várkonyi P4. National Centre for Biodiversity and Gene Conservation – Institute for Interspecies hybrids as potential surrogate hosts for avian Farm Animal Gene Conservation, Gödöllő, Hungary cryopreservation programmes; investigation of the hybrids Keywords: developmental stages, primordial germ cells, germline obtained from crossing Guinea fowl and domestic fowl chimaera, goose Mariann Molnár 1, Bence Lázár 1,2, Nikoletta Sztán 1, Barbara Végi 1, Árpád Drobnyák 1, Elen Gócza 2, Michael J. McGrew 3, As female is the heterogametic sex (ZW) in birds, it was necessary Eszter Patakiné Várkonyi 1 to develop a method for the long-term conservation of the genetic 1 National Centre for Biodiversity and Gene Conservation, Institute for stock of the W chromosome and mitochondrial DNA. Since eggs Farm Animal Gene Conservation, Gödöllő, Hungary; 2 Hungarian Univer- cannot be frozen due to their special biophysical characters, the sity of Agriculture and Life Sciences, Institute of Genetics and Biotech- preservation and recovery of the donor genotype can be accom- nology, Animal Biotechnology Department, Applied Embryology and Stem plished by producing germline chimaeras using stem cells. Cell Biology Group, Gödöllő, Hungary; 3 The Roslin Institute and Royal In contrast to the 21-day hatching period of the domestic fowl, Dick School of Veterinary Studies, University of Edinburgh, Easter Bush goose hatches at around 28–30 days. In order to evaluate the Campus, Midlothian, EH25 9RG, UK optimal window period of PGC migration, assays of staging goose embryos need to be performed. This is the time, when PGCs circu- Related to the avian gene preservation activities, the aim of our late in the embryonic bloodstream and colonizing the gonads, since research was producing interspecific hybrids (recipient) to investi- this hasn’t been previously investigated in goose. We also proved by gate their ability hosting the primordial germ cells from native poul- immunohistochemical methods that the goose blastoderm already try breed (donor). contains PGCs and these cells are present in the bloodstream of To achieve our goals, Guinea fowl x Hungarian yellow chicken the developing embryo at the appropriate stage. A new method hybrids were produced, the crossing was repeated inversely and was elaborated, which combines the simplicity of isolation of donor offspring were investigated in relation to their ability to be potential blastodermal cells and the injection into the bloodstream of the surrogate hosts for cryopreservation programmes. developing recipient embryo during the „window” period, is less Crossing of Guinea fowl females with rooster resulted 6.65% via- expensive and easy to perform. ble offspring but crossing of domestic fowl hens with Guinea fowl A high variance was observed at each embryonic developmental males was unsuccessful (0.14% viable offspring). During the investi- stage in case of Hungarian goose as it is a native breed. We deter- gation of chromosomes 2.7% of the samples showed abnormalities. mined the migration period of the primordial germ cells in goose The hybrids hatched between the 21st and 27th day. Three kind of embryos and found it to be between 69-84 hours of development. phenotype (light brown, dark brown, white spotted) were observed The presence of PG cells at these stages and in the blastodermal cell and none of the hybrids had helmet, crest or facial wattles. suspension also was confirmed by immunohistochemical staining. Between the 16th and 30th week of growth the gonads of hybrids were asymmetrically positioned, their shape were bent and their P6. size did not increase really with the age. Based on the investiga- tions, gonads of the hybrids may be suitable for hosting of donor Comparison of relative gene expression of porcine parthenotes PG cells and producing gametes. cultivated in group or single embryo culture 1 1 1,2 Fluorescently labelled chicken primordial germ cells were injected Matej Murin , Lucie Nemcova , Alexandra Bartkova , 1,2 1 into 3 days old hybrid embryos and their integration rate was meas- Jozef Laurincik , Radek Prochazka 1 ured on 7.5th, 14.5th and 18.5th day. 13.6% of the injected hybrid The Czech Academy of Sciences, Institute of Animal Physiology and 2 embryos survived and 85% of the examined gonads contained flu- Genetics, Libechov, Czech Republic; Faculty of Natural Sciences, Con- orescent labelled donor cells. stantine the Philosopher University in Nitra, Nitra, Slovak Republic This research work shows that these hybrids can be used in gene Keywords: parthenotes, pig, relative gene expression conservation as a universal host for PGCs of avian species. In vitro cultivation of porcine embryos still could be improved. The goal of this study was to analyse relative gene expression (GREM1, NANOG, POU5F1 and NANOG) in blastocysts from group embryo culture (G group) and single embryo culture (S group). Cumu- lus-oocytes complexes isolated from 3-6 mm follicles of prema- ture gilts were incubated in the TCM199 medium for 44 hours

(38.5°C, 5%CO2). The matured oocytes were parthenogenetically activated and incubated in PZM3 medium for 168 hours as G group (30-50 parthenotes/1 ml) or as S group (1 parthenote/20

µl covered with oil) (38.5°C, 5%CO2). Cleavage of parthenotes was ABSTRACTS | POSTER PRESENTATIONS 21

analysed on day 2 and blastocyst rate after 168 hours (three rep- P8. licates). Total RNA was isolated using Ambion RNAqueous-Micro Assaying mitochondrial cell number, morphology and respira- Kit. RT-qPCR was conducted in a RotorGene 3000 cycler using the tion status in the emerging cell lineages of the preimplantation QIAGEN OneStep RT-PCR Kit (4 replicates). Data are expressed as stage mouse embryo the mean±SD. P<0.05 was recognized as statistically significant. Valeriy Kutsyna, Alexander W. Bruce, Pablo Bora, Rebecca Collier, The cleavage in the G group (86.61±4.93%) did not differ from Martina Stiborova the S group (76.63±3.15%). The blastocyst rate from the G group Laboratory of Early Mammalian Developmental Biology, Department of (28.72±2.85%) did not differ from the S group (24.36±5.24%). We Molecular Biology and Genetics, Faculty of Science, University of South found significant differences in relative gene expression of Gremlin1 Bohemia, Ceske Budejovice, Czech Republic (GREM1). In conclusion, higher relative gene expression of GREM1 in Keywords: preimplantation embryo, mitochondria, mouse, blasto- G group could be connected with higher proliferation in this group cyst, lineages compare to S group. Also, we found no differences in embryo devel- opment of these experimental groups. Mitochondria play crucial cellular roles ranging through oxida- Acknowledgement: This work was supported by the project tive phosphorylation and generation of ATP, control of intra-cellu- “EXCELLENCE in molecular aspects of the early development of lar Сa2+ signaling cascades, regulation of redox cellular potential, vertebrates”, CZ.02.1.01/0.0/0.0/15_003/0000460 from the Oper- maintenance and balance of reactive oxygen species levels and are ational Programme Research, Development and Education. strongly associated with apoptosis, among many other physiolog- ical processes. In mammalian preimplantation development, it is P7. claimed the quality of inherited mitochondria is directly responsible for embryo integrity and the ability of blastocyst stage embryos to Detection of apoptosis in porcine oocytes after lissamine implant; as well as influencing development through to adulthood green B staining and association with certain pathologies. However, systematic and 1,2 2 2 Alexandra Bartkova , Veronika Kinterova , Lucie Nemcova , comprehensive research into mitochondrial cell number, morphology 1 1 1 Frantisek Strejcek , Martin Morovic , Michal Benc , and function in relation to the emerging three blastocyst stage lin- Amelie Bonnet-Garnier 3,4, Matej Murin 2, Radek Prochazka 2, eages (i.e. trophectoderm/TE, primitive endoderm/PrE and epiblast/ Jozef Laurincik 1,2 EPI) is still relatively scarce. Accordingly, using a immuno-fluores- 1 Faculty of Natural Sciences, Constantine the Philosopher University cence based confocal microscopy imaging based approach, we have in Nitra, Nitra, Slovak Republic; 2 Institute of Animal Physiology and assayed both the number/morphology and respiration status (uti- Genetics, Czech Academy of Sciences, Libechov, Czech Republic; lizing MitotrackerTM Red CMX-ROS) of mitochondria at all mouse 3 Université Paris-Saclay, UVSQ, INRAE, BREED, 78350 Jouy-en-Josas, preimplantation embryo development stages, and specifically within France; 4 Ecole Nationale Vétérinaire d’Alfort, BREED, 94700 Mai- the evolving blastocyst lineages (as delineated by marker protein sons-Alfort, France expression). Preliminary results suggest restricted active mitochon- drial respiration in differentiating TE cells (associated with a tubular Keywords: oocyte quality, Lissamine Green B, TUNEL micro-tubular network morphology), accompanied by comparatively Apoptosis plays an important role as a quality control mecha- inactive mitochondria in the blastocyst inner cell mass lineages nism which led to removing damaged and dysfunctional cells. In (including the pluripotent EPI). Reliable derivation of baseline data the embryotechnology, apoptosis correlate with oocyte quality. describing preimplantation mouse embryo mitochondrial status will Apoptotic oocytes are unable to develop and become fertilized. act as a foundation for future functional studies related to identi- Nowadays, there is currently no vital method available to detect fied cell signalling pathways and candidate mitochondrial structural apoptosis in porcine oocytes without cells harming. genes implicated in the derivation of specific blastocyst cell lineages. The evaluation of porcine oocytes quality is mainly based on the subjective evaluation of the morphology. Due to the controversy P9. of morphological evaluation, we evaluated the quality of individual DDX21 is a p38-MAPK sensitive nucleolar protein necessary COCs by Lissamine Green B (LB). The aim of this project was deter- for mouse preimplantation embryo development and cell-fate mined correlation between LB staining and apoptosis in in vitro specification matured porcine oocytes. Pablo Bora 1, Lenka Gahurova 1,2, Andrea Hauserova 1, For apoptosis detection, specifically DNA fragmentation, of porcine Martina Stiborova 1, Rebecca Collier 1, David Potěšil 3, oocytes after LB staining, we used Click-it Tunel staining (Ther- Zbyněk Zdráhal 3 and Alexander W. Bruce 1 moFisher, C10247). Porcine oocytes were stained by LB in three inde- 1 Laboratory of Early Mammalian Developmental Biology (LEMDB), pendent experiments. The apoptosis was detected after 44h of IVM in Department of Molecular 7 Biology & Genetics, Faculty of Science, 10.3% of oocytes from the control group. In the experimental groups University of South Bohemia, Branišovská 31, 37005 8 České Budějovice, after vital LB staining, we observed apoptosis in 1.8% of LB- oocytes Czech Republic; 2 Laboratory of Biochemistry and Molecular Biology of and up to 30.5% in LB+ oocytes. These results concluded that vital Germ Cells, Institute of Animal Physiology 10 and Genetics, CAS, Rum- staining and subsequent oocyte selection contribute to the identifica- burská 89, 27721 Liběchov, Czech Republic; 3 Central European Institute tion of high quality COCs. We have also found that selection of COCs of Technology, Masaryk University, 62500 Brno, Czech Republic. by LB leads to separation of proapoptotic oocyte complexes and has Keywords: DDX21, p38-MAPK, preimplantation development, cell no toxic effect on subsequent early embryogenesis. fate specification Acknowledgement: This work was supported by the projects Slovak Reasearch and Development Agency under the Contract no. SK-FR-19- During mouse preimplantation embryo development three cell 0010 and was co-funded by the projects VEGA 1/0001/19, 1/0167/20. lineages arise, that either give rise to extraembryonic tissues (i.e. 22 ABSTRACTS | POSTER PRESENTATIONS

trophectoderm (TE) and primitive endoderm (PrE)) or represent Bu-1+/CXCR4+/IgMlow expression pattern, and has a CSF1R+/ progenitor cells for the future foetus (i.e. epiblast (Epi)). It has been TIM4+/Lep100+ macrophage population; 3) the scaffold of the cor- shown that p38 mitogen-activated-kinases (p38-MAPK) have an tex is composed of mesenchymal reticulum cells that produce ECM; important role in TE development and blastocyst cavity formation. 4) In vivo and in vitro experiments show that tenascin-C provides an Moreover, our previous studies have shown p38-MAPK is involved inhibitory environment for B cell migration. in PrE specification, being functionally required for normal rRNA Grant: NKFI-124740 processing, polysome formation and translation during the early blastocyst stages. Here we describe the role of an identified can- P11. didate p38-MAPK effector protein, RNA helicase protein DEAD-box Investigating the regulatory role of Tead4 in blastomere posi- RNA helicase 21 (DDX21), during preimplantation mouse embryo tioning and cell fate during mouse preimplantation embryo- development. DDX21 expression starts after zygotic genome acti- genesis vation and reaches maximal levels at 8-cell stage, which is then Rebecca Collier 1, Aleksandar I. Mihajlovic 1,2, Martina Stiborova 1, maintained throughout the preimplantation period. At the early Lenka Gahurova 1 and Alexander W. Bruce 1 blastocyst stage (E3.5), DDX21 protein localisation changes from 1 Laboratory of Early Mammalian Developmental Biology, Department of most nuclear to exclusively nucleolar. The shift can be impaired by Molecular Biology and Genetics, Faculty of Science, University of South chemical inhibition of p38-MAPK activity in the same developmen- Bohemia, Ceske Budejovice, Czech Republic; 2 Current address: Research tal window, which indicates a p38-MAPK-DDX21 functional link. Centre at the University of Montreal Hospital (CRCHUM), 900 Rue St We also show siRNA mediated clonal Ddx21 gene downregulation Denis, Montreal, Quebec, H2X 0A9, Canada. causes reduced blastocyst cavity volume and defects in preimplan- tation embryo cell proliferation, which results in lower overall cell Keywords: Preimplantation mouse embryo, trophectoderm, api- number. Moreover, while Ddx21 down-regulation results in lower cal-basolateral polarity, Tead4. ICM cell numbers per se, we demonstrate this disproportionately In the preimplantation mouse embryo, trophectoderm (TE) specifi- affects the derivation of GATA4 expressing PrE cells as compared to cation is essential for the formation and hatching of the blastocyst, NANOG expressing Epi cells. Thus, these data support the impor- uterine implantation and development. Combined, apical-basolat- tance of DDX21, as a potential p38 MAPK effector, during mouse eral polarity acquisition and positional cues mediate the progres- preimplantation development and lineage specification. sion of individual blastomeres towards the TE cell fate. Whilst these processes certainly reinforce each other to provide the appropri- P10. ate conditions for TE differentiation and maturation, the extent Characterization and development of the lymphoid follicular and mechanics of this functional interaction remain poorly under- cortex of the bursa of fabricius stood. Here we report, clonal knock-down (KD) of Tead4 expres- Ádám Soós, Emőke Szőcs, Nóra Fejszák, Dalma Jancsovics, Viktória sion increases contribution of manipulated progeny to the ICM Halasy, Tamás Kovács, Nándor Nagy without effecting the extent of blastomere polarisation. Such cells Laboratory of Stem Cells and Experimental Embryology, Department of abscise, rather than dismantle, the apical domain; thus, function- Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis ally uncoupling polarity and outer-cell position, prior to internali- University, Budapest, Hungary sation. Accordingly, we have generated mRNA-seq datasets from both non-manipulated and Tead4-KD outer cell clones. A number of Keywords: bursa of Fabricius, cell migration, tenascin, RCAS-virus potential regulators of this apical abscission phenotype have been The bursa of Fabricius (BF) is a primary lymphoid organ, unique in identified and are currently under functional investigation. Together, birds, that is responsible for the development of B cells within its fol- these studies are uncovering the endogenous molecular mecha- licular microenvironment. The bursal lymphoid follicles consist of two nisms that homeostatically integrate both intra-cellular polarity and histologically and embryological distinct compartments: the ectoder- appropriate blastomere positioning, in a manner responsible for the mal medulla and the cortex of mesodermal origin. Despite the fact derivation of the TE lineage. that the histology of the follicular medulla is well characterized, the morphology of the ontogenetically later emerging cortex is less clear. P12. In this study, we describe the molecular composition and ontog- Detailed characterization of developing bursa of fabricius eny of the BF follicular cortex. In contrast to medulla, the adult reveals a novel lymphoid follicle inducer cell type cortical CD45+/chB6+ B-lymphocytes express surface CXCR4. In Emőke Szőcs, Ádám Soós, Viktória Halasy, Dalma Jancsovics, the cortex the reticular cells produce extracellular matrix (ECM) rich Nándor Nagy in proteoglycans, collagens, and glycoproteins, unlike the medulla Laboratory of Stem Cell and Experimental Embryology, Department of where there is no detectable ECM. Characterization of the ECM Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis showed that compared to most of the ECM proteins, expression University, Budapest, Hungary. of tenascin-C can be first observed on 16th day of embryogenesis surrounding the developing follicle buds. Tenascin-C blocked B cell Keywords: bursa of Fabricius, lymphoid follicle, avian embryo, mAbs migration in the explant cultures of embryonic BF. Similarly, RCAS- The avian bursa of Fabricius (BF) is a primary lymphoid organ, crit- Shh retroviral vector-induced overexpression of tenascin-C inhibits ical to normal B-lymphocyte development. During embryogenesis B-cell colonization of developing follicles. the epithelial anlage of the BF emerges as a diverticulum of the In summary: 1) development of the follicle cortex starts before cloaca surrounded by undifferentiated mesenchyme. While it is hatching; 2) the B-cells of the cortical follicle shows a specific believed that CD45+ hematopoietic stem cells colonize the epithe- ABSTRACTS | POSTER PRESENTATIONS 23

