YEN 2014

Acknowledgments

Thank you to all sponsors, invited speakers, judges and all the people involved in the organisation of the 6th Young Embryologist Meeting (YEM:2014).

Programme

9.30- 9.55       Registration

First Session: Cell fate specification
Chair: Sara Pozzi
10.00                Dr Dang Vinh Do (Gurdon Institute, University of Cambridge)
A Genetic and Developmental Pathway from STAT3 to the OCT4-NANOG Circuit is Essential for Maintenance of ICM Lineages in vivo
10.20                Mr Mubeen Goolam (PDN, University of Cambridge)
Satb1 regulates the early cell fate decisions in the preimplantation mouse embryo 
10.40                Miss Madeleine Pope (MRC Mammalian Genetics Unit)
Map3k4 is a dose dependent modifier of sensitivity to B6-YPOS gonadal sex reversal in mice
11.00                Dr Rie Saba (Queen Mary University of London)
Sox17 expression in the cardiac progenitor cells regulates the endocardium development
11.20                Dr Catarina Vicente (The Node Community Manager, Cambridge)
                           The Node: your community blog

Second Session: Patterning the embryo 
Chair: Hong-Ting Kwok
11.50                Miss Erica Namigai
The establishment of left-right asymmetry during spiralian development in the serpulid annelid Pomatoceros lamarcki
12.10                Dr Daniele Soroldoni
A doppler effect in embryonic pattern formation
12.30                Mr Joseph Grice
Conserved Homeodomain Binding Syntax associated with Hindbrain Patterning and Evolution
12.50                Miss Anneliese Norris
                  Morphogenesis of the chick eye: cellular and molecular mechanisms

Q&A Session: Working in Science: is it fun, well paid and rewarding?
Chair: Oleksandr Nychyk

Third Session: Biomechanics 
Chair: Matteo Mole
3.00                  Miss Elina Tsichlaki
Regulation of nuclear size in mammalian embryos
3.20                  Miss Elena Scarpa
Spatiotemporal dynamics of cadherin junctional complex and actomyosin assembly during contact inhibition of locomotion
3.40                  Dr Graham Sheridan
Investigating the role of mechanical cues in axon guidance

The Sammy Lee Memorial Lecture
Chair: Naiara Bazin
4.20                  Prof Bill Harris (Professor of Physiology, Development and Neuroscience, University of Cambridge)
The forever embryonic eyes of frogs and fishes

