KEYNOTE SESSIONS

Conserved programs for producing and maintaining high quality oocytes

Steven DeLuca, Ethan Greenblat, Matthew Sieber, Liang-Yu Pang, Megha Ghildiyal and Allan Spradling Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, MD USA 21204.

Our laboratory has studied multiple mechanisms used during oocyte development that are significantly conserved between Drosophila and mice. Among these are mechanisms controlling the formation and function of germ cell chromatin. By introducing reporter genes at more than 100 sites throughout the genome, and by carrying out ChiP analyses on FACS-isolated germ cells we have investigated how the functional and modification states of chromatin facilitate Drosophila female germ cell development. Eggless-dependent formation of H3K9me3-enriched heterochromatin occurs prior to the onset of meiosis and may repress non-homologous recombination. E(z)-dependent modification of Polycomb target gene loci blocks the ectopic activation of male-specific germline genes, while CBP-dependent H3 acetylation is essential for nurse cell function. We have also characterized how oocytes store lipids following an ecdysone pulse at stage10, and transition to a quiescent state following down-regulation of InR/Akt signaling. Disassembly of mitochondrial respiratory complexes mediated by Gsk3 stimulates gluconeogenesis and glycogen accumulation, followed by metabolic quiescence. Using ribosome profiling we have characterized the residual translational program of quiescent, mature eggs that maintains egg quality while they are stored in the ovary. Over an approximately two-week lifespan at 25 oC, oocytes modulate the translation of mRNAs that are largely associated with large RNP complexes. Several abundant proteins, including E3 ubiquitin ligases and chaperones, function to maintain oocyte viability during this period. Eggs with suboptimal nutrients, incomplete mitochondrial shutdown, or with reduced proteostasis capacity do not immediately become inviable, but produce defective embryos at an elevated frequency. Our studies emphasize the critical importance during oogenesis of the multiple sophisticated mechanisms that generate and maintain oocyte quality.

Evolutionary conservation and divergence in function of a homologous neuron underlying a complex behavior

David Stern Janelia Research Campus

The neural basis for behavioral evolution is poorly understood. Functional comparisons of homologous neurons can reveal neural differences underlying novel behavior patterns, but homologous neurons cannot be identified and manipulated in most taxa. Here, we take a new strategy to this problem by introducing genetic reagents from Drosophila melanogaster into other Drosophila species, which allowed us to compare the anatomy, physiology, and function of homologous song neurons between species that exhibit both conserved and derived song types. We found that a descending neuron with conserved electrophysiological properties is required for different song types in D. melanogaster and D. yakuba, although these songs are produced in similar behavioral contexts. Thus, this neuron has retained a functional role in producing a courtship song in a specific context, but functionally diverged in the song type it produces. This experimental approach can be generalized to other neurons and therefore provides an experimental framework for studying how the nervous system has evolved to generate behavioral diversity.

On growth and form in petals

Vivian Irish Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520

Petals are an excellent system for studying plant organogenesis; they have a simple structure with relatively few cell types. Over the years, my group has focused on various aspects of petal development, from characterizing homeotic genes required for petal identity, to dissecting gene regulatory networks necessary for petal initiation, growth and differentiation. More recently, we have focused on cell type differentiation, with a particular emphasis on understanding the mechanisms involved in shaping conical petal epidermal cells.

PLATFORM SESSIONS

NRPM, novel signaling regulators at the plasma membrane

Xueyi Xue and Juan Dong Waksman Institute of Microbiology, Rutgers the State University of New Jersey, Piscataway, NJ 08854

Plants are rooted in the soil, but generally well adapted to a wide range of environmental conditions. The plasma membrane integrates the cell with its extracellular environment. Behand the remarkable ability of plants to cope with external stresses and still strive, sophisticated and efficient mechanisms to establish and maintain plasma membrane homeostasis is involved. However, how sophisticated and dynamic signaling across the plasma membrane is achieved by a dedicated balance between positive and negative regulations remains a major challenge in understanding plant signal transduction. Here, we will discuss a newly identified signaling regulator at the plasma membrane, NRPMs (Novel Regulator at the Plasma Membrane). We characterize their loss- of-function mutant phenotype and their subcellular localization and functional inter-dependence with the receptor-like kinases. The NRPMs represent a new class of regulators at the plasma membrane in plants.

Rapid phenotyping of the developing mouse nervous system by single-cell mass cytometry.