lial-mesenchymal primordium that would provide a selective micro- At the end of culture, evident changes in cell morphology were environment for B cell precursor expansion, it is more likely that observed as well as the significant expression of specific neuronal separate B-cell, macrophage, dendritic cell precursors colonize the markers (ENO2 and MAP2) was detected using qPCR, flow cytom- mesenchyme, and some precursors migrate to the surface epithe- etry and confocal microscopy in all induced rabbit MSCs lines and lium initiating lymphoid follicle bud formation. also EPCs lines. The goal of this project is to characterize the developmental mech- This work was supported by the grants: APVV-14-0348, APVV-18- anisms of lymphoid follicle formation using a large panel of mono- 0146 and VEGA 1/0160/18. clonal antibodies (mAbs) specific for leukocytes (CD45), B-cells Tirpáková, M. et al. (2021). Phenotypical Characterization and Neu- (chB6, EIVE12), macrophages (TIM4), bursal dendritic cells (CSF1R). rogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchy- The staining of embryonic BF by these mAbs helps to distinguish mal Stem Cells. Genes, 12(3), 431. between three different lineages of hematopoietic cells. CD45+/ Vašíček, J. et al. (2021). Molecular Profiling and Gene Banking of EIVE12+ cells were first observed in the BF rudiment, many of them Rabbit EPCs Derived from Two Biological Sources. Genes, 12(3), 366. enter the surface epithelium to induce follicle bud formation. This will be colonized by the second cell type that belong to the CSF1R+/ P14. TIM4+ population, followed by chB6+ B cell precursors. In conclusion, we could determine three different types of precur- Exceptional interactions of neurodegeneration-related sors which colonize the embryonic BF, indicating that there is a pre- beta-amyloid with rotifer-specific exogenic biopolymers in bursal segregation between these blood-borne cell lineages. Using vitro 1 2 1 3 chick-duck chimeras, we demonstrate that the first cell types which Evelin Balazs , Zsolt Bozso , Janos Kalman , Tibor Hortobagyi , 1 1,* enter the bursal epithelium are not the dendritic/macrophages or B Zita Olah , Zsolt Datki 1 cell precursors, but are a transient lymphoid bud inducer cell popu- Department of Psychiatry, Faculty of Medicine, University of Szeged, 2 lation whose primary role is to induce follicle bud formation. Korányi fasor 8-10, H-6725, Szeged, Hungary; Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Semmelweis u. 6, 3 P13. H-6725, Szeged, Hungary; Department of Old Age Psychiatry, Institute of Psychiatry Psychology & Neuroscience, King’s College London, Box Induced neurogenesis in rabbit mesenchymal stem and PO70, De Crespigny Park, Denmark Hill, SE5 8AF, London, UK endothelial progenitor cells Jaromír Vašíček 1,2, Andrej Baláži 1, Miroslav Bauer 1,3, Keywords: Rotifer, Rotimer, Biopolymer, Beta-amyloid, Aggregation Mária Tirpáková 4, Andrea Svoradová 1,5, Peter Chrenek 1,2 Neurodegenerative diseases are predominantly initiated and inten- 1 NPPC - Research Institute for Animal Production Nitra, Lužianky, sified by the systemic enzyme resistance of toxic aggregated Slovakia; 2 Faculty of Biotechnology and Food Science, Slovak University proteins and related pathological consequences. Application of of Agriculture in Nitra, Nitra, Slovakia; 3 Faculty of Natural Sciences, neurodegeneration-related aggregates in microinvertebrate roti- Constantine the Philosopher University in Nitra, Nitra, Slovakia; fers is a novel interdisciplinary approach in our in vivo research. 4 AgroBioTech Research Center, Slovak University of Agriculture in The ability to utilize various types of native conglomerates and Nitra, Nitra, Slovakia; 5 Faculty of AgriSciences, Mendel University in aggregates is a phylogenetically selected property of these ani- Brno, Brno, Czech Republic mals. In current experiments, we applied the Euchlanis dilatata and Keywords: rabbit, stem cells, neuronal induction Lecane bulla monogonant rotifer species with a special biopolymer secreting capability. We demonstrate a special phenomenon that Mesenchymal stem cells (MSCs) are known to possess multipotent the rotifer-specific biopolymer, namely Rotimer, can serve as the ability and can be differentiated into adipocytes, chondrocytes and subject of in vitro research where it is able to bound specifically osteocytes. Moreover, a neurogenic potential of MSCs has been also the human-type neurotoxic beta-amyloid 1-42 (H-Aβ). In this work observed (Tirpáková et al., 2021). On the other hand, endothelial was reapplied the aggregated H-Aβ and its artificially designed progenitor cells (EPCs) might be defined as unipotent stem cells, scrambled 1-42 (S-Aβ) version on Rotimer. Previously we proved since they are closely similar to stem cells in terms of self-renew- that the rotifers are capable of using these toxic aggregates as an ability, clonogenicity, and their plasticity. Besides their unipotent exclusive energetic source (as ‘food’). In contrast to H-Aβ, the S-Aβ character, a possible trans-differentiation to neuron-like cells has version was detected as a relevant negative control and an inactive been previously reported (Vašíček et al., 2021). Here, we compare molecule in aggregation-related experiments. The biopolymer-se- the neurogenic differentiation potential of rabbit MSCs derived from cretion-capacity of rotifers is protective against H-Aβ aggregates; bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and adipose furthermore, we found that the exogenic Rotimer exudate is a spe- tissue (AT-MSCs), and rabbit EPCs derived from peripheral blood cial anti- and disaggregating agent against the oligomer and fibril- (PB-EPCs) and bone marrow (BM-EPCs). lar form of H-Aβ in optical imaging and fluorescent-based assays. Briefly, cells from each biological sources were isolated from healthy The above mentioned species shows different reactions to the drug and sexually mature rabbits of New Zealand White line. Cell lines treatments in all type of measurements. Our results may offer new were cultured in specific media till the passage 3 as described pre- pharmaceutical perspectives on the human relevance of neurode- viously. Then, the commercial medium for the neurogenic differen- generative diseases. tiation of cells (Mesenchymal Stem Cell Neurogenic Differentiation Medium; PromoCell) was applied and cell were cultured for 3 days according to the producer’s manual. 24 ABSTRACTS | POSTER PRESENTATIONS

P15. P16. Effect of the equilibration time on post-thaw quality of ram Development of dentin microstructure is controlled by the type sperm of adjacent epithelium Vozaf Jakub 1, Makarevich Alexander 2, Balazi Andrej 2, Marcos Gonzalez-Lopez 1, Josef Lavicky 1, Magdalena Kolouskova Vasicek Jaromir 1,2, Svoradova Andrea 2,3, Dujickova Linda 2, 1, David Prochazka ², Vladislav Rakultsev 1, Klara Steklikova 3,4, Olexikova Lucia 2, Chrenek Peter 1,2 Jozef Kaiser 2, Pavel Porizka 2, Maria Hovorakova 3, Mina Mina 5, 1 Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Jan Krivanek 1 Food Science, Tr. A. Hlinku 2, 94901 Slovak Republic; 2 NPPC, Research 1 Department of Histology and Embryology, Faculty of Medicine, Masaryk Institute for Animal Production Nitra, Hlohovecka 2, 95141 Lužianky- University, Brno, Czech Republic; 2 Group of Advanced Instrumentation near-Nitra, Slovak Republic; 3Department of Animal Morphology, Phys- and Methods for Materials Characterization, CEITEC Brno University of iology and Genetics, Faculty of AgriSciences, Mendel University in Brno, Technology, Brno, Czech Republic; 3 Institute of Histology and Embryol- Zemědělská 1, 613 00, Brno, Czech Republic. ogy, First Faculty of Medicine, Charles University, Prague, Czech Republic; 4 Department of Cell Biology, Faculty of Science, Charles University, Keywords: sheep, sperm, programmable freezing, equilibration, Prague, Czech Republic; 5 Department of Craniofacial Sciences School of motility, viability Dental Medicine, University of Connecticut, Farmington, Connecticut. The aim of our research was to improve ram sperm post-thaw qual- Keywords: teeth, dentin microstructure, odontoblast, odontogen- ity through optimization of cryopreservation regimen. We tested esis, dentinogenesis different lengths of equilibration (EL; 0, 2, 4, 6 and 8 hours) and storage in liquid nitrogen (LN; 1, 10 and 20 days) of ram sperm The research focused on dentin mineralization and odontoblasts´ dif- cryopreserved using programmable device. The ejaculates were ferentiation have emerged in the last decade due to the potential collected from clinically healthy and sexually mature rams (n = 12) applications in the dental tissue engineering field. Despite this, deeper of Wallachian sheep breed (2.5-5 years) twice a week by an elec- study about the differential role (functionally and spatially) of dental tro-ejaculation within the autumn season. Sperm doses were epithelium during odontoblasts’ development is still needed. extended in a Triladyl diluent containing glycerol, egg yolk and anti- In this poster, we compared the morphology and function of the biotics. Following thawing, the sperm samples were analysed for dentin in mice crown and roots. Using a reporter (DSPP-cerulean/ the motility (CASA), viability (SYBR-14/PI) and in vitro penetration/ DMP1-cherry) mouse strain and knockout mice with ectopic enamel fertilization (P/F) assays. It was found that the EL of 6 hours (EL-6) (Spry2+/-; Spry4-/-) we showed how contrasting microstructures of ensured higher post-thaw sperm total (84.8±1.48%) and progressive dentin are initiated since the early dentin matrix production and it is (74.8±2.17%) motility compared to other lengths tested. Moreover, maintained along with the dentin growth. This event is controlled by at 6 h of EL, the duration of storage in LN did not affect the sperm the different inductive role of adjacent epithelium. Thus, depending motility. Using a SYBR-14/PI assay, the proportion of viable versus on the type of interacting epithelium we propose two new dentin dead spermatozoa (52.85% vs. 32.89%, resp.) in the cryopreserved types: cementum vs. enamel-facing dentin. Finally, we showed cal- group significantly differed from those in the control group (79.86% cium and magnesium distribution in these two kinds of dentin by and 14.07%, resp.). Fertilizing ability of cryopreserved sperm (EL-6), utilizing spatial element composition analysis (LIBS). examined by in vitro P/F test on bovine mature oocytes (60.44%), Consequently, variations in dentin inner structure and composition was slightly lower compared to the control group (72.82%). How- are the outcome of different developmental origin since the very ever, in the percentage of fertilized zygotes (two pronuclei) the beginning of tooth development. Taken all together, our results elu- EL-6 group (57.14%) did not differ from the control (52.26%). These cidate the different effect of two main types of dental epithelium, results provide evidence that extension of equilibration time up to 6 important for either crown or root formation, on adjacent odonto- h can improve post-thaw quality of ram sperm. blasts which give rise to dentin of different quality. Acknowledgements: This research was funded by the Slovak Research and Development Agency (grant no. APVV-17-0124, P17. APVV-20-0006) and by the Scientific Grant Agency of the Ministry Role of Headcase in Drosophila hematopoiesis: what we know so far of Education, Science, Research and Sport of the Slovak Republic Bayan Kharrat 1,3, Gergely István Varga 2, Ferenc Jankovics 4, and Slovak Academy of Science (grant no. VEGA 1/0049/19). Rita Sinka 2, Enikő Sutus 1, Erika Gábor 1, Viktor Honti 1 1 Drosophila Blood Cell Differentiation Group, Institute of Genetics, Biolog- ical Research Centre, Szeged, Hungary; 2 Department of Genetics, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; 3 Doctoral School of Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; 4 Laboratory of Drosophila Germ Cell Differentiation, Institute of Genetics, Biological Research Centre, Szeged, Hungary Keywords: Headcase, Drosophila, hematopoiesis, blood cell, differ- entiation Drosophila melanogaster is an excellent model to study hemato- poiesis, since its signaling pathways controlling blood cell differen- tiation events are highly conserved. During our search to identify new regulators of hematopoiesis in Drosophila, we isolated Head- ABSTRACTS | POSTER PRESENTATIONS 25

case (Hdc), the orthologue of HECA, a tumor suppressor in humans. developed method based on ex vivo blood cell transdifferentia- We found that by modulating the Hedgehog and Decapentaplegic tion observed by cell-type specific transgene expression patterns. signaling pathways, Hdc controls differentiation of hemocyte pre- Morphological features are monitored with time-lapse microscopy, cursors in the lymph gland, a major hematopoietic organ in the Dro- images are analyzed using a deep learning-based algorithm. We sophila larva. Recent findings also suggests that as a component in render precursors, intermediate cells and terminally differentiated the insulin receptor/mTOR pathway, Hdc plays a role in the protec- cells to a regression plane that reveals differentiation routes and cell tion against cellular stress. fates. Using an extended regression plane for hemocytes isolated So far, no conserved functional domains were described in Hdc, from immune-induced and tumorous larvae will help understand and only two interacting partners of the protein were identified. To the differentiation events under various conditions. reveal the molecular function of Hdc, we generated HA-tagged Hdc Our research is funded by the OTKA K-131484 (VH) and NKFIH-871- isoforms, and using LC-MS/MS, we identified a list of new potential 3/2020 grants. interacting partners. Two of the candidates, unkempt and SUMO also showed genetic interactions with Hdc, which suggests that P19. these proteins contribute to progenitor maintenance in the lymph Actin based membrane nanotubes in developing zebrafish gland. In addition, we identified a phosphorylated peptide of the embryos Hdc protein, indicating that post-translational modification plays a Katalin Türmer 1, Miklos Nyitrai 1,2, Edina Szabo-Meleg 1,2 role in the regulation of Hdc function. 1 Department of Biophysics, Medical School, University of Pés, Pés, Hun- We aim to better understand the mechanisms via which Hdc pro- gary; 2 Szentagothai Research Centre, University of Pés, Pés, Hungary tects hematopoietic progenitors against cellular stress, and main- tains their state by the inhibition of premature differentiation. Keywords: actin, zebrafish embryos, membrane nanotubes Funding: this work was supported by the OTKA K-131484 (VH) and Membrane nanotubes (NT) were first reported in 2004 between rat NKFIH-871-3/2020 grants. pheochromocytoma (PC12) cells. These actin-based cell protrusions seem to be channels for intercellular communication [1]. Membrane P18. nanotubes were described both in vitro (in cell cultures) and in vivo A machine learning based approach to identify blood cell fates between zebrafish embryos [2]. We focused on the in vivo charac- in Drosophila melanogaster terization of membrane nanotubes in developing zebrafish embryos. Aliz Géczi 1, Erika Gábor 1, Bayan Kharrat 1, Ede Migh 2, Attila Laser-scanning confocal microscope and transmission electron Beleon 2, Péter Horváth 2, Bence Széplaki 3, Márton Enyedi 3, Lajos microscopy (TEM) were applied to examine the NTs between the Pintér 3, Lajos Haracska 4, Viktor Honti 1 epiblast cells of the embryos. 1 Biological Research Centre, Institute of Genetics, Drosophila Blood The characterization of membrane nanotubes shows that their Cell Differentiation Group, Szeged, Hungary; 2 Biological Research Cen- length in 4-5 hours old embryos is approximately identical with the tre, Institute of Biochemistry, Lendület Laboratory of Microscopic Image individual cell diameters. In the case of 6-7 hours old embryos, the Analysis and Machine Learning, Szeged, Hungary; 3 Delta Bio 2000 Ltd., tubes are considerably longer and their occurrence is pronounced in 4 the animal pole of the embryo. Szeged, Hungary; Biological Research Centre, Institute of Genetics, Lab- Our data clearly support the in vivo existence of NTs, but further oratory of Mutagenesis and Carcinogenesis Research, Szeged, Hungary experiments are needed to reveal their possible function in zebraf- Keywords: Drosophila, blood cell, transdifferentiation, machine ish embryo. learning, regression plane References: [1] Gerdes, H.-H. (2008) Intercellular transfer medi- The fruit flyDrosophila melanogaster has been widely used to study ated by tunneling nanotubes. Curr Opin Cell Biol 20(4):470–475. immunity and blood cell differentiation. Although, according to our [2] Caneparo L., Pantazis P., Dempsey W., Fraser S. E. (2011) Inter- recent knowledge, Drosophila lacks an adaptive immunity, the func- cellular Bridges in Vertebrate Gastrulation. PLoS One 6(5):e20230. tions of its immune cells (the hemocytes), such as phagocytosis, P20. encapsulation and coagulation are similar to those of vertebrate Rabbit inner cell mass retains potential to differentiate into the myeloid cells. Besides these mechanisms, the signaling pathways, trophectoderm lineage transcription and epigenetic factors playing key roles in blood cell Katarzyna Filimonow 1, Anna Chołoniewska 1, Jan Chołoniewski 2, differentiation are highly conserved. Therefore,Drosophila is an Zofia Eliza Madeja3 , Katarzyna Barłowska 1, ideal model organism for the study of the regulation of hemato- Elżbieta Wenta-Muchalska 1, Berenika Plusa 4, Anna Piliszek 1 poiesis and tumor formation caused by misregulated blood cell fate. 1 Department of Experimental Embryology, Institute of Genetics and Animal Previously, we showed that phagocytic larval plasmatocytes, Biotechnology of the Polish Academy of Sciences, Jastrzebiec, Poland; which were believed to be terminally differentiated effector cells, 2 Center of Excellence for Complex Systems Research, Faculty of Physics, are capable of transdifferentiating into encapsulating lamellocytes. Warsaw University of Technology, Warsaw, Poland; 3 Faculty of Veter- Recently, novel cell types were discovered in the larva, however, inary Medicine and Animal Sciences, Poznan University of Life Sciences, their differentiation routes are not well characterized. The signaling Poznan, Poland; 4 Division of Developmental Biology & Medicine, Univer- pathways and transcriptional regulatory events that contribute to sity of Manchester, Manchester, United Kingdom the activation of plasmatocytes and lead to lamellocyte differentia- tion are only partially understood. Keywords: lineage specification, trophectoderm, rabbit Our aim is to characterize the possible differentiation routes from In the course of mammalian development initial state of totipo- plasmatocytes to lamellocytes. For this, we are utilizing our newly tency has to be lost to allow acquisition of a specific cell fate. The 26 ABSTRACTS | POSTER PRESENTATIONS

first differentiation event results in the formation of trophectoderm P22. (TE) and the inner cell mass (ICM). In the mouse embryo the cell Organoids as a novel powerful in vitro tool for ovarian cancer fate of these two compartments is set quickly after formation of research a blastocyst. Here we present a detailed analysis of development Jana Kramářová 1, Anna Kotrbová 1, Veronika Bosáková 2, Markéta and cell fate of rabbit isolated ICMs. Our study points to significant Bednaříková 3, Jitka Hausnerová 4, Vít Weinberger 5, Jan Frič 2, inter-species differences, most notably en extended period of ICM Vítězslav Bryja 1, Vendula Pospíchalová 1 potency to differentiate into TE lineage. 1 Department of Experimental Biology, Faculty of Science, Masaryk Univer- We first identify GATA3 as an early marker of rabbit TE and CDX2 sity, Kotlářská 2, 611 37, Brno, Czech Republic; 2 International Clinical as a marker of more mature TE, as CDX2 expression is initiated at Research Center, St. Anne’s University Hospital Brno, Pekařská 63, 656 mid-blastocyst stage. We then analyse developmental potential 91, Brno, CzechRepublic; 3 Department of Internal Medicine - Hematol- of rabbit ICMs isolated by immunosurgery and subsequently cul- ogy & Oncology, University Hospital Brno and Medical Faculty, Masaryk tured in vitro. ICMs originating from early- to mid-blastocyst stage University, Jihlavská 20, 625 00, Brno, Czech Republic; 4 Department embryos are able to re-form a blastocyst-like structure, with a func- of Pathology, University Hospital Brno and Medical Faculty, Masaryk tional TE, and an ICM containing both SOX2-positive epiblast cells University, Jihlavská 20, 625 00, Brno, Czech Republic; 5 Department of and SOX17-positive primitive endoderm cells. We further observe Obstetrics and Gynecology, University Hospital Brno and Medical Faculty, that rabbit ICMs isolated form later blastocyst stages lose the ability Masaryk University, Jihlavská 20, 625 00, Brno, Czech Republic for TE specification. Instead, these ICMs form a halo-like cavity with an outer layer of SOX17-positive cells, indicating that the potential Keywords: ovarian cancer, high grade serous carcinoma, organoids, for TE differentiation gives way to formation of a different type of ascites epithelial extraembryonic layer. Ovarian cancer (OC) is a complex disease comprising several diverse subtypes varying in cell of origin, prognosis and progression. High P21. grade serous ovarian carcinoma of the ovaries, fallopian tube and Deciphering enamel decussation patterns by mapping of line- peritoneum (HGSC) is the most common (70 %) and lethal subtype age-traced epithelial stem cells of ovarian cancer. HGSC represents the most outstanding clinical Vladislav Rakultsev, Josef Lavicky 1, Jan Krivanek 1 challenge in gynecological oncology due to its propensity to relapse Department of Histology and Embryology, Faculty of Medicine, Masaryk following chemotherapy and rapidly metastasize. University, Brno, Czech Republic Mechanisms of HGSC pathobiology are still poorly understood and patient survival is not significantly improving. Particularly in Keywords: tooth, enamel, amelogenesis, decussation, enamel rods advanced HGSC, extensive seeding of the peritoneal cavity by OC Tooth enamel is an extremely hard and resilient complex tissue, cru- cells is often associated with the formation of malignant ascites, cial for mastication. It is formed in a process of amelogenesis, by which indicates poor prognosis for the patient but represents a con- specialized cells – ameloblasts. As ameloblasts develop, grow and venient source of tumor cells for research. secrete matrix components, distinct structures, known as enamel 3D in vitro tumor organoids capture disease hallmarks, recapitu- rods, emerge. It has long been known that enamel rods alternate late patient tumor characteristics, and have emerged as an efficient positions and overlap with each other, forming peculiar decussation model for cancer development research. However, the protocol for patterns. However, there is still no sufficient explanation of how OC organoid derivation was published only recently. Hence, we focus these patterns form, and how that process is initiated and regulated here on generating the organoid cultures from malignant ascites of during odontogenesis. In this study, we’ve visualized the formation OC and their characterisation using e.g. confocal microscopy and of decussation patterns using lineage tracing in a model system of flow cytometry. We believe this may be useful for evaluating the mouse incisors. We genetically traced epithelial stem cells, marked pathophysiology and propensity of OC dissemination. The organ- them with fluorescent reporter proteins. Using this state-of-the-art oids pave the way for personalized approach as they can serve as system, we were able to observe the enamel growth and map the important tools for improving treatment stratification of patients. development backwards in time. We suppose that ameloblasts’ fate and growth direction is decided in the very beginning of amelogen- P23. esis, in the apical part of the tooth – cells split and continue moving In vitro evaluation of apoptotic effect of polyphenol-rich in two distinct trajectories. Our results give a better understanding pomegranate peel extract on human ovarian cells of the aforementioned processes. We anticipate these results to be Simona Baldovská 1, Ladislav Kohút 2, Michal Mihaľ 3, followed by further functional research, which will be relevant for Adriana Kolesárová 3 reparative dentistry and generally broaden the existing understand- 1 AgroBioTech Research Centre, Slovak University of Agriculture in ing of tooth development. Nitra, Nitra, Slovak Republic; 2 Department of Small Animal Science, Faculty of Agrobiology and Food Resources, Slovak University of Agri- culture in Nitra, Nitra, Slovak Republic; 3 Department of Animal Physi- ology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic Keywords: pomegranate peel, ovarian cells, cancer, apoptosis Pomegranate peel presents a rich source of polyphenols with potential anti-cancer properties. Tumor suppressor proteins p53 ABSTRACTS | POSTER PRESENTATIONS 27