5:35                  Closing Address
5:45                  Reception at Marquis Cornwallis pubTalk AbstractsSession 1: Cell fate specification
A Genetic and Developmental Pathway from STAT3 to the OCT4-NANOG Circuit is Essential for Maintenance of ICM Lineages in vivo
Dang Vinh Do1, Barbara B. Knowles2, Davor Solter2 and Xin-Yuan Fu3
; 2 Institute of Medical Biology, A*STAR, Singapore; 3 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, USA.Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro the upstream signals that regulates this circuit during early development in vivo have not been identified. In this report, we demonstrate, for the first time, a hierarchical genetic pathway from STAT3, which directly regulates the OCT4-NANOG circuit to induce formation of the inner cell mass (ICM), the source of in vitro-derived ESCs. We now show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the 4-cell, and later stage, embryos. Using Lif-null embryos we find STAT3 phosphorylation is dependent on LIF in 4-cell stage embryos. In blastocysts, IL-6 acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by JAK1, a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm (TE) formation. Indeed, STAT3 directly binds to the Oct4 (Pou5F1) and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem (iPS) cells. These results provide a novel genetic model of cell-fate determination operating through STAT3 in the preimplantation embryo and in pluripotent stem cells in vivo.
Satb1 regulates the early cell fate decisions in the preimplantation mouse embryo 
Mubeen Goolam, Magdalena Zernicka-Goetz
Department of Physiology, Development and Neuroscience, University of Cambridge.Within the first four days of mouse embryonic development two cell fate decisions occur which establish the correct morphological and molecular events to form an implanting blastocyst. The first cell fate decision is a result of asymmetric divisions initiated at the 8-to 16-cell stage which gives rise outer cells, the extraembryonic trophectoderm (TE), as well as inside cells, the inner cell mass (ICM). The second cell fate decision segregates the ICM into the epiblast (EPI), which will form the embryo proper, as well as the second extraembryonic tissue, the primitive endoderm (PE). Through deep sequencing analyses we have identified that Satb1 is differentially expressed between inside and outside cells at the 16-cell stage. Depletion of both maternal and zygotic Satb1 altered ICM cell fate by depleting the number of PE cells in the blastocyst. This phenotype could be rescued by co-knockdown of Satb2 underlining the antagonistic effect of these two proteins. Clonal knockdown of Satb1 indicated that blastomeres lacking Satb1 preferentially form EPI cells within the ICM. Furthermore, increasing Satb1 levels resulted in the opposite phenotype with an increase in PE cells and a subsequent decrease in EPI. Together these results show a role for Satb1 in the preimplantation embryo.
Map3k4 is a dose dependent modifier of sensitivity to B6-YPOS gonadal sex reversal in mice
Madeleine Pope, Nick Warr, Gwenn-ael Carre, Pam Siggers, Andy Greenfield
MRC Mammalian Genetics Unit, Harwell, Oxfordshire.In mammals the Y-linked gene Sry is a dominant male determinant, whose expression initiates the cascade of events that drive differentiation of the testis. XY embryos lacking Map3k4 exhibit delayed, insufficient expression of Sry and develop ovaries on the C57BL/6J (B6) background. We have exploited a phenomenon termed B6-YPOS sex reversal to investigate the relationship between Map3k4 and Sry in the mouse.
When the Y chromosome from Mus poschiavinus (YPOS) is introduced onto the B6 background all XY animals undergo complete or partial sex reversal associated with delayed Sry expression. Gain-of-function experiments using a Map3k4 BAC transgene have revealed that overexpression of Map3k4 is sufficient to restore testis determination in B6-YPOS embryos. Furthermore, loss of a copy of Map3k4 results in more severe sex reversal. Comprehensive transcriptional profiling shows that overexpressing Map3k4 in B6-YPOS embryos partially restores the normal expression profile of Sry and establishes MAP3K4-dependent control of Sry expression timing as a key player in B6-YPOS sex reversal. We are currently exploring the epigenetic landscape of the Sry locus in B6-YB6 and B6-YPOS gonads during the critical period of testis determination, in order to elucidate epigenetic changes that could account for the differences in Sry expression underlying this phenomenon and, more genrally, shed light on how signalling to the epigenome controls cell fate.
Sox17 expression in the cardiac progenitor cells regulates the endocardium development
Rie Saba1, Ioannis Kokkinopoulos1, Hidekazu Ishida1, Keiko Kitajima2, Chikara Meno2, Yoshiakira Kanai3, Masami Azuma-Kanai4, Peter Koopman5, Yumiko Saga6, Yukio Saijou7, Ken Suzuki1 and Kenta Yashiro1
1 Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London; 2 Department of Developmental Biology, Graduate School of Medical Sciences, Kyushu University, Japan; 3 Department of Veterinary Anatomy, The University of Tokyo, Japan; 4 Center for Experimental Animal, Tokyo Medical and Dental University, Japan; 5 Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Australia; 6 Division of Mammalian Development, Genetic Strains Research Center, National Institute of Genetics, Japan; 7 Department of Neurobiology and Anatomy, The University of Utah, USA.During mouse development, cardiac progenitor cells (CPCs) in the heart field deliver many types of cells to form cardiac tissues. Several CPC marker genes were identified, however the regulatory mechanism underlying the fate determination has remained to be elucidated. Our single cell cDNA analysis showed that a pan-endodermal marker Sox17 was expressed in some proportion of CPCs from the early bud stage, and that the expression was highly correlated to that of the endothelial markers at the early somite stage. Sox17-positive mesodermal cells appeared in heart field from early head fold stage to early somite stage, and contributed only to the endocardium in the heart tubule, implying the cell-autonomic function in the endocardium development. We conducted the gain of function and loss of function studies by the mice with BAC Nkx2-5(Sox17-ires-lacZ-BghA) transgene and Mesp1Cre/+/Sox17flox/flox alleles, respectively. The results showed that Sox17 is not necessary or sufficient to induce the endocardial fate in CPCs, but can bias it toward the endocardium in some extent. Further Mesp1Cre/+/Sox17flox/flox embryos showed the typical defects of Notch signaling pathway and Notch1 was down-regulated in the mutant heart tubule. Thus we conclude that Sox17 expression in CPCs regulates the endocardium development via Notch signaling pathway.