Eli Zunder Department of Biomedical Engineering, School of Engineering and Applied Science, University of Virginia, Charlottesville, VA 22908

Mass cytometry is a flow cytometry variant that uses isotopically pure rare earth metals conjugated to antibodies or other affinity reagents, permitting measurement of over 40 markers simultaneously at the single cell level. This approach has proven highly successful in the study of immune cell types, but has not yet been used to examine cells of the nervous system, perhaps because neural tissue is challenging to dissociate into a single-cell suspension. In this presentation, we describe single-cell dissociation of microdissected neural tissues, and the development of neuronal and glial-specific antibody panels that pave the way for developing a proteomic cell atlas of the developing nervous system. The neural mass cytometry platform described here can be readily applied by other research groups to perform high-throughput neural phenotyping.

Macrophages directly alter fibroblast activity during epimorphic tissue regeneration and fibrotic healing

Jennifer Simkin University of Kentucky, USA

Injury culminates in tissue regeneration for some tetrapods and fibrotic healing for others, and there is mounting evidence that an initial immune response is essential for both processes. Macrophages, key orchestrators of the immune response, are necessary for proper tissue regeneration across epimorphic regeneration models including mammals. Macrophages are also known to promote fibrotic healing in models of wound repair thus prompting the following question: how do macrophages direct tissue regeneration in one context and fibrotic healing in another? In this study, we use a model of mammalian tissue regeneration, the African spiny mouse (Acomys cahirinus) to investigate macrophage activity during regeneration. Acomys are able to regenerate tissue of the external ear pinnae whereas the common lab mouse (Mus musculus) forms a scar. We hypothesized that unique macrophage activity in Acomys promotes regeneration over scar formation. To test this hypothesis, we assessed the ability of macrophages from both species to respond to classic stimulation paradigms in vitro. Analyzing gene expression and cytokine production from macrophages we found species-specific pro-inflammatory profiles. To test how ear fibroblasts respond to these species-specific macrophage profiles we analyzed changes in extracellular matrix production by Acomys and Mus fibroblasts cultured with macrophage-conditioned media. Our results suggest that pro-inflammatory macrophages from Acomys are able to reduce collagen production and increase matrix metalloproteinase production in ear fibroblasts from both species. Finally, we quantify macrophage phenotypes in vivo and observe specific differences in spatiotemporal localization of macrophage subtypes that respond to each injury. Together our findings suggest the existence of a unique pro-inflammatory macrophage in Acomys that can reduce scar matrix production by fibroblasts and enhance the production of remodeling enzymes important for regeneration.

Opposing action of Hedgehog and Insulin signaling controls proliferation and autophagy to determine Follicle Stem Cell Lifespan

Tanu Singh1,2, Eric Lee1, Tiffiney Hartman1, Dara Ruiz-Whalen1, Alana O'Reilly1 1Fox Chase Cancer Center, USA; 2Drexel University College of Medicine, USA

Egg production declines with age in many species, a process linked with stem cell loss. Diet-dependent signaling has emerged as critical for stem cell maintenance during aging. Follicle Stem Cells (FSCs) in the Drosophila ovary are exquisitely responsive to diet-induced signals including Hedgehog (Hh) and Insulin (IIS), entering quiescence in the absence of nutrients and initiating proliferation rapidly upon feeding. Although highly proliferative FSCs generally exhibit extended lifespan, we find that constitutive Hh signaling drives FSC loss and premature sterility despite high proliferative rates. This occurs due to Hh- mediated induction of autophagy in FSCs via a novel Ptc-dependent, Smo- independent mechanism. Hh-dependent autophagy increases during aging, triggering FSC loss and consequent reproductive arrest. IIS is necessary and sufficient to suppress Hh-induced autophagy, promoting a stable proliferative state. These results suggest that opposing action of diet-responsive IIS and Hh signals determine reproductive lifespan by modulating the proliferation- autophagy balance in FSCs during aging.