and p21 play a key role in suppressing carcinogenesis by induc- ysis and found that fliI mutant myofibrils were indistinguishable ing cell cycle arrest and triggering apoptosis. Apoptosis-inducing from control myofibrils during the early steps of myogenesis. How- factor is a mitochondrial protein, which shown to mediate caspas- ever, after initial assembly of the sarcomeres at the beginning of es-independent cell death. This in vitro study aimed to examine in myofibrillogenesis, sarcomere growth seems to be arrested in fliI vitro effect of pomegranate peel extract at a concentration range mutants. Furthermore, we observed progressively growing ectopic from 12.5 to 100 µg/ml (24 h) on human ovarian carcinoma cell actin aggregates, which later led to the complete disruption of the line (OVCAR-3) and human ovarian granulosa cells (HGL5) as cell organized IFM structure. Overexpression of truncated forms of FliI models. Tumor suppressor p53, cyclin-dependent kinase inhibitor 1A demonstrated that the gelsolin 1-3 domains play a critical role in (CDKN1A; p21), and apoptosis-inducing factor (AIF) were evaluated radial growth of the sarcomeres. Based on confocal and dSTORM by the enzyme-linked immunosorbent assay (ELISA). The pome- measurements of growing and fully mature sarcomeres, FliI is local- granate peel extract significantly increased p53 and p21 levels in ized to the ends of the actin based thin filaments in the Z disk, OVCAR-3 cells at the concentrations of 50 µg/ml (P≤0.05) and 100 where it most likely regulates the assembly and/or turnover of the µg/ml (P≤0.001) in comparison to control. Moreover, AIF levels thin filaments. were significantly (P≤0.05) increased at the concentrations 25, 50, and 100 µg/ml of pomegranate peel extract. On the other hand, P25. non-cancerous HGL5 cells were not affected by pomegranate peel Effects of sea buckthorn extract on human ovarian granulosa extract. In conclusion, the results show an increase in tumor protein and cancer cells in vitro p53, p21, and AIF levels in cancer cells OVCAR-3 after pomegranate Michal Mihaľ 1, Simona Baldovská 2, Erika Mňahončáková 3, peel treatment in a dose-dependent manner. Our data suggest the Adriana Kolesárová 1 potential role of pomegranate peel polyphenols for the regulation of 1 Department of Animal Physiology, Faculty of Biotechnology and Food apoptotic signaling pathway and prevention or treatment of ovar- Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 ian cancer by induction of apoptosis. However, further research is 76 Nitra, Slovak Republic; 2 Research Centre AgroBioTech, Slovak Uni- warranted to determine and better understand the full potential of versity of Agriculture in Nitra, Nitra, Slovak Republic; 3Botanical Gar- pomegranate peel as a promising agent in therapeutic applications den, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic in the future. Acknowledgement: This work was supported by the Ministry of Keywords: sea buckthorn, ovarian cells, apoptosis, steroid hormone Education, Science, Research and Sport of the Slovak Republic Sea buckthorn is an important medicinal plant with positive effects projects APVV-18-0312, DS-FR-19-0049, VEGA 1/0266/20, KEGA on human health. Recent studies have demonstrated the ability 033SPU-4/2021, The Excellent scientific team “Center of Animal of sea buckthorn and its flavonoids to regulate anti-apoptotic and Reproduction (CeRA)”, the Operational Program Integrated Infra- pro-apoptotic members in cancer cells. The aim of this study was to structure within the project: Demand-driven research for the determine the effect of ethanol sea buckthorn extract (Slovakia) at sustainable and innovative food, Drive4SIFood 313011V336, co-fi- concentrations 0; 12,5; 25; 50; 100; 200 µg/ml (24 h) on the viability nanced by the European Regional Development Fund, and AgroBi- of human ovarian carcinoma cell line (OVCAR-3) and human ovar- oTech Research Centre built in accordance with the project Building ian granulosa cell line (HGL5), as well as caspase-3 levels and also ,,AgroBioTech” Research Centre ITMS 26220220180. secretion of steroid hormones (17ß-estradiol and progesterone). Cell viability was evaluated by AlamarBlueTM cell viability assay, caspase-3 P24. levels, and the release of steroid hormones was assayed by ELISA Flightless-I regulates sarcomere growth by promoting actin methods. In our research, we did not observe any significant changes assembly in viability of ovarian cells HGL5 and OVCAR-3. On the other hand, Péter Görög 1,2, Szilárd Szikora 1, Dávid Farkas 1, József Mihály 1 the caspase-3 level was significantly (P≤0.05) increased at concen- 1 trations 50 and 100 µg/ml in cancer cell line OVCAR-3, but not in Biological Research Centre, Eötvös Loránd Research Network, Szeged, HGL5 cells. Progesterone release by HGL5 cells was not significantly Hungary; 2 Doctoral School of Multidisciplinary Medical Science, Fac- affected. However, a significant (P≤0.05) decrease of the 17ß-es- ulty of Medicine, University of Szeged, Szeged, Hungary tradiol secretion by extract at concentration 25 µg/ml in HGL5 cells Keywords: Flightless-I, Indirect flight muscle, dSTORM was observed. The understanding of the mechanisms of action of Flightless-I (FliI) have long been identified as a key factor of flight bioactive substances present in sea buckthorn is essential due to its pro-apoptotic effect on ovarian cancer cells, and inhibitory effect on muscle development, as fliI mutant flies are unable to fly due to 17ß-estradiol secretion by the ovarian granulosa cells. Further stud- myofibrillar abnormalities. FliI is an evolutionary conserved protein ies are needed to fully understand the impact. composed of leucine-rich repeats and gelsolin homology domains, Acknowledgment: This work was supported by the Ministry of Edu- which are characteristics of two separate protein families. These cation, Science, Research and Sport of the Slovak Republic projects domains are generally known for protein-protein, protein-lipid APVV-18-0312, DS-FR-19-0049, VEGA 1/0266/20, KEGA 033SPU- interaction, and for actin binding, capping and severing activity, 4/2021, The Excellent scientific team “Center of Animal Reproduc- respectively. However, characterization of the molecular mech- tion (CeRA)”, the Operational Program Integrated Infrastructure anisms of FliI is completely missing. To address this question we within the project: Demand-driven research for the sustainable performed a detailed loss of function analysis which revealed that, and innovative food, Drive4SIFood 313011V336, co-financed by the in addition to the previously observed disordered indirect flight European Regional Development Fund, and AgroBioTech Research muscle (IFM) organization, the intact sarcomeres are significantly Centre built in accordance with the project Building ,,AgroBioTech” smaller in fliI mutants. We also performed a developmental anal- Research Centre ITMS 26220220180. 28 ABSTRACTS | POSTER PRESENTATIONS

P26. are differences in their substrate specificity. We have focused our Comparison of asymmetric RNA localization within sterlet studies on DUSP6, DUSP7 and DUSP9, three closely related cyto- (Acipenser ruthenus) and African clawed frog (Xenopus laevis) solic phosphatases which preferentially dephosphorylate extracel- oogenesis lular signal-regulated kinases (ERK). We have observed that mouse Iegorova Viktoriia 1, Naraine Ravindra 1, Abaffy Pavel1 , embryonic stem cells depleted of any of these phosphatases do Psenicka Martin 2, Sindelka Radek 1 retain some of their basic characteristics, however during the in 1 Institute of Biotechnology of the Czech Academy of Sciences - BIO- vitro differentiation and the formation of early cardiac mesoderm CEV, Prumyslova 595, Vestec, 252 50, Czech Republic; 2 University of repressed, indicating that the studied DUSPs play a role in normal South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of cardiomyocyte development. Since the formation of cardiomyo- Waters, South Bohemian Research Center of Aquaculture and Biodiversity cytes is a complex process which involves correct spatiotemporal of Hydrocenoses, Zatisi 728/II, 389 25 Vodnany, Czech Republic activation/inactivation of MAPK as well as other signalling path- ways we focus our attention on studying how lack of selected Keywords: oogenesis, RNA localization, TOMO-seq, sterlet, Acip- DUSPs affects these pathways at different time points during the in enser ruthenus, Xenopus laevis vitro differentiation of cardiomyocytes. Organism development is a complex process that starts with bio- molecule organization, axis formation, development of the germ lay- P28. ers and then tissue specification. Most of the RNAs and proteins are Determination of proteins whose degradation is necessary for accumulated during the first stages of oogenesis within the vegetal embryonic genome activation in cattle pole, where they are shifted by METRO and late pathways. During Veronika Kinterova 1, Jiri Kanka 1, Alexandra Bartkova 1,2, oocyte development, RNAs and proteins can be found throughout Tereza Toralova 1 the whole egg and form animal/vegetal (A/V) axis, where they are 1 Institute of Animal Physiology and Genetics, Czech Academy of synthesized and stored for use during embryogenesis. However, the Sciences, Libechov, Czech Republic; 2 Faculty of Natural Sciences, Con- principles behind their distribution within the developing egg have stantine the Philosopher University in Nitra, Nitra, Slovak Republic still not been completely determined. To better understand these Keywords: maternal proteins, preimplantation development, mechanisms, we studied the oocytes from the African clawed frog embryonic genome activation (Xenopus laevis) and sterlet (Acipenser ruthenus) which belong to different classes:Amphybia and Actinopterygii respectively. Early embryonic development is driven by maternal mRNAs and Sterlets are ray-finned bony fishes that share strong cytological proteins synthesized during oogenesis. These reserves are used similarities during oogenesis with Amphibians. During early embry- until embryonic genome activation (EGA), when transcription onic development, sterlet represent a completely dividing embryo from embryonic genome starts. Maternal mRNAs are gradually with its stored yolk located in the vegetal hemisphere. The vegetal degraded, but there is still not much information about maternal pole blastomeres do not differentiate into embryonic tissue. In con- protein degradation. Some maternal proteins are stored through- trast, Xenopus embryos vegetal hemisphere develops into meso- out the whole preimplantation development, but it is assumed that dermal and endodermal tissues. at least some of them need to be degraded for successful EGA. In this research, we utilized RT-qPCR and TOMO-seq analysis to Recently, first proteins were found to be necessarily degraded determine RNA localization along the animal-vegetal axis of oocytes before EGA in mouse, Xenopus and Drosophila (TAB1, TOPB1, from stages III-VI. We were able to detect the maternally localized DRF1/DRF4B, CDC6, and CDC25A). But to the best of our knowl- genes that remained permanently localized within the extremely edge, no maternal protein whose degradation is necessary for EGA vegetal, vegetal, animal and extremely animal poles and other local- in cattle was found up to now. ized genes that migrated later during oocyte development. The candidate proteins were selected in two different ways – mam- malian homologs of proteins whose degradation is necessary for P27. MBT in lower organisms (Xenopus, Drosophila) and by mass spec- Sweeping DUSP out of the time window trometry. Up to now results from expression profiles of DBF4B, Martina Bőhmová, Stanislava Sladeček, Katarzyna Anna Radasz- TOPB1, CDC25A and CDC6 showed that in all cases a substantial kiewicz, Tomáš Gybeľ, Tomasz Witold Radaszkiewicz, Jiří Pacherník decrease of protein amount occurred around EGA (TOPBP1 and Department of Experimental Biology, Faculty of Science, Masaryk Univer- CDC25A at early 8-cell stage; DBF4B and CDC6 at L8c i.e. EGA of sity, Brno, Czech Republic cattle). This can suggest that degradation of these proteins start before EGA and is for the successful EGA necessary. Up to now Keywords: DUSP, MAPK, cardiomyogenesis results from mass spectrometry showed around 70 proteins exclu- Animal development is a complex process which relays on the inter- sively expressed before EGA and not detected after. Proteins for play of numerous signalling pathways, mitogen activated protein further study will be selected based on their function - necessity for kinases (MAPK) pathway being one of the most vigorously studied. EGA, transcription repressors or initiators of replication. Members of the MAPK protein family are active when phospho- Supported by IGA IAPG Kinterová. rylated and their regulation can be controlled through dephos- phorylation done by several phosphatases including family of dual specificity phosphatases (DUSP). Mouse genome encodes more than twenty DUSPs which differ in their structure, cellular localiza- tion and although they generally are specific towards MAPKs, there ABSTRACTS | POSTER PRESENTATIONS 29

P29. closed by two monolayer epithelial plates approaching each other. Different effects of natural and synthetic glucocorticoids on This late embryogenetic process is based on dynamic cell shape mouse preimplantation development changes which requires dynamic cytoskeletal rearrangements. Štefan Čikoš, Janka Babeľová, Alexandra Špirková, Ján Burkuš, Among others, these changes are regulated by the rearrangements Veronika Kovaříková, Zuzana Šefčíková, Dušan Fabian, Juraj Koppel of the F-actin network. Institute of Animal Physiology, Centre of Biosciences of the Slovak Acad- Of the many actin regulatory proteins, our research group focuses emy of Sciences, Šoltésovej 4-6, 04001 Košice, Slovakia on the functional analysis of the formin protein family. These pro- teins are involved in actin nucleation and elongation. Four of the six Keywords: preimplantation embryo, corticosterone, dexametha- formins in the Drosophila are expressed in the tissues involved in sone dorsal closure. Based on our results it appears that all these formins, Glucocorticoids are released into the bloodstream in a circadian notably DAAM (Dishevelled associated activator of morphogene- manner or in response to stress stimuli. Moreover, synthetic glu- sis), FRL (Formin related in Leukocytes), Formin3 and Dia (Diapha- cocorticoids are widely used drugs in human as well as veterinary nous) affect some dynamic parameter of dorsal closure, presumably medicine, and can further elevate glucocorticoid levels in the organ- affecting cytoskeletal organization. In addition, the possibility of ism. We compared the effects of corticosterone (a major endog- redundancy between the afore-mentioned formins also arises. Fur- enous glucocorticoid in rodents) and dexamethasone (a synthetic ther studies are on the way to determine the cytoskeletal changes glucocorticoid used for therapeutic purposes) on mouse preim- and explore the potentially redundant formin roles. plantation embryos in vitro. Our results showed that no embryos developed to the blastocyst stage in culture medium containing P31. 150 μM corticosterone. Lower corticosterone concentrations sig- Lack of RYBP disrupts mesoderm and cardiac progenitor cell nificantly inhibited embryo development (the proportion of higher formation developmental stages, cell number in blastocysts), but we found no Lilla Kókity 1,2, Surya Henry 1,2, Melinda Pirity 1 significant influence on apoptosis incidence. On the other hand, a 1 Biological Research Centre, ELKH, Szeged, Hungary; 2 Doctoral School significant proportion of embryos developed to the blastocyst stage in Biology, Faculty of Science and Informatics, University of Szeged, in culture medium containing 150 μM dexamethasone. Dexameth- Szeged, Hungary asone decreased cell number in blastocysts, and the effect was Congenital heart disorders (CHD) often arise due to mutations in stronger in ICM cells than in TE cells. In contrast to corticosterone, key transcription factors governing embryonic heart development. dexamethasone significantly increased the proportion of dead cells RING1 and YY1 binding protein (RYBP) is an epigenetic regula- in blastocysts at all applied concentrations (starting from 1.5 μM). tor and a core member of the non-canonical Polycomb repressive By analyzing the presence of active caspase-3 in the dead cells, complexes (ncPRCs), which play crucial role in regulating lineage we detected active caspase-3 staining in 16.3% of the dead cells specific gene expression during development. Previously, our lab- in the control group, in 25% of the dead cells in the corticosterone oratory demonstrated that mouse embryonic stem cells lacking group, and in 63% of the dead cells in the dexamethasone group. Rybp could not form contractile cardiomyocytes (CMCs) upon in Different effects of corticosterone and dexamethasone found in our vitro cardiac differentiation. By whole genome transcriptome analy- study might be associated with activation of different glucocorti- sis of the wild type and Rybp null mutant embryonic stem cells and coid receptor subtypes in early embryonic cells. derivative CMCs, we found that several genes important for cardiac Acknowledgement: This work was supported by the Slovak Acad- progenitor formation including the earliest cardiomyogenic marker emy of Sciences project VEGA 2/0092/19 and the Slovak Research Mesoderm posterior 1 (Mesp1) are under-represented in the mutant and Development Agency project APVV-18-0389. cells. To further dissect the molecular mechanism behind this, we characterised the expression kinetics of mesoderm progenitors P30. Brachyury, Eomesodermin, Gooscoid and the cardiac mesoderm The role of Formins during Drosophila embryonic dorsal clo- markers Mesp1 and Flk-1 in the wt and Rybp-/- cells at the early sure cardiac differentiation phases and determined the protein level of 1, 2 1 1 Krisztina Tóth , István Földi , József Mihály MESP1 in the wt and Rybp-/- cells. We also analysed the binding 1 2 Biological Research Centre, Szeged, Hungary; Doctoral School of of RYBP and its Polycomb cofactor RING1B to the promoter region Multidisciplinary Medical Scienses, Faculty of Medicine, University of of early cardiac marker genes using available ChIP-Seq data from Szeged, Szeged, Hungary cardiac progenitor cells. These results demonstrate the implications Keywords: Drosophila, dorsal closure, formin of RYBP during early mesoderm formation and cardiac progenitor specification during heart development and possibly contributing Epithelial fusion processes are of paramount importance for both for the identification of future medical therapies for conditions like development and wound healing. An excellent model for under- CHD. standing the regulation of epithelial fusion is the Drosophila embry- Acknowledgement: This work was supported by GINOP-2.3.2-15- onic dorsal closure. Although more and more has been understood 2016-00001 and GINOP-2.3.2-15-2016-00039, Hungarian State fel- about the various cellular movements and their coordination since lowship for PhD studies (LK), Stipendium Hungaricum and National the first characterization of the process, a comprehensive analysis Young scientists fellowship (SH). of the proteins involved remains to be seen. During the Drosophila embryonic development an extraembryonic tissue (amnioserosa) protected dorsal hole is formed, which is later 30 ABSTRACTS | POSTER PRESENTATIONS