Session 2: Patterning the embryo 
The establishment of left-right asymmetry during spiralian development in the serpulid annelid Pomatoceros lamarcki
Erica Namigai, Sebastian Shimeld
Department of Zoology, University of Oxford, Oxford.The establishment of body axes is a fundamental process of metazoan development. However, how the left-right (LR) axis is established remains poorly understood, especially in organisms outside of the Deuterostomia and Ecdysozoa. I am studying the mechanisms underlying LR asymmetry establishment in the Lophotrochozoa using Pomatoceros lamarcki, a serpulid annelid, as a model. To observe the first instance of symmetry breaking, we are using live imaging to understand biases during axis establishment at early cleavage stages, including the cellular architecture and dynamics of early spiral cleavage. We have also sequenced the P. lamarcki genome, and key genes with a conserved function in LR asymmetry (i.e. Nodal) are being analyzed. This research addresses key questions concerning body axis establishment during spiralian development, and is valuable for exploring the dynamic aspects of early development and to understand the evolution of LR asymmetry within the Lophotrochozoa.
A doppler effect in embryonic pattern formation
Daniele Soroldoni1,3,4†, David J. Jörg2†, Luis G. Morelli1,5, David L. Richmond1, Johannes Schindelin2,6, Frank Jülicher2, Andrew C. Oates1,3,4*
1Max Planck Institute of Molecular Cell Biology and Genetics, Germany; 2Max Planck Institute for the Physics of Complex Systems, Germany; 3MRC-National Institute for Medical Research, Mill Hill, UK; 4University College London, London; 5Departamento de Física, FCEyN UBA and IFIBA, Ciudad Universitaria, Argentina; 6University of Wisconsin at Madison, USA.
† equal contributionIn sequentially segmenting animal, the rhythm of segmentation is reported to be controlled by the time scale of genetic oscillations that periodically trigger new segment formation. However, here we present systematic, real-time measurements of genetic oscillations in zebrafish embryos showing that the time scale of genetic oscillations is not sufficient to explain the temporal period of segmentation. We find that a second time scale, the rate of tissue shortening, contributes to the period of segmentation through a Doppler effect. In addition, this contribution is modulated by a gradual change in the oscillation profile across the tissue. We conclude that the rhythm of segmentation is an emergent property controlled by the time scale of genetic oscillations, the change of oscillation profile, and by tissue shortening.
Conserved Homeodomain Binding Syntax associated with Hindbrain Patterning and Evolution
Joseph Grice, Laura Doglio, Greg Elgar
Division of Systems Biology, National Institute for Medical Research, Mill Hill, London.Understanding the function of regulatory elements is required to enrich our understanding of development, disease and evolution. The sequence features that mediate these are often unclear and the prediction of tissue-specific expression patterns from sequence alone have produced only modest results. The hindbrain is a vertebrate shared-derived structure segmented in to 7 rhombomeres, and offers an interesting model for the study of spatially restricted enhancer activities.
We first identified a regulatory signature within conserved hindbrain enhancers (syntax), consistent with published data, consisting of spatially co-occuring PBX, HOX and MEIS/PREP transcription factor binding motifs. We then use this syntax to accurately predict hindbrain enhancers in >90% of cases (61/64 tested elements). Finally, mutagenesis of the sites causes ectopic expression, demonstrating their requirement for segmental enhancer activities.
Our approach proves informative for the elucidation of motif grammars within conserved elements. These data support the theory that hundreds of CNEs are constrained by their functions as regulatory elements underlying the development of the hindbrain. It is likely that sequences of this kind contributed to the co-option of new genes to the hindbrain gene regulatory network during early vertebrate evolution by linking patterns of hox expression to downstream genes involved in segmentation and patterning.
Morphogenesis of the chick eye: cellular and molecular mechanisms
Anneliese Norris & Andrea Streit.
Department of Craniofacial Development and Stem Cell Biology, King’s College London, London.The vertebrate eye has dual origin: the lens arises from the surface ectoderm, while the retina, retinal pigment epithelium and optic nerve originates from the central nervous system. The coordination of cell fate and morphogenetic processes is crucial to form a functional eye. While cell fate decisions are a well studied, little is known about the cellular mechanisms that control eye formation in amniotes, and how both relate to each other. Here, we characterise the cellular events such as cell shapes from open neural plate stages to optic vesicle formation, along with proliferation and orientated cell division, and examined the contribution of each to the development of the eye. Using confocal microscopy and live imaging we provide the first detailed analysis of these processes in higher vertebrate. The main mechanisms that accompany epithelial morphogenesis are under the control of the non-canonical planar cell polarity (PCP) Wnt pathway. We have examined members of the PCP pathway, as well as Notch pathway components and we find that these are expressed in a restricted pattern from open neural plate stages to optic vesicle stages. We are currently investigating their role and how they interact. Ultimately this will allow us to establish the molecular mechanisms that control vertebrate eye morphogenesis and link them to cell fate allocation.