Comparative analysis of the response to Notch signaling perturbation across Xenopus species during embryonic development

Mark Pownall, Ronald Cutler, Margaret Saha College of William and Mary, USA

Embryonic development is a robust process during which embryos must respond and compensate for changes in order to achieve consistent patterning; however there are still questions about the limits and mechanisms of this robustness. Using tetraploid Xenopus laevis as a model, we have shown that embryos respond to perturbations of the highly-conserved Notch signaling pathway in a compensatory manner. RNA-seq analysis of Notch perturbed X. laevis embryos revealed that homeologs respond differentially to this perturbation, suggesting that the polyploidy of X. laevis may contribute to the compensatory abilities. To address this question, we have perturbed Notch signaling in X. borealis, a tetraploid species that is closely related to X. laevis, and characterized the response over time. Similarly to X. laevis, a compensatory response is seen in X. borealis, but embryos appear morphologically deformed throughout development, suggesting that X. borealis embryos may be more severely affected by this perturbation. RNA-seq was performed on Notch perturbed X. borealis embryos to quantitatively assess the response over time and compare changes in homeolog expression in X. laevis and X. borealis embryos following Notch perturbation. A de novo assembly of the X. borealis transcriptome was generated for further analysis. These data have revealed differences in the response to Notch perturbation between X. borealis and X. laevis.

Physical Constraint Induces Cell Division During Neurulation.

Lance Davidson1,2,3 and Deepthi Vijayraghavan1 1 Department of Bioengineering, 2 Department of Developmental Biology, 3 Department of Systems and Computational Biology, University of Pittsburgh, Pittsburgh PA 15213

Tissue cohesion is critical during morphogenesis where cell sheets move en mass. Cohesion is maintained through cell-matrix or cell-cell affinities while cell cycle checkpoints prevent tissue dissolution when shear, tension, or compressive stresses are highest. Checkpoints such as the one maintained by Wee2 in the mesoderm inhibit cell division during gastrulation (Leise and Mueller, 2002). We have uncovered a potential G2/M checkpoint that operates during neurulation. Here we show most cells in the Xenopus laevis neural plate complete S-Phase after gastrulation. Consistent with a generic block of cell division during morphogenesis, only a few cells in the neural pate complete M-phase during neural convergence and extension. Since cell proliferation can be sensitive to mechanical cues, e.g. in MDCK and zebrafish tail epidermis (Gudipaty et al., 2017), we tested whether neural plate cells could be driven through the G2/M checkpoint by mechanical perturbation. We placed isolated dorsal tissues, containing the neural plate, into confining stiff agarose cells. In response, neural plate cells under confinement increased their rate of cell-division from less than 2% per hour to more than 12% per hour. Dorsal isolates in confinement exhibit 3- fold greater stress than those in low confinement (Zhou et al., 2015) suggesting increased stress in the dorsal isolate release neural cells from a G2/M block. We are using the gel confinement assay to identify pathways that integrate environmental mechanical cues with cell cycle control systems to increase the numbers, and potential diversity of neural progenitor cells in the neural tube.

Characterization of epithelial cell rearrangements during lens placode invagination

Nathalie Houssin, Sung Yoon, Timothy Plageman Ohio state University, United States

Development of the vertebrate eye serves as a valuable model for epithelial morphogenesis. At the onset of lens formation, the surface ectoderm thickens to form the lens placode (LP) that undergoes a series of cell rearrangements and shape changes that drive invagination of the tissue. Currently, the cellular mechanisms driving epithelial invagination remain incompletely characterized. The contribution of apical constriction (AC) and mechanically constrained tissue growth only partially explain this process. To better characterize it, cellular junctions of mouse and chick embryos were visualized using fluorescent live microscopy. It was observed that LP cells behave differently from their surrounding neighbors. Central LP cells reduce their apical area isotropically and maintain their junctions with neighboring cells whereas peripheral LP cells are anisotropic and dynamically rearrange themselves resulting in a central converging movement. During this process, epithelial rosettes form through the contraction and elimination of bicellular junctions oriented circumferentially while rosettes resolution form through the generation and elongation of bicellular junctions oriented radially. Also, junctional contraction appears Rho-kinase dependent and occurs coordinately with several junctions of adjacent cells aligned in arcs. As Par-complex protein Par3 has previously been implicated to regulate invertebrate epithelial rosette resolution, its role during vertebrate placode invagination was determined. While Par3 protein appears to be isotropically localized in LP peripheral cells, it is localized to the vertices of rosettes. Static analysis of fixed embryos lacking Par3 demonstrated that rosette structure and anisotropic distribution depend on Par3. Further analysis of LP cell behaviors lacking Par3 using live imaging and pattern analysis tools will shed light into the role of rosette resolution, radial cell movement, and Par3 function during epithelial invagination.