P32. erable amount of testis-specific transcript synthesised before meio- Differentiation of pluripotent stem cells using odonto- sis, and their transcripts persist in later spermatogenic stages. Here blasts-specific transcription factors we present the structural and functional specializations of mito- Josef Lavický, Tomáš Bárta, Vendula Fridrichová, Lucie Pešková, chondria in testis and some of the key elements of these changes. Aleš Hampl, Jan Křivánek We aim to reveal the role of testis-specific components of mito- Department of Histology and Embryology, Faculty of Medicine, Masaryk chondria; enzymes and structural proteins and describe the simi- University, Brno, CZ larities and differences between the somatic and germ-cell specific mitochondria. Keywords: Odontoblasts, pluripotent stem cells, differentiation, Funding: NKFIH_OTKA 132155, GINOP 2.3.2-15-2016-00032, reprogramming GINOP-2.3.2-15-2016-00035 Teeth are made of three types of tissue: dental pulp, enamel and dentin. Tooth’s morphology, function and mechanical properties P34. are determined by dentin, which is the most abundant hard den- Sirtuin 1 protects oocyte quality within the maternal age and tal tissue. While being indespensible, dentin has only a very lim- is required for early embryonic development ited reparative regenerative capacity. Currently, there are no widely Jan Nevoral 1,2, Ladan Monsef 1, María Iniesta-Cuerda 1, used dental treatments utilizing native biological mechanisms for Hedvika Řimnáčová 1, Tereza Žalmanová 3, Šárka Prokešová 3, facilitating tissue regeneration or reparation. Additionally, attempts Milena Králíčková 1,2 to obtain odontoblasts – the dentin producing and repairing cells in 1 Biomedical Center, Faculty of Medicine in Pilsen, Charles University, vitro remain generally unsuccessful, likely due to their highly spe- Pilsen, Czech Republic; 2Department of Histology and Embryology, Fac- cialized, postmitotic phenotype. We designed a novel approach of ulty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic; direct differentiation of pluripotent stem cells into odontoblasts by 3 Institute of Animal Sciences, Prague-Uhříněves, Czech Republic harnessing developmental trajectories constructed using single-cell Keywords: oocyte, embryonic genome activation, Sirtuin 1, epige- RNA-seq data from mouse continuously growing incisor. Here we netics show the changes in the expression patterns of mouse pluripotent stem in response to overexpression of several, recently described, Sirtuin 1, a member of NAD+-dependent histone deacetylases, odontoblast-specific transcription factors after both short- and has been described as an impactful molecule and reproduc- long-term exposure using qPCR and immunohistochemistry. We tion-protective factor, regulating gamete maturation via epigenetic anticipate our results to highlight the role of the overexpressed and non-epigenetic targets. Indeed, our previous findings elu- transcription factors in odontoblast development (differentiation) cidated spindle tubulin and histone code as Sirtuin 1 substrates, and introduce their possible utilization in regenerative dentistry. deacetylated in matured oocytes. In addition to the contribution to oocyte maturation, we postulated Sirtuin 1 role in fertilization P33. and early embryonic development. For elucidation of hypothesis, Mitochondrial dynamics and metabolic remodelling in animal conditional knock-out was used in accordance with Act No. Drosophila spermatogenesis 246/1992 Coll. In authorized animal facility. Females of Zp3::Sirt- Viktor Vedelek 1, Gabor Juhasz 2, Péter Lőrincz 2, Rita Sinka 1 1loxPloxP genotype were mated with wild-type or Sirt1+/- males 1 Department of Genetics, University of Szeged, Szeged, Hungary; or used as Sirt1null oocyte donors following hormonal stimulation. 2 Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd Alternatively, Sirt1null oocytes were subjected to parthenogenetic University, Budapest, Hungary activation and immunocytochemistry. Our observation revealed that at least mono-allelic expression of the Sirt1 gene is required Keywords: Drosophila, spermatogenesis, mitochondria, metabolism for offspring production (Sirt1null x Sirt1+/-). However, biallelic Sirt1 Mitochondria are versatile cellular organelles with the capability to excision leads to the termination of parthenogenetically-activated adapt to the function of host cells. A good example of this is the Sirt1null oocytes, whilst the development cannot pass beyond mitochondrial adaptation in the sperm. The morphology of mito- the 2-cell stage. The phenotype of Sirt1null oocyte was amplified chondria in sperm generally shows striking differences compared to alongside advanced maternal-age, leading to even indolence to hor- mitochondria in somatic cells. Many components of the cell orga- monal stimulation. Taken together, Sirtuin 1 contribution to embry- nelles that present in sperm have testis-specific paralogs, such as onic genome activation is noteworthy, as well as the age-protective the ones that encode mitochondrial components in Drosophila. Two effect of Sirtuin 1 on oocyte quality and female fertility. Obviously, differently specialized mitochondrial derivatives run along the entire several significant targets of Sirtuin 1 remain to be described, length of the 1.8 mm sperm tail. The major derivative is filled with essentially for knowledge transfer to reproductive medicine. paracrystalline material while the minor derivative has a considera- This study was supported by the Czech Health Research Council bly smaller volume, without paracrystalline material accumulation. (NV18-01-00544), the Charles University Research Fund (Progress This type of differentiation requires a series of consecutive develop- Q39), by the MEYS CR, due to project No. CZ.02.1.01/0.0/0.0/16_01 mental steps, with strict regulation. The exceptional morphological 9/0000787, and the grant SVV–2020-2022 No 260 536. changes assume changes in molecular composition and metabolism of mitochondria. Mitochondrial alterations to the greatest extent happen after meiosis. Since post-meiotic transcription is negligible, regulation and realisation of post-meiotic development mainly rely on gene products accumulated in primary spermatocytes. A consid- ABSTRACTS | POSTER PRESENTATIONS 31

P35. altering the length of thin filaments are linked to nemaline myop- Differentiation potential analysis ofDrosophila blood cells athy and dilated cardiomyopathy. Despite a wealth of knowledge Enikő Sutus 1, Nikolett Virág 1,2, Ferenc Jankovics 3, Rita Sinka 4, on various actin regulatory proteins, the molecular basis of thin fil- Viktor Honti 1 ament assembly and length specification in muscle cells remained 1 Drosophila Blood Cell Differentiation Group, Institute of Genetics, poorly understood. Biological Research Centre, Szeged, Hungary; 2 Biology BSc, Faculty of To address this question and follow the growth of thin filaments, we Science and Informatics, University of Szeged, Szeged, Hungary; performed ‘pulse chase’ experiments by expressing GFP-labeled actin 3 Laboratory of Drosophila Germ Cell Differentiation, Institute of Genet- monomers in a temporally regulated manner and assessed their incor- ics, Biological research Centre, Szeged, Hungary; 4 Department of Genetics, poration into the thin filament array. Furthermore, using G-actin spe- cific probes, we characterized the distribution of the myofibrillar actin Faculty of Science and Informatics, University of Szeged, Szeged, Hungary monomer pool in growing and mature sarcomeres. Collectively, this Keywords: Drosophila blood cells, cell cultur, transdifferentiation dataset allowed us to outline a model in which the sarcomeric thin Drosophila melanogaster has been widely used as a model organism filaments grow by a pointed end elongation mechanism, which is sup- to study the immune system and the blood cell associated tumor for- ported by an actin monomer pool in the H-zone of growing sarcomeres. mation. Similarly to vertebrate blood cells, Drosophila immune cells We also identified numerous factors that are involved in the elon- (hemocytes) differentiate in spatially separated compartments in mul- gation of thin filaments and using a single-molecule localization microscopy based approach we found that they accumulate into tiple waves. In the circulation of the naive larva, two types of hemo- two clearly distinct spatial domains of the H-zone. Our localization cytes were identified: the phagocytic plasmatocytes (95%) and the studies provide strong support for an annealing mechanism where melanizing crystal cells (5%). Upon immune induction and in tumorous short actin oligomers form in the midline of the H-zone and subse- larvae, a third type of hemocytes, the lamellocyte differentiates. quently fuse to the pointed ends of existing thin filaments. Interestingly, plasmatocytes – which were previously believed to be terminally differentiated cells – are able to transdifferentiate into P37. lamellocytes in case of immune induction and in tumorous larvae. The latest single-cell transcriptome data also revealed a remarkable Generation of CRISPR/Cas9 knock out mouse model of autism heterogeneity of plasmatocytes, which led to their categorisation spectrum disorder 1 1 1 1 into functional clusters. Tímea Pintér , Zoltán Gál , Nándor Lipták , Orsolya I. Hoffmann , 2 1 To better understand the plasmatocyte-lamellocyte conversion and László Hiripi, Peter Dovc , Lilla Bodrogi 1 its regulation, we established an ex vivo cell culturing method to Hungarian University of Agriculture and Life Sciences, Institute of Genet- 2 study naive, immune induced and tumorous hemocytes. We found ics and Biotechnology, Gödöllő, Hungary; University of Ljubljana, Bio- that hemocytes are easily maintained in cell cultures, especially technical Faculty, Department of Animal Science Ljubljana, Slovenia in the presence of lamellocytes, i.e. isolated from wounded larvae Keywords: DIP2C, CRISPR/Cas9, knock out mouse, autism or from larvae carrying a gain-of-function mutation in the JAK2 homologue hopscotch, which results in hemocyte overprolifera- Autism spectrum disorder (ASD) is a group of early-onset heteroge- tion and melanizing tumours. Slow, but continuous conversion of neous neurological disorders with strong genetic predisposition. The plasmatocytes into lamellocytes, was also observed in our cultures, purpose of our project was to develop gene knock out (KO) mouse which suggests that plasmatocytes maintain their plasticity during model with new generation CRISPR/Cas9 genome editing technology culturing. We use co-cultures of hemocytes expressing cell type to functionally analyze the associations between molecular, func- specific transgenic markers to reveal the cell-autonomous and non- tional and behavioural factors of ASD. Our gene of interest is Dip2C cell autonomous manner of proliferation and transdifferentiation (disconnected interacting protein 2C). Three knock-out founder ani- events of activated plasmatocytes. mals have been produced. Initial characterization of the knock-out This work was supported by OTKA K131484 (VH) and NKFIH-871-3/2020. mouse lines has already started. Mutation analyses of founder ani- mals led to the exclusion of two founder females with 15 basepair P36. deletion and also mosaicism was revealed in a male founder animal. F1 heterozygous animals have been reevaluated with sequencing and Nanoscopic analysis of actin assembly in developing myofibrils only one subpopulation with a 14 basepair deletion was further bred Szilárd Szikora 1,2, Tamás Gajdos 2, Péter Görög 1,3, Péter Bíró 2, Tibor to produce homozygous individuals. CRISPR-Cas9 off target analyses, Novák 2, Csaba Kozma 1, Miklós Erdélyi 2, József Mihály 1,2 histology of the brain and characterisation of reproductive traits are 1 Institute of Genetics, Biological Research Centre, Szeged, Hungary; accomplished and behavioral analyses of mutant animals is planned. 2 Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary; 3 Doctoral School in Biology, Faculty of Science and P38. Informatics, University of Szeged, Szeged, Hungary A novel approach to silence p53 tumor suppressor gene in a Keywords: actin cytoskeleton, myofibril, nanoscopy, Drosophila, somatically transgenic mouse model IFM; dSTORM Anna Georgina Kopasz, Dávid Pusztai, Réka Karkas, Liza Hudoba, In muscle cells, the tropomyosin and troponin decorated actin fila- Gergely Imre, Abdullah Khaldoon Sadiq Ahmed and Lajos Mátés ments or thin filaments form highly organized and stable structures. Institute of Genetics, Biological Research Centre, Szeged, Hungary During myofibril assembly the thin filaments significantly increase RNA interference (RNAi) is a biological process in which small both in length and number. Optimal length of the thin filaments is non-coding RNA molecules silence gene expression transcriptionally precisely regulated, and it is critical for efficient activity. Mutations or post-transcriptionally. In the past decade, this cell physiological 32 ABSTRACTS | POSTER PRESENTATIONS

phenomenon has become a powerful tool to analyze loss-of-func- remained unclear how DAAM regulates the MT cytoskeleton. It can be tion phenotypes, for example the effect of tumor suppressor genes. either a direct effect of DAAM or the regulation can happen indirectly Of the various model systems, in vivo mammalian models can mimic via the modulation of the actin cytoskeleton. human physiological processes most faithfully and the laboratory Genetic interaction studies revealed that FRL has a negligible effect mouse is currently the predominant mammalian species in bio- on axonal growth, which suggests that FRL and DAAM do not have a medical research. However, in mice many genetically engineered redundant function in primary neurons. In order to clarify the role of mutations have embryonic lethal effects, especially those which DAAM in axonal growth, wild type (WT) and mutant transgenes of have cancer „driver” function. Nowadays, the greatest challenge DAAM were expressed to rescue the mutant phenotype of primary of cancer research is the identification of so called „driver” muta- neurons. Our results revealed that WT did, but an actin-processing tions facilitating disease progression, among the more abundant mutant form (I732A) of DAAM did not possess any rescue-capac- „passenger” mutations found in tumors. Each and every gene with ity. Overexpression of a constitutively active form of DAAM in the proved cancer „driver” role may open up new possibilities in the era embryonic nervous system had a strong negative effect which was of cancer treatment research. However, it is no longer possible to abolished by the presence of I732A mutation. clearly identify new “driver” genes using bioinformatics methods In summary, we found that DAAM and FRL do not play redundant based on mutation frequency analysis alone, due to that poten- roles during axonal growth in primary neurons. Since presence of tial candidates are mutated at lower frequencies in cancer samples. the I732A mutation eliminated the rescue-capacity of DAAM, we Thus, there is a growing demand for in vivo experimental systems can state that the actin-processing activity of DAAM is crucial for where cancer „driver” roles may be confirmed. In such a system, axonal growth. candidate oncogenes may be co-expressed with miRNAs that tar- get known or candidate tumor suppressor genes thereby potentiat- P40. ing tumor inductive effects. B cell developmental trajectory modeling based on complex By combining RNAi-based gene silencing and very efficient somatic immunophenotype using CyTOF transgenesis, we obtained an in vivo experimental system that Janovska Pavlina 1, Stancikova Jitka 2, Stuchly Jan 2, allows analysis of the function of potential „driver” genes. We Bakardjieva Marina 2, Valisova Martina 3,4, Plesingerova Hana 1,3, ensured the equalized co-expression of the potential oncogene, the Kotaskova Jana 3,4, Kalina Tomas 2 a Bryja Vitezslav 1 marker gene and the miRNA, by using a well-balanced bidirectional 1 Department of Experimental Biology, Faculty of Science, Masaryk Uni- promoter which was previously characterized in our laboratory. versity, Brno, Czech Republic; 2 CLIP Cytometry, Department of Pediatric Among known tumor suppressors TP53 is the most penetrant being Hematology and Oncology, 2nd Faculty of Medicine, Charles University mutated in 50 percent of human tumors. Therefore, we validated and University Hospital Motol, Prague, Czech Republic; 3 Department our experimental system by efficiently silencing the mouseTp53 of Internal Medicine – Hematology and Oncology, University Hospital gene. By silencing Tp53, we have the opportunity to study potential Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic; cancer „driver” effect of candidate oncogenes identified from large 4 Central European Institute of Technology (CEITEC), Masaryk Univer- human cancer databases. sity, Brno, Czech Republic Acknowledgement: This research work was conducted with the support of the ÚNKP-20-2 – New National Excellence Program Keywords: Mass cytometry, B cells, developmental trajectory of the Ministry for Innovation and Technology from the Source of Mass cytometry (cytometry by time-of-flight – CyTOF) is a tech- the National Research, Development and Innovation Fund and the nique that enables sensitive detection of a high number of protein Szeged Scientists Academy under the sponsorship of the Hungar- markers at single cell level. Importantly, it allows combination of ian Ministry of Innovation and Technology (FEIF/433-4/2020-ITM_ surface protein staining with detection of intracellular markers and SZERZ). specific phosphorylation events, analysis of rare cell subsets and multiplexing of several samples in one run, which is often problem- P39. atic when using standard flow cytometric approach. Role of formin proteins in the cytoskeleton coordination during In this study, we used multiplexing techniques to perform analysis axonal growth of 28 markers that reflect B cell lineage commitment in the early István Földi 1, Krisztina Tóth 1, Ioannis Zygouras 1,3, Péter Gaszler 2, progenitors in bone marrow (BM) and the subsequent maturation Beáta Bugyi 2, József Mihály 1 steps towards transitional, naïve, memory and plasma B cell sub- 1 Biological Research Centre, Szeged, Hungary; 2 University of Pécs, sets found in BM and in peripheral blood (PB) of healthy donors. To Pécs, Hungary; 3 Aristotle University of Thessaloniki, Thessaloniki, Greece avoid possible batch effects, the analysis always combined samples acquired from BM and PB of several donors in one run. Keywords: cytoskeleton, formin, axonal growth We applied in house data analysis pipelines, with modified dimension Axonal growth is carried out by coordinated changes of the actin and reduction tools, which are performing well on the large CyTOF data microtubule (MT) cytoskeleton, regulated by cytoskeleton-associ- and reflect also trajectory inference analysis, which is used to deter- ated proteins. Formins are actin nucleation factors having role in the mine the most probable maturation steps between analyzed B cell assembly of actin filaments. The Drosophila DAAM (dishevelled-as- subsets. This approach is suitable for applications where we do not sociated activator of morphogenesis) and FRL (formin-related pro- expect detection of individual cell clusters but more probably cells tein) formins have been proved to be important in the development transitioning between different stages of the developmental process. of the nervous system. Our preliminary studies showed that the MT Financial support: Czech Science Foundation EXPRO grant GX19- dynamics changed significantly in DAAM mutant neurons. However, it 28347X, MZ ČR – RVO (FNBr, 65269705). ABSTRACTS | POSTER PRESENTATIONS 33