Session 3: Biomechanics 
Regulation of nuclear size in mammalian embryos
Elina Tsichlaki and Greg FitzHarris
University College London, London.The mechanisms that dictate nucleus size are largely mysterious. Experiments in fission yeast suggest that nuclear size is dictated by the size of the cell, whereas studies in Xenopus indicate that nuclear import rates control nuclear volume. Here we set out to investigate factors regulating nucleus size in mammalian embryos.
We generated 3-dimensional confocal images to determine Nuclear/Cytoplasmic (N/C) volume ratio. Fluorescent Recovery After Photobleaching (FRAP) of GFP-tagged nuclear localization signal (GFP-NLS) was used to establish nuclear-import rates. Micromanipulation was used to probe the influence of cell volume upon nuclear size. Nuclear size decreases with successive cleavage divisions and N/C ratio was relatively constant at any given developmental stage, suggesting a direct relationship between nuclear and cell size. Experimental cytoplasmic removal reduced significantly nuclear size, further validating this relationship. However, the N/C ratio set-point increases progressively through development, revealing other factors present influencing nuclear size. Nuclei in experimentally-generated embryos with ‘double-sized’ blastomeres were of normal size, also suggesting a developmental programme involved. However, the rate of nuclear import was identical through development revealing that nuclear import does not dictate nuclear size. Rather, cell size in conjunction with developmentally-regulated cytoplasmic factors dictate nuclear volume in mammalian embryos.
Spatiotemporal dynamics of cadherin junctional complex and actomyosin assembly during contact inhibition of locomotion
Elena Scarpa1, Maddy Parsons2, Andras Szabo1, Roberto Mayor1
1Department of Cell and Developmental Biology, UCL, London. 2Randall Division for Cell and Molecular Biophysics, King’s College London, London.When mesenchymal cells interact they experience contact inhibition of locomotion (CIL): cells enter in contact, stop moving, repolarize in opposite direction and separate. Neural crest cells are a model to study CIL. During their migration, CIL is required for their directional migration. CIL induces cell polarization by controlling protrusions via RhoGTPases: when NCCs collide, RhoA activity increases at the contact while Rac1 decreases. These events depend on NCadherin.
Whether a cadherin adhesion complex is assembled during CIL and how cell-cell interactions dynamically control Rho GTPases and actomyosin cytoskeleton has not yet been characterized.
By imaging live Xenopus NCCs we show that a transient cadherin complex is formed upon CIL. NCadherin, p120, alpha- and beta-catenin are recruited at NC-NC junctions when the lamellipodia of two cells enter in contact. Live FRET imaging of collisions using RhoA and Rac1 probes shows that RhoA is active at the junction until cells separate while Rac activity remains higher at the cell leading edge. Finally, we investigated actomyosin organization: during collisions actin and myosin are poorly recruited at the cell cortex underlying the junction, and contractile structures are assembled at the trailing edge of contacting cells seconds before separation. These results suggest that upon CIL a functional cadherin complex is organized in NCCs and Rho GTPases and actomyosin cytoskeleton are dynamically regulated.
Investigating the role of mechanical cues in axon guidance
G.K. Sheridan1, H. Svoboda1, D. E. Koser1, A. Dwivedy1, A. J. Thompson1, M. P. Viana3, L. F. Costa3, J. Guck2, C. E. Holt1, K. Franze1
1Department of Physiology, Development and Neuroscience, University of Cambridge, 2Dept. of Physics, University of Cambridge, Cambridge, 3Institute of Physics at Sao Carlos, Univ. of Sao Paulo, Sao Paulo, Brazil.During Xenopus embryogenesis, development of the visual system involves the projection of retinal ganglion cell (RGC) axons from eye primordia to correct synaptic contacts in the optic tectum. There are well-characterised chemical cues that guide axons to their target locations. During chemotactic migration, however, growing axons also physically interact with their surrounding environment. Therefore, it is possible that mechanical interactions could also influence their guidance to the tectum.
To probe RGC mechanosensitivity, we cultured Xenopus eye primordia ex vivo on compliant hydrogels with increasing rigidities. We found clear morphological differences in RGC axons grown on soft and rigid substrates. This includes differences in: 1) axon length; 2) axon velocity and directionality; 3) axon fasciculation and 4) growth cone turning on stiffness gradients. This suggests that RGC axons are mechanosensitive which may impact axon path-finding.
Using in vivo atomic force microscopy-based stiffness mapping of Xenopus exposed brains, we found stiffness gradients along the path RGC axons grow. We reveal how RGC axons migrate through relatively soft brain structures until they reach the more rigid optic tectum, where they terminate. Exposing developing Xenopus embryos to inhibitors of mechanosensitive ion channels causes RGC miswiring; suggesting that mechanical properties of the extracellular environment may contribute to axon guidance in the developing brain. 

Poster Titles