Cell escape during pollen germination in the Brassicaceae

Anna Edlund Lafayette College, Easton, PA 18042

Despite attempts to degrade the sporopollenin in pollen walls, this material has withstood a hundred years of experimental treatments and thousands of years of environmental attack in insects and soil. Nonetheless, sporopollenin degrades locally only minutes after pollination in ~25% of Brassicaceae species. We have described a two-part pollen germination mechanism in Arabidopsis thaliana involving both chemical weakening of the exine wall and swelling of the underlying intine, and have identified naturally occurring components from pollen and stigma surfaces that when mixed experimentally, or at pollination, are sufficient to degrade sporopollenin. Our work on native biochemistry for breaching sporopollenin suggests new research directions in pollen aperture evolution and could aid efforts to analyze sporopollenin’s composition.

Quantification of tissue biomechanics during neural tube closure of mouse embryo with optical method

Jitao Zhang, Giuliano Scarcelli University of Maryland, United States

Embryonic development involves the interplay of driving forces that sculpt the tissue and the mechanical resistance that the tissue offers in response. While increasing evidence has suggested the crucial role of physical mechanisms underlying embryo development, tissue biomechanics remain to be understood due to the lack of techniques that can quantify the stiffness of tissue in-situ with 3D high-resolution and in a non-contact manner. In this work, we demonstrated the capability of Brillouin microscopy in quantifying the tissue biomechanics of mouse embryo in situ. Brillouin microscopy is an all-optical method that probes the mechanical properties of material via light scattering, and thus is non-contact and noninvasive. In addition, it does not need to deform the sample by external load during measurement, and can achieve sub-micron resolution when using high numerical-aperture objective lens. Here, we applied this optical technique to quantify the tissue stiffness of neural tube during the development of mouse embryo. We found the tissue stiffens significantly after the closure of the neural tube at cranial regions by comparing embryos at E 8.5 and E 9.5. In addition, we observed that the region of fusion following neural tube closure is softer than the adjacent neural folds, and the neural folds show a stiffness gradient along dorsal- ventral direction. Furthermore, we found the overlaying ectoderm is much softer and more pliable than the closed neural tube, and thus can be clearly distinguished based on its mechanical properties. In conclusion, we demonstrated the capability of Brillouin microscopy to quantify tissue stiffness during neural tube closure of mouse embryos. In general, this approach will be helpful in providing for a better understanding of how tissue biomechanics regulates embryonic development.

Modeling a Mammalian Nervous System in vitro through Instruction of Aggregates of Mouse Embryonic Stem Cells

Jinwoo Lee University of Virginia, USA

In order to establish novel experimental strategies to study mammalian neural development and neurological disorders, our lab is currently building a model of nervous system in vitro. To do so, we intend to develop an embryo mimetic system that will contain a properly patterned central nervous system. Our lab conceived an innovative approach in which we initiate embryonic development by instructing aggregates of mouse embryonic stem cells (embryoid bodies - EBs) with spatially localized morphogen activity centers. The resulting embryoids form the three germ layers through gastrulation and then proceed to organogenesis. At the end of gastrulation, these embryoids display a pseudostratified columnar epithelium expressing neural ectoderm markers. This neural plate shows proper apico-basal polarity with mitosis occurring at the apical surface. During neurulation, this neural plate folds and in some cases, closes. This neural tissue is patterned along the anterior-posterior axis from the hindbrain to the caudal spinal cord. It also displays expression of roof plate and neural crest markers along its lateral borders. Moreover, neurons are generated within the neural plate and extend their axons from the developing neural tube to the non-neural part of the growing embryoids.Altogether, these results demonstrate that the embryoid generated in vitro recapitulate development of a spatially patterned mammalian nervous system in vitro. This embryo mimetic system is a promising model for studying development of mammalian nervous system both in wild-type and in pathological conditions and will also be used in screens for drug discovery.

Long-range signaling that coordinates adult pigment pattern development in zebrafish

David Parichy Department of Biology, University of Virginia, Charlottesville, VA 22908

Zebrafish pigment pattern is a useful system for dissecting controls of morphogenesis and differentiation during the development of adult form. In this species, three different classes of neural crest derived pigment cells organize into a series of alternating light and dark stripes. Here I present recent findings that identify critical roles for thyroid hormone in promoting adult pigment pattern development, and in eliciting cell-type specific responses across pigment cell lineages that are essential for stripe formation. Our work provides insights into how local and global factors interact to generate species-specific pigmentary outcomes, and may shed light on the development and evolution of other adult derivatives of embryonic neural crest.