P41. strated a similar learned decrease of feeding behavior when food was Exploring the role of cephalic furrow formation in Drosophila available. Conditional inhibition of a single chemosensory neuron with Marina Belen Cuenca 1, Bruno Cossermelli Vellutini 1, avoidance stripe assays ruled out the possibility of habituation and Pavel Tomancak 1, 2 provided evidence for aversive associative learning. 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dres- Our findings reveal a long-term aversive memory in response to den, Germany; 2 Central European Institute of Technology, Brno, Czech starvation which might help avoid impending adversity. We pro- Republic pose that re-encountering olfactory cues associated with previous episodes of starvation might paradoxically elicit avoidant feeding Keywords: drosophila, cephalic furrow, tissue mechanics behavior by retrieving the stressful memory. Cephalic furrow formation is a major morphogenetic process in Dro- sophila gastrulation, and yet, it remains poorly understood. The fur- P43. row demarcates the head-trunk boundary, a conserved feature of Ontogeny of cortical B cells in bursa of Fabricius bilaterians, and it is established by precise molecular patterning and Nóra Fejszák, Viktória Halasy, Katalin Kocsis, Emőke Szőcs, highly stereotypic morphogenesis. Importantly, the furrow formation Ádám Soós, Nándor Nagy overlaps in time with other gastrulation events and local cell divisions Semmelweis University, Department of Anatomy, Histology and Embryol- (in so-called mitotic domains) that occur in the vicinity of the invagi- ogy, Budapest, Hungary nation site and might influence each other. Later, the furrow unfolds, Keywords: bursa of Fabricius, B lymphocytes, CXCR4 molecule, raising questions about its developmental role. In this work we inves- migration tigated the function of the cephalic furrow using photomanipulation techniques and mutants that fail to initiate the furrow. Performing The avian model provides an excellent experimental system for recoil analysis after laser ablation we found that the invagination studying the development of B lymphopoises because it takes place process is an active mechanism of tissue-pulling, absent in mutants. in a separate organ the bursa of Fabricius (BF). During embryonic In these mutants the tissue buckles creating folds that diverge in development the BF is colonized by CD45+/chB6+ B cell precur- position and dynamics from wild-type furrows. We observed that sors (prebursal stem cells) to form the follicle buds which ultimately the buckling might be a response to tissue compression coming develop into lymphoid follicles. After hatching the B cells exist from from the germband extension and mitotic domains’ enlargement. By the BF via fine capillary network of follicles cortex in order to colo- stopping germband extension with laser cauterisation the folds were nize the peripheral lymphoid organs. As an effort to learn how lym- inhibited in mutants. We hypothesized that the furrow could play phoid cell diversification is regulated in the birds we have produced the role of priming the folding position to avoid chaotic buckling and a panel of monoclonal antibodies (mAbs) against cell suspension symmetry breaking in the embryo morphology during gastrulation. of BF. One of these mAbs (clone: 7H3) recognized a cell surface antigen (MW:~80kDa) expressed by CD45+/chB6+ /CXCR4+ B cell P42. precursors which colonize the bursal anlage. During the embryonic The fear of hunger: starvation-induced aversive associative period, all B cells of BF express the 7H3 antigen. After hatching 7H3 memory in C. elegans expression of the lymphoid follicles gradually diminished, except a Blénesi Szilvia, Hajdú Gábor, Sőti Csaba subpopulation of cortical B cells. Double immunolabeling demon- Semmelweis University, Department of Biochemistry and Molecular Biology strated that 7H3+ B cells occur exclusively in the inner part of the follicular cortex where the B cells show low expression of the cell Keywords: starvation, aversive learning, associative memory, che- migration mediator molecule, CXCR4. Antibody 7H3 blocked follicle mosensory neuron, maladaptive behavior formation in the chorioallantoic explant culture of embryonic bursa. Maladaptive behaviors are linked to eating and metabolic disorders, Our result demonstrate that 7H3 mAb may recognize a novel anti- but how memories of past starvations shape feeding behaviors is gen involved in the migration of lymphoid cells and its expression unclear. The roundworm Caenorhabditis elegans with an entirely is accompanied by different maturation phases of B cells from the mapped connectome, learning and memory capacities is a versa- precursor stage to the emigration from BF. tile model of neuroscience. It is well-known that nematodes reduce OTKA grant: 124740 their attraction towards olfactory cues previously paired with star- vation. Here, we investigated the mechanism of the starvation-in- P44. duced behavioral change. Behind neural progenitors: Possible new role of RYBP and Pau- We employed benzaldehyde (BA) and diacetyl (DA), naturally attrac- par in Pax6 regulation tive food-derived odorants sensed by different chemosensory neu- Lili Adorján 1,2, Enikő Sutus 1,3, Surya Henry 1,3, Melinda Katalin Pirity 1 rons. We confirmed that after a 4-hour starvation in the presence 1 Biological Research Centre, ELKH, Szeged, Hungary; 2 Faculty of of BA or DA, worms significantly reduced their chemotaxis to both Science and Informatics, University of Szeged, Szeged, Hungary; 3 Doc- odorants. Starvation alone or the odorant paired with food did not toral School in Biology, Faculty of Science and Informatics, University of affect naive behavior, excluding olfactory adaptation as a mediator. Szeged, Hungary Further, the persistent change after 24-hour starvation and 4-hour Keywords: neural differentiation, neural progenitors, long non-cod- recovery suggested learned alteration and long term memory forma- ing RNA, luciferase reporter assay tion in food searching behavior. Worms also reduce feeding behavior in the lasting absence of food. Strikingly, reduced rate of pharyngeal Mouse embryos harbouring homozygous mutation for the RING1 pumping in the presence of starvation-associated odorants demon- and YY1 binding protein (RYBP) die in utero while heterozygous 34 ABSTRACTS | POSTER PRESENTATIONS

mutants often exhibit neural tube defects and disorganised neo- We also found a temporary maximum value or even decreased cell cortex indicating the importance of RYBP in vivo in neural devel- number on Day5/Day6 in the serum-treated male lines. Since the opment. Previously, our laboratory has also shown that during in medium replacement was on Day6, this could mean that the cells vitro neural differentiation the expression of key neural genes was depleted some medium component contained by the chicken serum impaired when RYBP was absent. One of the most striking increase until Day 5. However, there was no such proliferation stop in the was in the level of Paired Box 6 (Pax6) mRNA expression. Pax6 is case of the serum-free medium cultures. a key neural transcription factor. Timed and developmentally regu- lated expression of Pax6 is critical for corticogenesis and the initia- P46. tion of neuroprogenitor differentiation. Increase inPax6 expression Elucidating the Role of Planar Cell Polarity in Amoeboid Cell is a prerequisite for neural progenitor formation but high level of Migration via Automatic Image Analysis and Tracking Pax6 prevents consequent terminal differentiation. Štěpán Čada, Olga Vondálová Blanářová, Vítězslav Bryja Pax6 expression drives neural development by regulating key neu- Department of Experimental Biology, Faculty of Science, Masaryk Univer- ronal transcription factors and this mechanism is affected by PAX6 sity, Brno, Czech Republic Upstream Antisense RNA(Paupar), a long non-coding RNA in the Pax6 locus. The mechanism of how Pax6 and Paupar together influ- Keywords: cell polarity, image analysis, cell migration, lymphocytes ence neural differentiation is poorly understood. Planar cell polarity core (PCP core) is a highly evolutionarily con- To unravel this molecular mechanism, we (1) examined the expression served set of proteins involved in development of virtually all Meta- kinetics of Paupar during in vitro neural differentiation of both wild zoa. Existence of this pathway have been most completely described type and Rybp-/- cells, (2) identified and clonedPax6PI promoter for in Drosophila wing epithelia model, where it coordinates orientation reporter assays, (3) performed luciferase reporter assay to determine of actin bristles. While a similar function has been observed in ver- the activity of RYBP and Paupar at the Pax6PI promoter. tebrates i. e. in orientation of inner ear hair cells, the most profound These results highlight the role of Rybp in neural development and effect of PCP core disruption in vertebrates affects developmental establishes a connection amongst transcription factors such as processes requiring active rearrangement of cells or migration, such Pax6, their regulators such as Rybp and a long non-coding RNA as cortical extension and neural tube closure. Furthermore, altered Paupar. expression of PCP core proteins and their proposed activators, non-canonical Wnt ligands, have been shown to promote invasive Acknowledgement: This work was supported by GINOP-2.3.2-15- and migratory character of various types of cancer. Despite studies 2016-00001 and GINOP-2.3.2-15-2016.00039, Stipendium Hungar- showing polarized distribution of these proteins in migratory cells, icum and National Young scientists fellowship. their precise function in these processes remains unresolved. We set out to elucidate the role of PCP core in migration and polar- P45. ity of lymphocytes using automated image analysis pipelines. We The effects of serum-containing and serum-free media on believe this intrinsically polarized model can provide information chicken PGCs about the immediate conserved function of PCP core and thus can András Ecker 1, Bence Lázár 1, Roland Tóth 1, Martin Urbán 1, Nikolett lead to better understanding of this signalling mechanism in other Tokodyné Szabadi 1, Eszter Patakiné Várkonyi 2, Elen Gócza 1 biological contexts. 1 Hungarian University of Agriculture and Life Sciences, Gödöllő, Hun- This work was funded by Ministry of Education, Youth and Sports of gary; 2 National Centre for Biodiversity and Gene Conservation, Gödöllő, the CR (CZ.02.1.01/0.0/0.0/16_025/0007381). Hungary P47. Keywords: PGC, proliferation, chicken, serum The effects of T-2 toxin and zearalenone on the embryonic Primordial germ cells (PGCs) are a powerful tool for gene preserva- development of rabbit and chicken tion, gene modification and several animal healthcare technologies. Urbán M. 1, Tóth R. 1, Szőke Zs. 1, Ebbin N.A. 1, Pintér T. 1, Bodrogi L. 1, However, in most cases, we need many cells to reach the optimal Gócza E. 1 success rate. To accomplish this, we have to optimize the in vitro 1 Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary culture of the PGCs. A suitable method for this is the development of new media and compare it with the existing media. Keywords: mycotoxin, embryo, in vitro culture Our experiment examined the effects of two culture media on the The production of good quality food is one of the most critical aspects male and female cell lines’ proliferation rate and cell number. We used of animal husbandry. However, mould infection is a global problem in the 4ZP male (ZZ) and the 5ZP female (ZW) cell lines. The cells were grains. In our experiment, we examined the effect of T-2 toxin and isolated from HH-14 stage embryos via blood sampling and cultured zearalenone on chicken and rabbit embryonic development. under optimal condition. We used serum-containing and a serum-free Rabbit embryos were washed out one day after insemination. The medium for culturing. On Day 2-7 of culture, we measured the pro- embryos were cultured for three days. In the 2.5 ng/mL T-2 toxin liferation rate of cell lines by Image Xpress’ Pico cell counting device. containing medium, only 63% of the embryos developed into a Our results demonstrated that in the serum-containing medium, blastocyst. At 5ng/mL concertation, the rate of development was we could count significantly higher cell number on Day4, Day5 and 56%. We could detect the T-2 toxin integration in the cytoplasm and Day6 in male lines and on Day4 and Day5 in female lines. However, nucleus of cells of blastocysts. the serum-containing medium 5ZP female cell line had the lowest We observed a significant decrease in the development of embryos proliferation rate on Day7 compared to Day6. compared to the control group. At the 5 ng/mL zearalenone con- ABSTRACTS | POSTER PRESENTATIONS 35

centration, 60% of the embryos reached the morula stage. At P49. the ten ng/mL concentration, 50% of the embryos continued to Creation of a multiallele knockout genotype in rabbit using develop, and at the 25 ng/mL concentration, the embryos did not CRISPR/Cas9 develop further. However, none of the embryos from the three Tímea Pintér 1, Miklós Geiszt 2, Gábor L. Petheő 2, Máté Mihálffy 2, groups reached the blastocyst stage. Gabriella Skoda 1, Nándor Lipták 1, Andrea Kerekes 1, Zsuzsanna Embryos from Partridge colour Hungarian chicken breed were Bősze 1, László Hiripi 1, Lilla Bodrogi 1 injected with T-2 toxin after three days of incubation. Then the eggs 1 Institute of Genetics and Biotechnology, Hungarian University of Agri- were opened on the Day10 of development, and the embryos were culture and Life Sciences (MATE), Gödöllő, Hungary; 2 Department of dissected. At 1ng/mL, T-2 toxin concentration, there was no signif- Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hun- icant decrease in embryo growth; however, at a concentration of gary 5ng/mL, the embryos were 28.6% less developed than the animals in the control group. However, at a concentration of 8ng/mL, only Keywords: NADPH oxidase 4; CRISPR/Cas9; germline multiallelic 48.57% of the embryos developed further after injection. knockout rabbit Recently, non-rodent species have become increasingly important P48. in preclinical studies. The requirement for these animal models is to Generation of induced pluripotent stem cell lines and neural produce reliable and predictable results in addition to representing progenitors from identical diabetic twins complex etiologies and physiology of a genetically diverse patient Katalin Vincze 1,3, Eszter Szabó 1, Dóra Reé 1, Bálint Jezsó 1, Gábor population. Földes 2, Andrea Á. Molnár 2, János M. Réthelyi 3,4, Ágota Apáti 1 In our study we created NADPH-oxidase 4 (NOX4) knockout trans- 1 Institute of Enzymology, Research Center for Natural Sciences, Eötvös genic rabbits using CRISPR / Cas9 technology. This approach can be Loránd Research Network, Hungary; 2 Heart and Vascular Center, Sem- useful for creating genetically divergent and homogeneous popula- melweis University, Budapest, Hungary; 3 Molecular Psychiatry Research tions for studies in translational medicine. Group, National Brain Research Program (NAP), Hungarian Academy of NOX4 is a promising therapeutic target, as it is linked to several Sciences and Semmelweis University, Hungary; 4 Department of Psychiatry pathologies. It may be associated with some neurodegenera- and Psychotherapy, Semmelweis University, Budapest, Hungary tive diseases, bone development, may be involved in immune and inflammatory processes, cell proliferation, the development of Keywords: induced pluripotent stem cells, type 2 diabetes, in vitro tumors, and their presence also affects fibrosis, but its role in many disease modelling other biological processes has been investigated in practice. NOX4 Aims: The main goal of our work is to generate induced pluripotent is present in almost all mammalian tissues, showing high expression stem cells (iPSc) and neural progenitors (NPCs) from monoclonal levels in the renal tubules as well as lower levels in endothelial and twin sisters diagnosed with type 2 diabetes for evaluation of the myocardial cells, suggesting an effect on the cardiovascular system. complications of the disease. We created NOX4 knockout (KO) rabbit lines in order to study the in Methods: We generated induced pluripotent stem cell lines from vivo effects resulting from a lack of NOX4 protein and loss of gene peripheral blood samples of the twins (66 years old) with DM2. We function. One of the knockout founders was a germline multiallelic established two clonal lines from each patient. The samples were knockout male. Its offspring segregated into three distinct NOX4 reprogramed via Sendai viral transduction of the four Yamanaka knockout and a wild-type lines. factors (Oct3/4, Sox2, klf4, cMyc). Determine the cell lines’ identity Although mosaicism is a relatively common phenomenon in rabbit by short tandem repeat analysis, and the genetic integrity by kar- transgenesis, it is even possible to exploit it in preclinical studies yotyping. Our second validation step was to prove pluripotency by with careful planning and evaluation. flow cytometry analysis of surface marker Stage Specific Antigen-4, and the assessment of morphology immunofluorescent staining of P50. Oct4 and Nanog (endogenous pluripotency transcription factors). Patterns of energy utilisation during early animal embryonic In vitro spontaneous differentiation potential was examined via development embryoid body formation. To generate neuronal progenitor cells we Vinca Yadav 1, Pavel Tomancak 1,2, Jonathan Rodenfels 1 used a validated induction protocol. Identified our neuronal progen- 1 Max Planck Institute of Molecular Cell Biology and Genetics (MPI- itors by immunflorescent staining for Sox2 and Nestin. CBG), Dresden, Germany; 2 Central European Institute of Technology Results: Two validated IPS cell lines were generated from both of (CEITEC), Brno, Czech Republic the twins. Future plans: Differentiation of neuronal cell lines from DM2 iPSCs, Keywords: embryonic energetics, isothermal calorimetry, metab- and comparative studies with healthy, sex- and age-matched con- olism trols to characterize the effects of DM2 on neural cultures. Early animal developmental trajectories are diverse, and yet, share Funding: This study was funded by the National Brain Research the common feature that a developing embryo must mobilise its Program (NAP) of Hungary (grant numbers: 2017-1.2.1-NKP-2017- maternally deposited energy stores to fuel its initial cell divisions 00002), National Research, Development and Innovation Office and early morphogenetic events. Typically, the bulk of intracellu- (OTKA- K128369) and PRIME Consortium (H2020 Programme): Pre- lar energy conversion to the form of adenosine triphosphate (ATP) vention and Remediation of Insulin Multimorbidity in Europe. Grant occurs in the mitochondria, highly dynamic organelles known to agreement ID: 847879. undergo rapid changes in morphology and localisation depend- ing on their activity. However, whether mitochondrial dynamics or ABSTRACTS | POSTER PRESENTATIONS

overall patterns of energy expenditure during early embryogenesis ZMYND-11 gene to pluripotent stem cells, then we differentiated are evolutionarily conserved or divergent remains unknown. them into neuronal cell types. We used two differentiation proto- Recent work by Jonathan Rodenfels has shown that heat dissipation cols, resulting in, prox-1 positive hippocampal neurons, and corti- measured by isothermal calorimetry (ITC) is a reliable indicator of cal neurons. With this method we have the possibility to monitor the overall metabolic rate of zebrafish embryos. His results reveal cellular phenotypes during neuronal differentiation, mirroring neu- a dynamic pattern of fish embryonic heat dissipation with specific rodevelopment; potentially discovering alterations relevant to the temporal features. In combination with biochemical and pharma- pathogenesis of schizophrenia. cological approaches, we have now adopted ITC to ask whether Methods: To monitor neuronal function, we use a multi-electrode these patterns of energy dissipation are also observed in embryos array. This device measures the extracellular field potential with with dramatically different developmental modes, starting with a high temporal and spatial resolution. We can detect extracellular marine annelid, Platynereis dumerilii. In addition, we are exploring action potentials, manifesting as spikes, burst, or network burst. the mitochondrial dynamics and distribution in these early embryos. The synchronisation of electric activity is a sensitive indicator of Furthermore, we are extending this approach to other species to network connectivity in a differentiating neuronal cell population. sample more nodes of the phylogenetic tree in order to enable By adding chemical stimuli (glutamate, potassium-chlorite, antipsy- inferences about the evolution of embryonic metabolism. A con- chotics) we can measure differences in reactivity. served pattern of energy expenditure would suggest that embryos Goals: We aim to measure and compare synchronicity of electric conform to a specific energy ‘budget’, which may be under a strict activity and reactivity during neuronal differentiation in cell cultures ancient evolutionary constraint, whereas a divergent pattern would from a schizophrenic patient and healthy controls. suggest that embryonic metabolism can adapt to accommodate Results: Our hypothesis is that the connectivity, and electric activ- differences in developmental modes across different lineages. ity of the neurons from schizophrenic patient is diminished com- pared to healthy controls. P51. Funding: NAP 2017-1.2.1-NKP-2017-00002 grant, Semmelweis The role of the transcription factor Meis1 during craniofacial 250+ Kiválósági PhD Ösztöndíj (EFOP-3.6.3-VEKOP-16-2017-00009) development in zebrafish Viktorie Psutková, Ondřej Machoň P53. Institute of experimental medicine CAS, Prague, Czech Republic Teeth are produced from progenitor cell niche in the absence of the dental lamina in the sterlet sturgeon Keywords: craniofacial, development, meis1 Štěpánka Novotná 1, Anna Pospíšilová 1, Jan Štundl 1,2,3, The TALE-class homeodomain transcription factor Meis1 is neces- Martin Pšenička 3, Robert Černý 1, Vladimír Soukup 1 sary for the correct function of Hox genes, which play an important 1 Department of Zoology, Faculty of Science, Charles University, Prague, role in establishing the antero-posterior axis during the embryo- Czech Republic; 2 California Institute of Technology, Pasadena, Califor- genesis. Neural crest cells migrate along the antero-posterior axis nia, USA; 3 South Bohemian Research Center of Aquaculture and Biodi- and form many tissues including craniofacial structures. Previous versity of Hydrocenoses, Vodňany, Research Institute of Fish Culture and results have demonstrated that the meis1 gene is important during the development of viscerocranium, heart outflow tract, and other Hydrobiology, Faculty of Fisheries and Protection of Waters, University neural crest cells derivates. We focus on the role of meis1 genes of South Bohemia, České Budějovice, Czech Republic during craniofacial development in the zebrafish(Danio rerio) model Keywords: fish, tooth development, replacement, progenitor cells, organism. Recently, we have generated zebrafish mutant lines, in dental lamina, vertebrates which two paralogues of meis1 gene, meis1a and meis1b, have been inactivated. Here, we describe differences in craniofacial pheno- Vertebrate teeth are usually formed in a sequential manner from type between meis1a and meis1b knockout mutants and meis1a/ the deeply invaginated epithelium, the successional dental lamina. meis1b double knockout mutants. Despite high sequence similarity Specialized population of Sox2+ progenitor cells are employed from between meis1a and meis1b paralogues, only meis1b is crucial for the so called taste/tooth junction i) to populate the dental lamina craniofacial development. and give rise to tooth buds or ii) stay superficially to produce taste buds. P52. Here we analyze tooth development in the sterlet sturgeon, a In vitro modelling of schizophrenia by induced pluripotent member of the basally branching lineage of ray-finned fishes that, stem cells using multi-electrode array unusually, produces teeth from the locally thickened epithelium. Csongor Tordai 1, Katalin Vincze 1,2, Hathy Edit 1, Máté Baradits 1, Despite the absence of the successional dental lamina, we found János Réthelyi 1*, Ágota Apáti 2* Sox2+ cells located at the superficial position in the close associ- 1 National Brain Research Project (NAP) Molecular Psychiatry Research ation of the developing tooth and taste buds. This position further Group, Hungarian Academy of Sciences and Department of Psychiatry and houses long-term label-retaining cells suggestive of the presence of Psychotherapy, Semmelweis University, Budapest, Hungary; tooth progenitor cell niche. 2 Molecular Cell Biology Research Group, Institute of Enzymology, We propose that tooth development in the sterlet employs progen- Research Centre for Natural Sciences, Budapest, Hungary itors present in the close association with taste buds. This relation- Background: The broad aim of our research group is to model ship suggests that presence or absence of the dental lamina are neurodevelopmental psychiatric disease in vitro using induced only two phenotypic expressions of the same system used for the pluripotent stem cells. We reprogrammed somatic cells from a development of vertebrate teeth. schizophrenic patient carrying functionally relevant mutation in the ABSTRACTS | POSTER PRESENTATIONS

P54. We used flow cytometry to determine the fluorescence intensity Symmetry breaking and morphogenesis in regenerating Hydra and number of positive cells in each electroporated cell culture. cell aggregates We used a male (1116 cell line) and a female (1125 cell line) Anaïs Bailles 1,2, Christoph Zechner 1,3,4, Pavel Tomancak 1,5 PGC line. After the electroporation, the cells were analyzed by a 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, CytoSmart machine. The machine could detect the movement and Germany; 2 Max Planck Institute for the Physics of Complex Systems, dilution rate of transfected cells. The cells collected and injected Dresden, Germany; 3 Center for Systems Biology Dresden, Dresden, back into Partridge-color Hungarian recipient breed embryo. The o Germany; 4 Cluster of Excellence Physics of Life, Dresden, Germany; injected embryos were placed back into 37.8 C incubator. 10-Days- 5 CEITEC, Brno, Czech Republic old embryonic gonads were dissected. The isolated gonads were embedded in gelatin and cryosectioned. The sections were immu- During development, genetically identical cells progressively com- nostained with TOPRO3 nucleus stain and DAZL stem cell-specific mit to distinct fates, forming patterns of differentiated cells. Impor- antibody. Final, the sections were analyzed by confocal microscopy. tantly, pattern formation is precisely controlled in space and time to lead to proper tissue morphogenesis. In turn, tissue morphogenesis P56. generates forces and deformations that can affect patterns of gene The endodermal contribution to the rostral head development expression. How cells self-organize to generate a heterogeneous in the basal fishes yet precisely patterned population, while undergoing morphogene- Agáta Horáčková, Rober Černý sis, remains unclear. To investigate this problem, we use the remark- Charles University, Prague able self-organizing capacities of the cnidarian polyp Hydra. A full animal can regenerate from a clump of 1000 to 100000 dissoci- The endoderm of the vertebrate head contributes to the developing ated epithelial Hydra cells. Within days, the cell aggregate recon- pharynx up to the oral opening. Our previous research has shown stitutes epithelial layers and junctions, forms a symmetric spheroid that a portion of the endoderm, called preoral gut (POG), protrudes that undergoes repeated cycles of inflation-deflation, then sponta- in front of the mouth in basal ray-finned fishes (the bichir, the stur- neously breaks symmetry and gain polarity with the reappearance geon and the gar). In the present study we focus on the posible of a pattern of proteins of the canonical Wnt pathway and the mor- patterning and migratory capacity of the POG epithelium. We have phogenesis of a new head. During this process, the initially disorga- used a detailed cell-tracing method to show the POG contribution nized cytoskeleton progressively aligns along the polarity axis, as it to the prechordal plate mesenchyme. We also show a close topo- is the case in the adult Hydra. We image the regeneration and mor- graphical relationship of the rostral endoderm and cartilages of the phogenesis of the aggregates over time, using light-sheet micros- skull. These findings demonstrate an unexpected role of the endo- copy and transgenic lines labelling the cytoskeleton. By doing so, we derm in the development of the rostral head. hope to gain insight into the cytoskeleton organization and its role during the inflation-deflation cycles and the subsequent aggregate P57. polarization. We also plan to systematically vary the cell population Development of three-dimensional cell culturing microfluidic composing the aggregates and use the regeneration outcome to platform distinguish between mathematical models of aggregate symmetry Michael Killinger 1, 2, Karel Klepárník 1 breaking. 1 Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry, Czech Academy of Science, Brno, Czech Republic; 2 Depart- P55. ment of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Examination of Venus and mCherry transfected primordial Republic germ cells Keywords: 3D cell cultures, microfluidics Roland Tóth 1, Stefanie Altgilbers 2, Wilfried A. Kues 2, Elen Gócza 1 The engineering of three-dimensional (3D) cell cultures is a prom- 1 Hungarian University of Agriculture and Life Sciences, Gödöllő, Hun- ising strategy for developmental biology and regenerative medi- gary; 2 Friedrich Loeffler Institut, Mariensee, Germany cine. With respect to this, the efficient vascularization of new 3D Keywords: primordial germ cells, transfection, integration rate cell structures is the major scientific and technical challenge, since The primordial germ cells integrate only in the gonads of recipient large tissue constructs or organs require a constant nutrient supply embryos. We created two fluorescent PGC line by Neon electropo- to survive in vitro. The most promising tool for increasing the sus- ration. First, we amplified the plasmid DNA in E. coli competent cells. tainability of 3D cell structure is a recent application of microfluidic The isolated DNA vectors were transfected into chicken PGCs by chips to control 3D cell culture development. Microfluidic technol- electroporation (1116 male and 1125 female PGC lines). Two types ogy allows to controll dynamic conditions for efficient continuous of plasmid vectors (SB-Venus, and mCherry), and SB transposase perfusion of nutrients and removal of the metabolic waste of the (pSB100, which contains the hyperactive variant of SB transposase cells, easy automatization, and reduced consumption of reagents under the CMV promoter) were used. pT2-Venus plasmid encodes and cells. Moreover, the flow of media also exposes the cell to shear the Venus fluorescent reporter under CAGGS promoter, flanked by stress similar to the tissue microenvironment. inverted terminal repeats (ITRs) of the SB transposase. We used Here, we designed and developed the prototype of a miniaturized different molar ratios of transposase and transposon (1:2; 1:6; 1:8). culture chamber providing continual perfusion of nutrients to min- After the electroporation we found approximately 70–80 % effi- imalize a necrotic core formation and support differentiation of 3D ciency of electroporation. The fluorescent transgenic cells were pre-osteoblast (MC3T3-E1) cultures. Moreover, the polydimethylsi- enriched by BD FACS Canto II (BD Bioscience). loxane based microfluidic platform inside the culture chamber can ABSTRACTS | POSTER PRESENTATIONS

be easily fabricated according to requirements and can be designed nally thought. Indeed, a screen using an in vivo translation reporter for a variety of applications such as long-term organoids cultivation has demonstrated that ~10% of mammalian mRNAs contain some or cell co-cultivation. This system helps to understand various cel- elements with IRES function (Weingarten-Gabbay et al., 2016). lular signaling pathways during the 3D cell culture formation and We created a transient reporter system to test putative IRES search for candidate drugs to cure pathologies including cancer and sequences into zebrafish (Danio rerio) embryos. The integration metabolic disease. of the reporter into the genome as well as translation from the The research was supported by the Czech Science Foundation putative IRES region is indicated by the formation of fluorescent (GA CR project 20–00726 S). M.K. is Brno Ph.D. Talent Scholarship proteins. This way we can determine how efficiently IRES-like Holder funded by the Brno City Municipality. sequences are recognized by the ribosome. Furthermore, the ribosomal RNA (rRNA) segments that are impor- P58. tant in the recognition of IRES sequences contain multiple con- Benchmarking investigation of RuX, a novel Mifepristone-in- served pseudouridine residues, raising the possibility that this duced transcriptional regulation system epitranscriptomic modification might play a role in IRES recognition. Áron Zsigmond 1, Katalin Paréj 1, Marcell Cserhalmi 1, To test this possibility we also injected our reporter constructs into Anne Meinzinger 2, Alexandra Kellenberger 2, Péter Horváth 2, dkc1 mutants, impaired in the function of dyskerin, one of the key Tiziano Tallone 3, Zsolt Lőrincz 1, Sándor Cseh 1, Doron Shmerling 2 enzymes in the pseudouridine synthase ribonucleoprotein complex 1 TargetEx Biosciences Ltd., Budapest, Hungary; 2 PolyGene AG, responsible for rRNA pseudouridylation. Rümlang, Switzerland; 3 Cardiocentro Ticino, Lugano, Switzerland Finally, using the sequences for the 5′ untranslated regions of the annotated zebrafish genes, we predicted additional IRES elements Keywords: Gene regulation, Synthetic biology, Mifepristone in silico using the IRESpy XGBoost model. Inducible gene regulation methods are indispensable in diverse bio- logical applications, yet many of them have severe limitations in P60. their applicability. Limitations include inducer toxicity, limited vari- Loss of Sprouty produces a ciliopathic skeletal phenotype in ety of organisms the given system can be used in, side effects of mice through elongation of primary cilia and upregulation of the induction method etc. Using a mutant ligand binding domain of Hedgehog signaling the glucocorticoid receptor (CS1/CD), used together with various Eva Hrubá 1,2, Michaela Kavková 3, Linda Dalecká 4,5, genetic elements such as Gal4 DNA binding domain and the VP16 Miloš Macholán 1, Tomáš Zikmund 3, Miroslav Vařecha 6, transactivator domain, we have recently created a novel inducible Michaela Kunová Bosáková 6, Jozef Kaiser 3, Pavel Krejčí 1,6, system, the RuX system. Mária Hovořáková 5, Marcela Buchtová 1,2 During our experiments we have benchmarked the fold change in 1 Institute of Animal Physiology and Genetics, Czech Academy of Sciences, reporter expression following induction in our system against the Brno, Czech Republic; 2 Department of Experimental Biology, Faculty of commercially available GeneSwitch system in three commercially Science, Masaryk University, Brno, Czech Republic; 3 Central European available mammalian cell lines: HeLa, HEK-293 and CHO. The RuX Institute of Technology, Brno University of Technology, Brno, Czech system is shown to be capable of over 1000-fold inducibility, it has Republic; 4 Department of Cell Biology, Faculty of Science, Charles Uni- flexible applications, and is offered for use in cell culture as well as versity, Prague, Czech Republic; 5 Institute of Histology and Embryology, in animal models. First Faculty of Medicine, Charles University, Prague, Czech Republic; Based on these results, our system is a suitable alternative or comple- 6 Department of Biology, Faculty of Medicine, Masaryk University, Brno, mentary tool for gene-expression studies in several mammalian cell Czech Republic lines, utilizing a non-toxic inducer. The modularity of the system means The Sprouty family is a highly conserved group of intracellular it is easily adaptable for each use case to achieve the highest possible modulators of receptor tyrosine kinase (RTK)-signaling pathways, signal-to-noise ratio between baseline expression and induction. which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders P59. called ciliopathies. Our aim was to evaluate Sprouty2 and Sprouty4 IRES-mediated translation in dyskerin mutant zebrafish gene dependent alterations of ciliary structure and to focus on the Noémi Borbély, Renáta Hamar, Máté Varga determination of its association with Hedgehog signalling defects in Department of Genetics, ELTE Eötvös Loránd University, Budapest, Hungary chondrocytes. Analysis of the skeletal phenotype of transgenic mice Keywords: Internal Ribosomal Entry Sites (IRESs), zebrafish, pseu- with different dosages ofSprouty2 and Sprouty4 genes revealed sev- douridylation eral defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, Initiation of protein synthesis in eukaryotic cells most often reduced bone thickness and trabecular bone mass, skull deformities occurs through the “cap-dependent” translation of messenger or chondroma like lesions were revealed. All these pathologies might RNAs (mRNAs). However, a different mechanism may be required be attributed to ciliopathies. Elongation of the ciliary axonemes in under stress conditions. Internal Ribosomal Entry Sites (IRESs) are embryonic and postnatal growth plate chondrocytes was observed sequences that can recruit ribosomes and allow the cap-independ- in Sprouty2-/- and Sprouty2-/-;Sprouty4-/- mutants compared to corre- ent translation of specific mRNAs under non-homeostatic condi- sponding controls. Also, cilia- dependent Hedgehog signaling was tions, like pathological or physiological stress. upregulated in Sprouty-deficient animals.Ptch1 and Ihh expression Multiple recent studies have demonstrated that the presence of were upregulated in the autopodium and in the proximal tibia of IRES sequences in eukaryotic mRNAs is more common than origi- Sprouty2-/-;Sprouty4-/- mutants. Increased levels of GLI3 repressor ABSTRACTS | POSTER PRESENTATIONS

(GLI3R) form was confirmed inSprouty2 -/-and Sprouty2+/-;Sprouty4-/- P62. primary cell cultures established from costal cartilages or from early Impact of environmental exposures on cardiac differentiation stages of limb buds. These findings demonstrate that mouse lines of pluripotent stem cells deficient in Sprouty proteins manifest phenotypic features resem- Federica Lamberto 1,2, Melinda Zana 2, András Dinnyés 1,2 bling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of 1 Department of Physiology and Animal Health, Institute of Physiology RTK and dysfunction of primary cilia during skeletogenesis. and Animal Health, Hungarian University of Agriculture and Life Sci- Acknowledgement: This study was supported by the Czech Sci- ences, Páter Károly Str. 1, H-2100, Gödöllő, Hungary; 2 BioTalentum ence Foundation (GA21-04178S), and by the Ministry of Education, Ltd., Aulich Lajos Str. 26, Gödöllő, H-2100, Hungary Youth and Sports of the Czech Rep. (CZ.02.1.01/0.0/0.0/15_003/00 00460 and project CEITEC 2020 - LQ1601). Keywords: DOHAD, human induced pluripotent stem cells (hiPSCs), cardiomyocytes, oxidative stress, epigenetics, environmental fac- P61. tors The impact of environmental exposures on the neuronal differ- The Developmental Origins of Health and Disease (DOHaD) hypoth- entiation of pluripotent stem cells esis has progressed widely in the research field during the last Alex Horánszky 1,2, Melinda Zana 2, Andras Dinnyes 1,2 decades. From the maturation of gametes throught the early 1 Department of Physiology and Animal Health, Institute of Physiology embryonic development, environmental factors can predispose the and Animal Health, Hungarian University of Agriculture and Life Sci- offspring to non communicable diseases (NCDs), such as diabetes, ences, Páter Károly Str. 1, H-2100, Gödöllő, Hungary; 2 BioTalentum cardiovascular diseases or neuronal disorders. Animal models and Ltd., Aulich Lajos Str. 26, Gödöllő, H-2100, Hungary observational human studies underpin much of the DOHaD field, showing that several epigenetics, cellular, metabolic and/or physio- Keywords: Brain development, DOHaD, Neuronal differentiation, logical processes are involved. Nevertheless, over the past 20 years, hiPSCs, Oxidative Stress the use of human induced pluripotent stem cells (hiPSC) are becom- Increasing evidence demonstrates that altered conditions during ing an attractive tool to study developmental biology. the periconceptional (PC) period of gamete maturation and early The present study aims to test the effects of environmental expo- embryonic development can have lasting effects on the health of sures and oxidative stress during early stage of cardiac develop- progeny. Such effects can result in the onset of neurological dis- ment, using in vitro hiPSC-derived cardiomyocytes model. The ease and neurodevelopmental disorders (‘Developmental Origins of acquired hiPSC-derived cardiomyocytes have been validated using Health and Disease (DOHaD) concept). The present study aims to immunocytochemistry and RT-qPCR with expression of major link mouse models and human clinical observations with the estab- cardiac development genes (e.g. NKX2-5, GATA4) and structural lishment of a human induced pluripotent stem cell (hiPSC)-derived markers (e.g. MYH6, TNNT2), both in 2D and 3D in vitro system. model for brain development. To assess the effect of early-life Additionally, spontaneously beating activity has been observed and stress on neurodevelopment, the impact of oxidative stress induc- analysed through video microscopy and image-analysis algorithms ing compounds will be determined experimentally using in vitro using Pulse imagin system. Moreover, based on our preliminary hiPSC-derived neuronal tissue with qualitative (Immunocytochem- results, the hiPSC-derived cardiomyocytes are a valid model for istry) and quantitative (RT-qPCR and Western Blotting) methods. toxicity testing of different compounds (e.g. Doxorubicin and Para- iPSC-derived neuronal cultures have been established and validated quat), suggesting their suitability for future analysis on cell viability, with positive expression of neuroectodermal (SOX1, PAX6) and oxidative stress and epigenetics alterations during the early stage neuron-specific markers (MAP2, TUB3) in 2D culture. Preliminary of cardiac development. toxicity testing including cell viability assays and reactive oxygen In conclusion, our in vitro model will help to investigate gene species measurements have been undertaken using oxidative stress expression and phenotypic changes during early cardiac develop- inducing compounds such as Menadione and Paraquat. This study ment, correlating human clinical observations and animal studies in will establish an in vitro hiPSC-derived model for embryonic brain the DOHaD field. development. Our model will be utilized to investigate gene expres- sion and phenotypic alterations, as well as epigenetic changes in P63. brain development induced by a chronic oxidative challenge. Nuclear import dynamics of the Drosophila Moesin protein Conclusively, this study will promote preventative measures and Zoltán Kovács, Ildikó Kristó, Péter Borkúti, Péter Vilmos potential therapeutic applications for neurological and neurode- Biological Research Centre, Szeged, Hungary velopmental disorders that can arise due to early-life stress, com- plementing animal studies and human clinical observations in the Keywords: Drosophila, nuclear transport, FRAP, cytoplasmic reten- DOHaD field. tion The actin-binding moesin protein is a member of the evolutionary conserved ERM (Ezrin-Radixin-Moesin) family and the only mem- ber present in Drosophila melanogaster. The cytoplasmic function of moesin is the crosslinking of the actin cytoskeleton and plasma membrane proteins. However, moesin is also present in the nucleus and under certain circumstances (e.g. heat shock, ecdysone treat- ment) it’s amount there can increase significantly. The nuclear ABSTRACTS | POSTER PRESENTATIONS

activity of moesin was found pivotal in Drosophila development by ment, and hypoplastic or missing craniofacial nerves in mutant our laboratory but the exact mechanisms, by which moesin enters embryos. Our results suggest an essential role of the CDK13 in and exits the nucleus are not known. craniofacial development, therefore future investigation focused Our goal is to elucidate the dynamics and the underlying mechanisms on molecular mechanisms underlying developmental processes can of the nuclear transport of Drosophila moesin. To this avail, our main help us to understand precise involvement of this kinase in regula- tool is the fluorescence microscopy technique called FRAP (Fluores- tion of morphogenetic processes. cence Recovery After Photobleaching). In these experiments confocal Acknowledgement: This work was supported by Czech Science microscopy is used to bleach the entire nucleus and then to monitor Foundation (grant 19-01205S) the fluorescence recovery from the cytoplasm, which process reflects the nuclear import activity of GFP-moesin. The nuclear import of P65. actin was analyzed by the same method earlier, therefore we used Role of LGR5-positive cells during development of craniofacial the import characteristics of GFP-actin as a reference. structures In our work we examined the nuclear import dynamics of wild type Olbertova K 1,2, Hrculak D 3, Kříž V 3, Vrlikova L 1, Lusková D 1, moesin under different conditions and investigated the behavior of Korinek V 3, Buchtova M 1,2 various mutant forms. Our results so far suggest a model, in which 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology moesin is withheld in the cytoplasm by a cytoplasmic retention sig- and Genetics, Brno, Czech Republic; 2 Department of Experimental Biol- nal located on the protein. Moesin can enter the nucleus in greater ogy, Masaryk University, Brno, Czech Republic; 3 Laboratory of Cell and amount only upon certain stimuli, which most likely cause the tem- Developmental Biology, Institute of Molecular Genetics, Praha, Czech porary cessation of cytoplasmic retention. Republic