Evolutionary loss of developmental plasticity in the pea aphid Gregory K. Davis Department of Biology, Bryn Mawr College, Bryn Mawr, PA 19010

The pea aphid, Acyrthosiphon pisum, exhibits remarkable developmental plasticity in response to seasonal changes in photoperiod. In spring and summer, aphids reproduce asexually and are viviparous, yielding large numbers of genetically identical female offspring. The longer nights accompanying the fall induce these asexual aphids to produce sexual males and females, which mate to lay frost-resistant eggs. These eggs diapause through the cold winter months and hatch into asexually reproducing females in the spring, founding new clonal populations. Pea aphid populations have been shown to exhibit latitudinal variation in this photoperiod response, presumably reflecting local adaptation to variation in the timing of the first frost. Populations from the southern United States have been reported to exhibit attenuated photoperiod responses or to have lost the ability to produce sexuals altogether. I will describe differences in the photoperiod response of strains from the Northern and Southern United States and describe our efforts to understand the mechanism of change underlying these differences.

Identification and characterization of key regulators of runner-based asexual reproduction in the wild strawberry

John Sittmann, Julie Carauana, Zhongchi Liu University of Maryland, United States

Strawberry undergoes two distinct modes of reproduction. They can either reproduce sexually through flowers or reproduce asexually through the production of stolen, a horizontal stem with new daughter plants produced at every other node. This horizontal stem is also called runner as if the daughter plants are running away from the mother plant. Importantly, current agricultural practices of strawberry propagation are all runner-based as such runner-based clonal propagation serves to maintain the superior hybrid genotype, which would otherwise be lost through sexual reproduction. Further, runner and flower- producing shoot represent two alternative developmental fates. The same axillary meristem in the axil of a leaf chooses to become a flowering shoot or runner in response to environmental factors such as day length and temperature. The molecular mechanisms underlying these processes remain unknown. Since most plants do not exhibit runners, strawberry also serves as an excellent system to understand runner as a unique developmental process. We performed an ENU mutagenesis screen of wild strawberry (Fragaria vesca) that normally does not make runner. A constitutive runnering strawberry mutant referred to as U230 was identified. Using mapping-by-sequencing, the causative mutation was shown to be gene06210 coding for the DELLA protein involved in Gibberellic acid (GA) signaling. DELLA proteins were previously shown in Arabidopsis and rice to repress GA response including stem growth. Upon exposure to GA, DELLA would be degraded, allowing downstream GA responses and stem elongation to occur. Further analysis of this DELLA protein in the context of strawberry runner development is ongoing and results will be reported.

Role of smooth muscle in shaping airway epithelium prior to fusion in developing chicken lung

Michael Palmer Princeton University, USA

The respiratory system of birds contains a network of connected airways that begin as terminal structures and subsequently fuse as embryonic development progresses. We used embryonic Gallus gallus as a model organism with which to investigate morphogenesis in the lung during the stages leading up to fusion of the airways. We used immunohistochemistry analysis to visualize morphogenesis of the epithelium and surrounding tissue. We found that prior to fusion, the tips of the airways first bend away from their fusion partner, then initiate new branches in the direction of their target. These new branches contain the cells that form the first points of contact between the fusing airways. These branching events occur in regions devoid of smooth muscle, implying a possible role for smooth muscle in shaping the airway epithelium prior to fusion. We hypothesize that smooth muscle wrapping is required for epithelial fusion, which would constitute a novel mechanism for building continuous tubes in the embryo. This mechanism would be consistent with the role of smooth muscle in forming the terminally branched airways of the mammalian lung during development, and thus suggest either a conserved evolutionary morphogenetic mechanism or possibly an instance of convergent evolution across these two classes of animals with dramatically distinct respiratory systems.