P64. Development of craniofacial structures is a complex process, which is controlled by various cellular and molecular mechanisms. Neural Cyclin-dependent kinase 13-deficiency leads to craniofacial crest cells migrate to developing head and contribute to numer- and neurogenesis defects during mouse embryonic develop- ous craniofacial structures. They proliferate and differentiate into ment the specific cell types. Also, they play a role in the adult tissue 1,2 1,2 4,5 2 Nela Jandova , Marek Hampl , Jan Prochazka , Jiri Kohoutek , repair due to their self-renewal capacity. In our study, we focused 1,2 Marcela Buchtova on the detection LGR5-positive cells (Leucine-rich repeat contain- 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology ing G-protein-couple receptor 5) during craniofacial development. 2 and Genetics, Czech Academy of Sciences, Brno, Czech Republic; Depart- LGR5 is a transmembrane receptor, which has been previously ment of Experimental Biology, Faculty of Science, Masaryk University, found to be expressed in stem cells of various developing as well 3 Brno, Czech Republic; Laboratory of Transgenic Models of Diseases, as adult tissues. In craniofacial area, the expression of LGR5 was Institute of Molecular Genetics, Czech Academy of Sciences, Prague, located in the area of mesenchyme adjacent to the nasal septum, Czech Republic; 4 Czech Centre for Phenogenomics, Institute of Molecu- in the mesenchyme of labial folds along the tongue, in the mes- lar Genetics, Czech Academy of Sciences, Prague, Czech Republic enchyme surrounding both non-sensory and sensory epithelium of Keywords: cyclin-dependent kinase 13, craniofacial, neurogenesis the vomeronasal organ. Interestingly, in these areas, there was not found overlap with other progenitor marker SOX2 or WNT recep- Cyclin-dependent kinase 13 (CDK13) is a transcriptional kinase tor FZD6 and expression patterns were rather complementary to with the ability to regulate transcription via phosphorylation of the each other. The fate of LGR5-positive cells by lineage tracing, which C-terminal domain of RNA polymerase II. People carrying muta- revealed the decrease of their number during progress of devel- tions within the CDK13 gene are generally delayed in development, opment and their continuous disappearance from the craniofacial exhibit congenital heart defects, skeletal defects and craniofacial tissues. Analysis of LGR5-/- embryos uncovered phenotype in the dysmorphism. Based on these observations, we generated two palatal area, lingual groove or nasopharyngeal duct, which display Cdk13 mouse mutants with different allelic forms: tm1a (hypo- LGR5-positivity during embryonic development. In conclusion, we morphic) with milder craniofacial phenotype including cleft palate detected the expression of LGR5 in several distinct areas of the and tm1d (complete knockout) with missing midline facial struc- craniofacial region, especially in the mesenchyme surrounding epi- tures leading to midline cleft. Craniofacial precursor cells migrate thelial folds and grooves indicating possibly a novel role of LGR5 in from tissues of their origin along the axons to their final destina- morphogenesis of these structures. tions to differentiate and form the specific structures. It has been Acknowledgement: This work was supported by the Czech Science shown that CDK13 is an important molecule for axon outgrowth in Foundation (19-01205S). vitro. Our goal was to reveal, whether the migration of craniofa- cial-forming cells is impeded and axonal outgrowth defective in the P66. Cdk13 mutant animals. Gene expression profiling using PCR Arrays Early developmental patterning of cranial placodes in for mouse neurogenesis revealed extensive changes in several non-teleost fish embryos markers. Furthermore, neurofilaments detection on whole-mount Brezarova, D., Horackova, A., Cerny, R. embryos uncovered defects in axon outgrowth in Cdk13-deficient Dept. Zoology, Charles University in Prague embryos and alteration of their branching in 3D, therefore it verified Craniofacial development of non-teleost fishes might serve as our hypothesis. In summary, our analyses uncovered significantly a unique model of the early vertebrate head evolution. In bichir, altered expression of neurogenesis markers specific for migration, sturgeon, and gar embryos we have previously discovered the exis- adhesion, and axon outgrowth during secondary palate develop- tence of the preoral gut (POG) as the early, rostral-most pharyn- ABSTRACTS | POSTER PRESENTATIONS

geal endoderm forming in front of the mouth (Minarik et al., Nature to malfunctions of primary cilia and patients display eye phenotype. 2017 ). POG arguably represents an ancient blueprint of deuteros- Here, we focus on the role of Tmem107. This protein is localized tome pharyngeal development integrated into head formation in in a transition zone of a primary cilia and mutation in this gene early stages of vertebrate evolution. Further characterization of was discovered in several patients with Joubert and Meckel-Gruber this rostral craniofacial domain might thus reveal configurations syndrome. First, we analyzed gene expression of Tmem107 in eye expected in early vertebrates. We recently identified that in bichir, structures during prenatal development and uncovered enhanced sturgeon, and gar embryos early patterning of the rostral cra- nial neural crest apparently involves direct interactions with POG expression in optic stalk and differentiating retina. To investigate the (Stundl et al., Dev. Biol. 2020). In this project we further characterize role of Tmem107 in eye development, we used a mouse model with early developmental patterning of the rostral cranial placodes in a complete knockout of Tmem107. Mice embryos lacking Tmem107 non-teleost fish embryos using gene expression, molecular mark- displayed eye defects such as anophthalmia or microphthalmia. The ers, and histological analyses. According to our data, embryos of examination of several markers typical for an eye development such non-teleost fishes provide complex as Sox1, Sox2, Pax2 and Pax6 uncovered their impaired expression craniofacial patterning unusual for any other living vertebrates. in all analyzed stages during eye development. Furthermore, we analyzed the effect of overexpression and knock-out of Tmem107 P67. in human ARPE19 cell line. In both experimental approaches, we Migration and skeletogenesis of trunk neural crest in non-tel- observed differences in formation of primary cilia in ARPE19 cells. eost fishes Furthermore, qPCR analysis of CRISPR/Cas9 KO cells revealed aber- Tomáš Suchánek, Robert Cerny rant expression of ciliary as well as Hedgehog pathway genes. In Department Zoology, Charles University in Prague, Czech Republic addition, western blot analyses determined changes in Gli2 and Gli3 expression in Tmem107-deficient cells in comparison wild-type cul- Keywords: trunk neural crest, neural crest migration, skeletogene- tures. Altogether, our data suggest that Tmem107 plays a role in sis, scales, non-teleos fishes, bichir early stages of vertebrate eye development, and it is also necessary The neural crest is a transient population of cells that arises from for normal ciliogenesis in the retina. the dorsal neural tube of vertebrate embryos. These cells extensi- Acknowledgement: This work was supported by Czech Science vely migrate into the periphery to contribute to a remarkable range Foundation (21-05146S). of structures and cell types such as odontoblasts, facial bone and cartilage, pigment cells, neurons, glia, or heart components. Whe- P69. reas cranial neural crest cells migrate in three streams and produce Studying light-regulated microRNAs using human retinal orga- most of head skeletal tissues, trunk neural crest cells migrate as single noid model cells and their skeletogenic potential has been questioned from both Canan Celiker 1,2, Denisa Jurčíková 2, Lucie Pešková 2, developmental and evolutionary view. Here we analysed migration Jana Šebestíková 1,2, Kamila Weissová 1,2, and Tomáš Bárta 1,2 and skeletogenic potential of trunk neural crest cells in embryos of 1 Institute of Animal Physiology and Genetics CAS, Veveří 97, 602 00 non-teleost fishes: Senegal bichir Polypterus( senegalus), European Brno, Czech Republic; 2 Department of Histology and Embryology, Fac- sturgeon (Acipenser ruthenus) and Tropical gar (Atractosteus tropicus) ulty of Medicine, Masaryk University, Kamenice 3, 62500 Brno, Czech using gene expression, molecular markers, and in vivo cell tracking. Republic We present data on emergence, migration, and early differentiation of trunk neural crest cells, but also a long term fate-mapping experiment Keywords: retinal organoids, microRNAs, pluripotent stem cells, to reveal possible contribution of trunk neural crest cells into ganoid light scale formation as a test of their skeletogenic potential. MicroRNA molecules (miRNAs) represent a class of small non-coding RNAs that act as post-transcriptional gene repressors in the biolog- P68. ical processes. The expression of miRNAs occurs in a tissue-spe- The Role of Ciliophaty Protein Tmem107 in the Vertebrate Eye cific manner and in this context the retinal miRNAs expression has Development unique profiles in the developing and adult retina. Key processes Marija Dubaic 1,2, Marek Hampl 1,2, Tomas Barta 3, Natalia A. Shylo 4, regulated by miRNAs in the retina include adaptation to differing Michaela Kavkova 5, Tomas Zikmund 5, Scott D. Weatherbee 4, and light intensities, rapid turnover of the phototransduction cascade, Marcela Buchtova 1,2 and maintenance of cellular homeostasis. Recently a class of con- 1 Institute of Animal Physiology and Genetics, CAS, Brno, Czech Republic; served, light-regulated miRNAs with not fully understood functions 2 Department of Animal Physiology and Immunology, Masaryk University, has been discovered. However, it is very challenging to study these Brno, Czech Republic; 3 Department of Histology and Embryology, Fac- miRNAs in human retinae, because of lack of human retinal tissues. ulty of Medicine, Masaryk University, Brno, Czech Republic ; 4 Depart- Here we show that retinal organoids derived from human pluripo- ment of Genetics, Yale University, School of Medicine, New Haven, CT, tent stem cells exhibit cellular heterogeneity and composition, are USA; 5 CEITEC–Central European Institute of Technology, Brno Univer- reactive to light, and represent applicable models for exploring the sity of Technology, Brno, Czech Republic functions of light-responsive miRNAs. We used our in-house devel- oped device to light stimulate retinal organoids in order to study Keywords: Tmem107, eye development, primary cilia light-regulated miRNAs. Primary cilia are projections from the surface of mammalian cells Acknowledgement: This work was supported by the Czech Sci- whose main purpose is the regulation of cell signalling, especially ence Foundation (21-05146S and 21-08182S), by the Grant Agency during development. Some human diseases have recently related of Masaryk University (GAMU) – MUNI/G/1391, and the European ABSTRACTS | POSTER PRESENTATIONS

Regional Development Fund – Project INBIO (No.CZ.02.1.01/0.0/0.0 tissues, the primary cilium coordinates a series of cellular signal- /16_026/0008451). We acknowledge the core facility CELLIM sup- ling pathways, such as FGF or Hedgehog. Defects in their function ported by MEYS CR (LM2018129 Czech-BioImaging) are tightly coupled with several developmental disorders and other metabolic diseases. Nowadays, significant attention is being paid P70. to the association between disruption of primary cilia function and Retinal organoids: The model to study the effects of light on cancer initiation. Because primary cilia are responsible for the tem- the retinal circadian rhythms poral and spatial integration of signalling pathways, loss of primary Kamila Weissová 1,2, Jana Šebestíková 1,2, Canan Celiker 1,2 and cilia can lead to tumor growth. In this project, we focused on the Tomáš Bárta 1,2 most common tumor of the oral cavity, the squamous cell carci- 1 Institute of Animal Physiology and Genetics CAS, Veveří 97, 602 00 noma (OSCC). Our goal was to determine the possible alterations Brno, Czech Republic; 2 Department of Histology and Embryology, Fac- in morphology and role of primary cilia in this tumor. Using fluores- ulty of Medicine, Masaryk University, Kamenice 3, 62500 Brno, Czech cent confocal microscopy and labelling of cilia by acetylated alpha Republic tubulin, we discovered a reduction of primary cilia numbers in OSCC cells or their complete absence. Next, we analyzed changes in gene Keywords: retinal organoids, circadian rhythms, light expression related to the primary cilia signaling in OSCC samples. The circadian clocks had been deeply studied at the broad level The expression of transcription factor Gli1, which plays a key role of different peripheral tissue as well as at the level of single-cell in Hedgehog signalling, was upregulated in OSCC in comparison to level. Clocks in different tissue are synchronized by different exter- healthy gingiva. On the other hand, the expression of multitrans- nal stimuli. However, all the peripheral clocks are synchronized with membrane receptor Ptch was slightly downregulated, compared the central pacemaker located in the suprachiasmatic nuclei. In to control tissues. Moreover, SHH protein was found to be trapped mammals, the crucial synchronizing input is provided by the light inside the cells. Our findings may help to closer understand the through the retinal hypothalamic tract. Moreover, light directly sets molecular processes contributing to OSCC initiation and thus lead local retinal circadian rhythms that are more independent from the to identification of new molecular therapeutic targets in future. central pacemaker than the rest of the peripheral clocks. This research was supported by the Ministry of Health CR (NV 19-08- The synchronization of the central pacemaker and the local reti- 00383). nal circadian rhythms with the light-dark cycle plays a vital role in physiology. Deregulation of the clock may contribute to the devel- P72. opment of several human diseases. Despite the recent research PORCN inhibition stimulates hypertrophic cartilage through advances in the circadian rhythms field, little is known about the IHH activation molecular mechanisms underlying the effects of light on circadian Szotkowska Tereza 1,2, Killinger Michael 1, Zezula Nikodem 2, clock regulation in the human retina. Bryja Vitezslav 2, Buchtova Marcela 1,2 In this study, we use retinal organoids derived from human pluripo- 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology tent stem cells to delineate the effects of light stimulation on the and Genetics, Czech Academy of Sciences, Brno, Czech Republic; expression of genes involved in circadian clock regulation. Using our 2 Department of Experimental Biology, Faculty of Science, Masaryk Uni- unique in-house-developed machine for light stimulation we show versity, Brno, Czech Republic that the exposure of retinal organoids to the light can synchronize Keywords: Porcupine, WNT, chondrocyte the expression of crucial genes that are coupled to the circadian clock machinery. Porcupine (PORCN) is a membrane-bound endoplasmic reticulum This work was supported by the Czech Science Foundation protein belonging to the O-acyl transferase superfamily. PORCN 21-05146S and 21-08182S, by the Grant Agency of Masaryk Uni- is necessary for the palmitoylation of WNT proteins, which subse- versity – MUNI/G/1391, and the European Regional Development quently leads to their secretion and proper signalling. Mutations or Fund - Project INBIO (No.CZ.02.1.01/0.0/0.0/16_026/0008451). loss of function of PORCN leads to significant skeletal abnormalities in human patients. We decided to analyse the possible effect of two P71. PORCN inhibitors on chondrogenesis with a focus on endochondral Alterations of primary cilia morphology and signaling in tum- bone formation. Moreover, we tested inhibitor PF670462 of WNT ors of the oral cavity pathway targeting casein kinase 1 to distinguish its direct effect Jana Filušová 1, Marek Horanský 1, Barbora Putnová 1,2, to PORCN inhibitors. Our analyses revealed enhanced growth of Jan Štembírek 3, Lucie Vrlíková 1, Marcela Buchtová 1 mesenchymal condensations and an increased number of cartilage 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physi- nodules in primary micromass cultures established from early limb ology and Genetics, Czech Academy of Sciences, Brno, Czech Republic; buds, which were treated with C59 and LGK974. This effect was 2 Department of Pathological Morphology and Parasitology, University of confirmed by Alcian Blue staining due to the enhanced production Veterinary and Pharmaceutical Sciences, Brno, Czech Republic; of extracellular matrix by chondrocytes. Furthermore, we observed 3 Faculty Hospital in Ostrava, Czech Republic increased expression of chondrogenic markers (Sox9, Col2a1) in the micromass cultures treated with WNT inhibitors. HH signalling, Keywords: primary cilia, tumors of the oral cavity, signalling dis- which is necessary for the maturation of chondrocytes, was signifi- orders cantly increased in cultures treated with PORCN inhibitors confirm- Primary cilia are solitary, microtubule-based organelles, which are ing relationship between these two pathways. Histological analysis found on the surface of most vertebrate quiescent cells. In normal of embryonic tibias cultures treated with PORCN inhibitors revealed ABSTRACTS | POSTER PRESENTATIONS

the enlargement of the hypertrophic cartilage zone that was con- P74. firmed by increasedCol10a expression through RNAScope analyses. The role of Satb2 gene mutation in the progression of late In contrast, we did not observe the alteration of chondrogenesis odontogenesis after PF670462 treatment, leading to conclusion that only PORCN Šulcová M. 1,2, Nevoránková P. 2, Kavková M. 3, Zikmund T. 3, inhibitors display a positive effect on chondrogenesis. However, fur- Buchtová M. 1,2 ther investigation of relationship between WNT with HH signalling 1 Department of Experimental Biology, Faculty of Science, Masaryk Uni- during individual phases of chondrogenesis will be needed. versity, Brno, Czech Republic; 2 Laboratory of Molecular Morphogenesis, The study was supported by the MEYS CR (CZ.02.1.01/0.0/0.0/15_ Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of Sci- 003/0000460). ences, Brno, Czech Republic; 3 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic P73. Keywords: Satb2, odontogenesis Role of Shh and Wnt signalling pathways during early incisor and vestibular development Satb2 is member of the family of special AT-rich binding pro- Huteckova Barbora 1,2, Sulcova Marie 1,2, Hovorakova Maria 3, teins, which are associated with nuclear MARs mediating higher Tucker S Abigail 3,4, Buchtova Marcela 1,2 order chromatin integration and therefore modulating target gene 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physi- expression. By such, Satb2 participate in the process of various ology and Genetics, Czech Academy of Sciences, Brno, Czech Republic; transcription factor regulation both during early embryogenesis 2 Department of Experimental Biology, Faculty of Science, Masaryk Uni- and later in process of cell differentiation. To describe the effect of versity, Brno, Czech Republic; 3 Institute of Histology and Embryology, Satb2 deficiency, Satb2 knockout mouse strain was generated. In -/- First Faculty of Medicine, Charles University, Prague, Czech Republic; Satb2 mice, the loss of Satb2 gene expression leads to defect in 4 Centre for Craniofacial and Regenerative Biology, King’s College craniofacial patterning coupled with shorter mandible and cleft pal- London, London, United Kingdom ate. Here, we focused on the role of Satb2 in odontogenesis. First, we evaluated protein expression of Satb2 in craniofacial area of wild- Keywords: vestibular lamina, cyclopamine, C59 type mouse revealing differences in the expression along the ros- The vestibular lamina originates from epithelial-mesenchymal inter- tro-caudal axis of the jaw, where stronger expression was located in actions during early embryonal development of the oral cavity. In the mesenchymal cells around forming incisors while the expression the lower incisor region, the vestibular and dental laminas form around molars was weak. To uncover the direct effect of Satb2-defi- from a common epithelial thickening that divides into two laminas ciency, we collected samples of Satb2-/- embryos ranging from E12,5 during the development. Our study focus on Shh and Wnt signal- till new-borns. Tooth development was evaluated by various histo- ling pathways and their potential role in the separation of the ves- logical approaches, immunohistochemistry and in situ hybridization tibular and incisor placode and the regulatory processes directing to uncover changes in expression of different odontogenesis-related cells towards their fate. First, we investigated gene expression of markers. Moreover, embryos at stage E16.5 and E18.5 were scanned Shh and Wnt members by RNAScope to reveal differences in their by Micro-CT to visualize hard tissue morphology surrounding tooth distribution in vestibular and dental thickenings during early devel- germ. Then, samples were stained in iodine solution and tooth germs opment. Next, we investigated the possible role of Shh and Wnt sig- were segmented in Avizo software to reconstruct 3D morphology nalling with slice cultures of mice embryos lower jaw and inhibition of incisors and molars. Our preliminary analyses uncovered distinct of these pathways using cyclopamine and C59. Shh gene expression changes in lower incisor development. However, exact effect of was specific to the initiation knot as a primary signalling centre. Satb2-deficiency on regulation of individual steps of odontogenesis After two days of inhibition of Shh signalling, the growth of both in both incisors and molars including odontoblast differentiation will analysed structures was affected. The vestibular lamina was signifi- be necessary to further uncover in future. cantly smaller and tooth germ displayed abnormal morphology. Wnt Acknowledgement: This work was supported by the Ministry of signalling regulates cell proliferation and some Wnt molecule mem- Health (grant NU20-06-00189). bers were expressed in the cells forming vestibular lamina. Our first experiments, however, revealed altered morphology of tooth germ P75. after two days of Wnt inhibition by C59 but the vestibular lamina Identification of a new pathogenic gene in a central nervous appeared to be unaffected, which opens new questions about the system, kidney and heart-related syndrome role of WNT signalling during placode separation. In conclusion, we Dávid Czimer 1, Magdolna Keszthelyi Tália 2,3, Dorottya Ralbovszki 1, uncovered differences of Shh and Wnt signalling pathways during Christine Bole 4, Corinne Antignac 4, Kálmán Tory 2,3 and Máté Varga 1,2 early oral cavity development. Next we plan to focus on uncover- 1 Department of Genetics, ELTE Eötvös Loránd University, Budapest, ing the possible contribution of these pathways to early cell fate Hungary; 2 MTA-SE Lendület Nephrogenetic Laboratory, Semmelweis decisions. University, Budapest, Hungary; 3 Ist Department of Pediatrics, Semmelweis Acknowledgement: This work was supported by the MEYS CR University, Budapest, Hungary; 4 INSERM, UMR 1163, Imagine Institute, (CZ.02.1.01/0.0/0.0/15_003/0000460) and the Ministry of Health Paris, France (MU20-08-00205). We recently identified in a seven-year-old boy with neurodevelop- mental, renal and cardiac involvement (cerebral atrophy, polycys- tic kidneys, truncus arteriosus communis) compound heterozygous TMEM260 mutations. This evolutionarily conserved, vertebrate-spe- ABSTRACTS | POSTER PRESENTATIONS

cific gene shows the sequence signatures of a transmembrane pro- also plays key role in gene regulation. During RNA export, specific tein and has two isoforms with undescribed functions. proteins are recruited to the transcribed RNA molecule where they We have generated a zebrafish Danio( rerio) knock-out line for the form an RNA-protein complex, called messenger Ribonucleopro- orthologous gene, which interestingly shows no overt phenotype tein Particle (mRNP). In our laboratory we are studying the func- and no major ciliary defects. This is unexpected as polycystic kid- tion of Moesin, the single cytoskeletal actin-binding ERM protein ney disease hitherto has been almost exclusively associated with in Drosophila melanogaster. In our work we demonstrated that the genes involved in ciliogenesis or ciliary function. Later experiments Moesin protein is present also in the nucleus where it is required demonstrated, however, that tmem260 deficient larvae are sensi- for proper mRNA export. Moesin showed direct protein interactions tive for heat-shock and display an array of phenotypic and behav- with Mediator and TREX-2 complex members in in vitro assays. Both ioral phenotypes, not seen in heat-shocked wild type and control complexes function in mRNA export, strongly suggesting a funda- mutant animals. mental role for Moesin in the process through these complexes. We Currently we are exploring if the chaperone function of Tmem260 confirmed the interactions alsoin vivo with nuclear protein com- can be linked to ciliary regeneration, or the observed phenotypes plexes isolated from cultured cells, where we detected the Moesin, develop independently of ciliary function. Med7 and Med15 proteins in the same nuclear protein complex. Moesin also showed co-localization with Mediator complex mem- P76. bers on the giant chromosomes of Drosophila larvae. Currently, we Therapeutic potential of Sertoli cells are performing Bimolecular Fluorescent Complementation Assays Tereza Tlapáková, Vladimír Krylov, Magdalena Krulová to further confirm the protein interactions, in order to better under- Charles University, Faculty of Science, Department of Cell Biology, Vin- stand the role and significance of Moesin in mRNA export. ičná 7, Prague 2, Czech Republic P78. Keywords: Sertoli cells, mesenchymal stem cells, inflammation Bloom syndrome helicase contributes to germ line develop- Impaired fertility is a major health problem worldwide with a strong ment and longevity in zebrafish impact on the human psychology and physiology. Men suffering Tamás Annus 1, Dalma Müller 1, Bálint Jezsó 2,3, György Ullaga 1, from various infections, such as mumps or chlamydia, are at risk Gábor M. Harami 4, László Orbán 5, Mihály Kovács 4,6, Máté Varga 1 of damage to testicular tissue associated with a reduced number 1 Department of Genetics, ELTE Eötvös Loránd University, Budapest, Hun- of spermatogonia and their functional properties. In the search for gary; 2 Institute of Enzymology, Research Centre for Natural Sciences, cells with strong regenerative properties, Sertoli cells (SCs) have Eötvös Loránd Research Network, Hungary; 3 Department of Anatomy, been shown to modulate the immune response in vitro and in vivo Cell and Developmental Biology, ELTE Eötvös Loránd University, Buda- models. As MSCs, SCs successfully underwent osteogenic, chondro- pest, Hungary; 4 ELTE-MTA „Momentum” Motor Enzymology Research genic and adipogenic differentiation and the expression profile of Group, Department of Biochemistry, ELTE Eötvös Loránd University, canonical MSC markers on the surface of SCs was determined. We Budapest, Hungary; 5 Frontline Fish Genomics Research Group, Depart- observed that SCs are able to rescue T helper cells from apopto- ment of Applied Fish Biology, Institute of Aquaculture and Environmental sis, promote their anti-inflammatory phenotype without regulating Safety, Hungarian University of Agriculture and Life Sciences, Georgikon their proliferation, and more effectively induce polarization of M2 Campus, Keszthely, Hungary; 6 MTA-ELTE Motor Pharmacology Research macrophages than MSCs. At the same time, we demonstrated the Group, Department of Biochemistry, ELTE Eötvös Loránd University, Buda- ability of SCs to transfer mitochondria to immune cells. In a mouse pest, Hungary model, we try to evaluate the protective immunomodulatory and Keywords: RecQ helicases, Bloom syndrome, zebrafish supportive properties of SCs in a model of testicular inflammation accompanied by loss of sperm and/or SCs and infiltration of immune RecQ helicases – also known as the ‘guardians of the genome’ – cells into testicular tissue. In the human model, we plan to create in play crucial roles in genome integrity maintenance through their vitro 3D testicular organoids from healthy donors as a physiologi- involvement in various DNA metabolic pathways. Aside from being cally relevant model system of the testicular microenvironment for conserved from bacteria to vertebrates, their importance is also the study of SSC niche interactions and cell-cell interactions in the reflected in the fact that in humans impaired function of multiple testes under various experimental conditions. RecQ helicase orthologs are known to cause severe sets of prob- lems, including Bloom, Werner or Rothmund-Thomson syndromes. P77. Our aim was to create and characterize a zebrafish Danio( rerio) Moesin is involved in mRNA export as a new member of the disease model for Bloom syndrome, a recessive autosomal disor- Mediator complex der. In humans, this syndrome is characterized by short stature, Ildikó Kristó, Zoltán Kovács, Péter Borkúti, Zoltán Lipinszki, skin rashes, reduced fertility, increased risk of carcinogenesis and Aladár Pettkó-Szandtner, Péter Vilmos shortened life expectancy brought on by genomic instability. We Biological Research Centre, Szeged, Hungary show that zebrafishblm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Keywords: Moesin, mRNA export, Mediator Moreover, similarly to other factors involved in DNA repair, some Accurate and precise control of gene expression is critical for cell functions of zebrafish Blm bear additional importance in germ line survival in order to respond to cellular stress and environmental development, and consequently in sex differentiation. Unlikefanc stimuli. Gene activity is tightly regulated at the level of transcription genes and rad51, however, blm appears to effect its function inde- and translation but mRNA export which links the two processes pendent of tp53. Therefore, our model will be a valuable tool for ABSTRACTS | POSTER PRESENTATIONS

further understanding the developmental and molecular attributes Cellular stress (in particular oxidative stress) is an important factor of this rare disease, along with providing novel insights into the role in various neurodegenerative and neurodevelopmental disorders of genome maintenance proteins in somatic DNA repair and fertility. affecting NPCs. Therefore, to examine the cytotoxic and cytophys- iological effects of oxidative stress we developed an in vitro model P79. system based on iPSC derived NPCs corresponding with the neural Exploring sorting nexin functions in Drosophila progenitors of the hippocampal gyrus dentatus. In our model sys- Tamás Maruzs 1, Dalma Feil-Börcsök 1,2, Enikő Lakatos 1,2, tem oxidative stress was induced using cobalt chloride and para- Gábor Juhász 1,3, Péter Lőrincz 4, András Blastyák 1 and quat treatments, and then the growth curve of cell cultures was Gábor Juhász 1,4 recorded, their metabolic activity was measured using PrestoBlue 1 resazurin-based cell viability assay, and the integrity of the mito- Biological Research Centre, Eötvös Loránd Research Network, Szeged, 2 chondrial network was examined using confocal microscopy. The Hungary; Doctoral School of Biology, University of Szeged, Szeged, 3 expression pattern of ABCG2 and ABCB1 as potent cytoprotective Hungary; Szeged Scientists Academy, University of Szeged, Szeged, Hun- agents were also examined by rtPCR and immunocytochemistry. gary; 4 Department of Anatomy, Cell and Developmental Biology, Eötvös As significant changes were observed in each of abovementioned Loránd University, Budapest, Hungary parameters, we tried to find some biologically active molecules, Keywords: sorting nexin, vesicular trafficking, endosome, Drosophila which are able to attenuate the effect of the oxidative stress. The endomembrane system of eukaryotic cells represent an intri- Because oxidative stress in the central nervous system is often cate network the members of which are connected with each other associated with neuroinflammation and vice versa, we investigated either via vesicular transport processes and permanent physical the effect of some inflammatory mediators. contacts. This complex, dynamic system plays a pivotal role in cell Our results showed no significant changes of the cell number or physiology and its proper function requires the concerted action of viability of the NPCs by the short treatment with the inflammatory several proteins. Main research focus of our group is the investiga- mediators and following oxidative stress compared to the stress tion of genes and proteins involved in vesicular trafficking routes conditions without inflammatory mediators. Of course, it does not chanelling to lysosomes, the central degradative organelles of cells. mean that no significant change could be detectable as a result of Members of the Sorting nexin (Snx) protein family play important the same treatments in a longer time period, or in other cellular roles in numerous locations of the endolysosomal system. All Snx parameters, included morphological and molecular properties. To proteins contain the lipid-binding PX-domain that enables their mem- clarify these questions, we are planning further investigations. brane association where they utilize other protein domains to take Supported by the National Brain Research Program of Hungary (NAP part in versatile molecular events. However, exact cellular functions 2017-1.2.1-NKP-2017-00002 and KTIA_NAP_13-2014-0011), and by of many Snx proteins are currently unknown. Most Sorting nexins are the National Research, Development and Innovation Office (OTKA: evolutionarily conserved, offering the possibility to investigate their K115375, K128369 and PD128830). functions in model organisms. We use various fruitfly tissues to study the molecular functions of the less characterized Snx proteins in the P81. endolysosomal system. Our current focus is the investigation of the Cytoskeletal changes and reparative processes in brain function of Snx25, a known membrane contact site protein involved exposed to metal nanoparticles in a human hereditary neurodegenerative disease. Our results show Adriena Jedličková 1, Daniela Kristeková 1, Jana Dumková 2, that the mutation of the fruitfly counterpart of this gene leads to Lucie Vrlíková 1, Denisa Lusková 1, Tereza Smutná 1, Pavel Mikuška 3, severe defects in the endosomal maturation process of the highly Tomáš Vaculovič 4, Marcela Buchtová 1,5 endocytic larval nephrocytes. Importantly, our findings highlight the 1 Laboratory of Molecular Morphogenesis, Institute of Animal Physi- involvement of Snx25 in the function of recycling endosomes, a pre- ology and Genetics, Czech Academy of Sciences, Brno, Czech Republic; viously unknown aspect of the protein’s function. 2 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 3 Institute of Analytical Chemistry, P80. Czech Academy of Sciences, Brno, Czech Republic; 4 Department of Chem- The effect of oxidative stress and inflammatory mediators on istry, Faculty of Science, Masaryk University, Brno, Czech Republic; 5 the viability of neural progenitor cells Department of Experimental Biology, Faculty of Science, Masaryk Uni- Bálint Jezsó 1, Gábor Erdős 1, Edit Hathy 1, János Réthelyi 2, versity, Brno, Czech Republic 1 1 1 László Homolya , Ágota Apáti , Zsuzsa Erdei Keywords: cytoskeleton, brain, reparation 1 Institute of Enzymology, Research Centre for Natural Sciences, Buda- pest, Hungary; 2 Department of Psychiatry and Psychotherapy, Semmelweis Lead nanoparticles (PbNPs) are posing a potential risk to all liv- University, Budapest, Hungary ing organisms and the negative effect of lead accumulation has been previously associated with neurodegenerative diseases. Here, Keywords: oxidative stress, neural progenitors we aimed to reveal the impact of PbNPs inhalation on brain tis- To the best of our current knowledge, three neurogenic zones within sues in mice model and reparative processes of brain, which are the brain remain active throughout our whole lives, the subventricular initiated during nanoparticle clearance. Mice were placed to whole- zone of the forebrain and hypothalamus and the subgranular zone of body chambers for 11-weeks-long inhalation period and exposed the hippocampal dentate gyrus. The neural progenitor cells (NPCs) to generated PbNPs of distinct characteristics. In addition, a group located these areas could have role in learning, spatial memory, emo- with elimination period was included to uncover speed of reparative tional functions and regeneration as well, and they are also a potential processes and mechanism of their action. In PbONPs treated ani- target of several biomedical and therapeutic approach. mals, pathological changes were observed mainly in the hippocam- ABSTRACTS | POSTER PRESENTATIONS

pal regions where dark, shrunken, necrotic neurons were located P83. in the pyramidal layer of Ammon’s horn. Spongiform changes were Signaling of receptor tyrosine kinases involves primary cilia revealed by neurofilament labeling and confirmed in white matter Alexandru Nita 1, Sara P. Abraham 1, Pavel Krejci 1,2,3, of brain tissue. Moreover, the presence of necrotic neurons was Michaela Bosakova 1,2,3 determined in stratum pyramidale. These findings indicate that 1 Department of Biology, Faculty of Medicine, Masaryk University, 62500 hippocampus belongs to the main targets of metal nanoparticles Brno, Czech Republic; 2 Institute of Animal Physiology and Genetics of within the brains. Specific intracellular non-membranous inclusions the CAS, 60200 Brno, Czech Republic; 3 International Clinical Research were found to be located in neuron cytoplasm of mouse cortex Center, St. Anne’s University Hospital, 65691 Brno, Czech Republic after PbONPs inhalation. These inclusions were identified as Hirano bodies according to the rod-shaped, multilamellar, and filamentous The primary cilium plays key regulatory functions in many cellular structure and they are often associated with neurodegenerative processes, either by spatial compartmentalization of the signaling disorders such as frontotemporal dementia, Parkinson´s disease, molecules, or precise fine-tuning of their action. This is highlighted Alzheimer disease or other tauopaties. In conclusion, we determined by the growing list of pathologies that are now being classified distinct changes in arrangement of cytoskeleton in brain tissue of under the umbrella term of ciliopathies. Ciliopathies are often sys- exposed animals, uncovered very slow clearance ability of brain tis- temic disorders, and commonly present with symptoms that include sue to remove lead and demonstrated that our clearance period was developmental disorders of the skeleton, congenital heart defects, not sufficient to restore all pathological features in hippocampus. renal malformations, cognitive disorders and obesity. Several mem- Acknowledgement: This work was supported by the Czech Science brane receptors and their downstream effectors have been shown Foundation (20-02203S). to reside in the primary cilium. In our study, we focused on 38 mem- bers of the receptor tyrosine kinase (RTK) family, and 24 of their P82. pathology-associated variants, and assessed their interaction with primary cilia. We found that a considerable number of RTKs localize Rho-associated kinase inhibition affects polarity and cavity to the ciliary compartment, while noting differences between wild- formation in the rabbit embryo type and disease-associated variants. Our data show importance of Katarzyna Michniak, Elżbieta Wenta-Muchalska, Anna Piliszek the ciliary RTK localization for a precise regulation of its signaling. Institute of Genetics and Animal Biotechnology, Polish Academy of Sci- These findings expand our understanding of the RTK signaling and ences, Jastrzębiec, Poland the primary cilium compartment in development and disease. Keywords: Rho-associated kinase, ROCK inhibiton, Hippo pathway, rabbit, trophectoderm, lineage specification, preimplantation devel- P84. opment Regulation of primary cilia by Ciliogenesis associated kinase 1 Preimplantation mammalian embryonic development leads to the (CILK1) 1 1 1,3 formation of the blastocyst, which consists of two cell types: the Sara P. Abraham , Alexandru Nita , Miroslav Varecha , Pavel 1,2,3 1,2,3 outer cell layer of trophectoderm (TE) involved in implantation and Krejci , Michaela Bosakova 1 formation of extraembryonic tissues and the inner cell mass (ICM), Department of Biology, Faculty of Medicine, Masaryk University, 62500 2 which further differentiates into the pluripotent epiblast and the Brno, Czech Republic; Institute of Animal Physiology and Genetics of 3 primitive endoderm. the CAS, 60200 Brno, Czech Republic; International Clinical Research The mouse research model uncovers the Hippo pathway involve- Center, St. Anne’s University Hospital, 65691 Brno, Czech Republic ment in TE differentiation in the preimplantation embryo. During Primary cilium is a microtubule-based organelle protruding outside preimplantation mouse embryo development, a differential Hippo of the cell, regulating cell-to-cell communication and intracellular pathway activity between outside and inside cells regulates proper signaling. Proper function of cilia is important in development as TE and ICM differentiation. well as homeostasis of the tissues. Production and maintenance of Here we present the evidence of Hippo pathway involvement in TE/ the cilia is a dynamic and complex processes, and loss-of-function ICM specification in the rabbit embryo, by detailed analysis of YAP, of the critical regulators produces signaling defects that manifest aPKC and F-actin localization in the rabbit embryo from the late in a pleotropic group of disorders collectively termed ciliopathies. morula to the expanded blastocyst stage. CILK1 (Ciliogeneis associated kinase 1), an RCK (ros cross-hybrid- Earlier studies found a connection between Rho-associated kinase izing kinases) family member, is an evolutionarily conserved and (ROCK) activity and correct first embryo lineage specification. ubiquitously expressed serine/threonine kinase that functions in Specifically, ROCK kinase has been shown to regulate Hippo path- development of multiple tissues. Loss of CILK1 activity leads to way activity in mouse preimplantation embryos and is essential for ciliopathies such as short rib-polydactyly syndrome and endo- mouse blastocysts formation, appropriate trophectoderm differen- crine-cerebro-osteodysplasia. CILK1 has been shown to regulate tiation, apicobasal polarization and cell adhesion. cilia formation, architecture and signaling. This knowledge triggered us to examine the influence of the chem- In this study, we used microinjection of small amounts of active CILK1 ical ROCK inhibition on rabbit embryonic development. to challenge primary cilia at the physiological level of CILK1 expres- Our investigation shows that ROCK inhibition by Y27632 disturbs sion. We also implemented a conditional knockdown system to study rabbit embryo cavitation and drives an abnormal distribution of the effects of CILK1 downregulation, and the molecular mechanism aPKC and F-actin. Immunofluorescent analysis of treated embryos of CILK1 action. Understanding these mechanisms would allow for indicates a decrease in YAP and GATA3 levels. better therapeutic options for the ciliopathy patients.