Pioneering Microinjection Studies in Cephalopods: Lineage Insights and Transgenic Opportunities

Karen Crawford St Mary's Col of Maryland, USA

The cephalopods: squid, octopuses, cuttlefish and Nautiloidea, have long fascinated developmental biologists and the embryonic stages of many members from this group have been well described, often in fine line drawings. In addition, recent molecular studies of organogenesis stage embryos, harvested from naturally laid jelly fingers, have emerged. Despite this interest and effort, a reliable microinjection method to label cells in early cleavage stage embryos for either lineage analysis or introduction of transgenes has proved elusive. Over the years, reliable in vitro fertilization and culture methods for the Longfin squid, Doryteuthis (Loligo) Peallii, have been developed. More recently, this work has expanded to include microinjection methods to introduce vital dyes (DiI) and mRNA transgenes into cleavage stage embryos. This report includes an overview of in vitro fertilization, early cleavage, and embryogenesis in Loligo, in addition to preliminary results from DiI lineage and transgenesis-RFP (red fluorescent protein) reporter analysis. These results have spawned a collaborative study at the Marine Biological Laboratory (MBL), Woods Hole, MA, to not only extend this work in Loligo to include CRISPR/Cas9 knockouts, but also expand them into two new model organisms, the Hawaiian bobtail squid (Euprymna scolopes) and the striped pyjama squid (Sepioloidea lineolate). Unlike Loligo, these developing model system organisms have recently been bred in culture through multiple generations at the MBL. These advancements not only create the opportunity to more effectively study the molecular mechanisms of cephalopod development year-round, but also to generate our first lines of transgenic cephalopods.

Integrating classical and molecular genetics using Mendel’s le mutant

Nick Kaplinsky Department of Biology, Swarthmore College, Swarthmore, PA 19081

Introductory Biology courses often use Mendel’s pea experiments as the basis for teaching classical genetics and then use other examples for teaching molecular genetics. The molecular mechanisms that underlie several of Mendel’s mutant phenotypes have been elucidated and provide an opportunity to integrate classical and molecular approaches. We have developed a multi-week lab that investigates the molecular basis of Mendel’s stem length trait which is controlled by the LE gene. In addition to exploring the relationship between phenotypes and genotypes, this lab can be used to teach students about signal transduction and biosynthetic pathways. The lab requires a minimal amount of plant care and uses commercially available pea lines.

Reimagining K-12 STEM Education by Incorporating the Arts

Merci N. Best, Robin R. Best, and Cheryl L. Dickter BIMS Graduate Program, School of Medicine, University of Virginia, Charlottesville, VA 22908

Females and racial minorities (referred hereafter as multicultural individuals) are underrepresented in the fields of science, technology, engineering, and mathematics (STEM). The current study is focused on exploring what relationships exist between STEM education programs and the formation of positive STEM-self-efficacy that sustains female and multicultural populations interest and participation in the STEM pipeline. Thirty-eight students from multicultural backgrounds at a medium-sized community center completed Race- Science IAT and Self Esteem IAT (Custom Items) measures. Results revealed students who regularly participated in STEAMtrix, LLC, a STEM education program that incorporates the arts, report increased levels of positive STEM-self- efficacy. The study offers insight into what can be done during out-of-school time by community organizations and/or school systems to promote early access and self-efficacy in relation to STEM educational experiences.

SEA-PHAGES: A model integrated Research Education Community (iREC)

Graham F, Hatfull. Department of Biological Sciences, University of Pittsburgh, Pittsburgh PA 15260

Involvement of scientific research at the undergraduate level has numerous educational benefits to the student. However, opportunities for apprentice-like research experiences are limited by the positions available, and competition for these opportunities can impede full inclusivity of the great student diversity. Course–based research experiences (CUREs) can involve larger number of students, but meaningful and productive experimental systems are few, largely because of limited time and resources. An integrated Research Education Community (iREC) offers an alternative system in which a discovery-based research project is supported by a robust programmatic infrastructure; this facilitates inclusion of diverse institutions from community colleges to R1 Universities, faculty with varied experiences and expertise, and large numbers of undergraduate students without prior selection based on academic performance. The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is a model iREC, in which first year undergraduates isolate and genomically characterize bacteriophages. Because the phage population is vast, dynamic, and old, their genomic diversity is massive, and thus there is a high likelihood that students will discover new genomes and new genes. The SEA-PHAGES infrastructure currently supports 110 participating institutions, and over 4,500 students in this current year. The program has generated a large collection of over 13,000 individual phage isolates, of which over 2,500 have been fully sequenced. Psychometric assessment using the Persistence in the Sciences (PITS) survey indicates the strong student impact on their intent to continue scientific study relative to a traditional\ introductory laboratory course, and the impact is broadly observed regardless of demographic, gender, and institutional variation, supporting the inclusive nature of the iREC model.

Coordinating collective cell behaviors from local interactions through the planar cell polarity pathway

Danelle Devenport Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions, and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles (HFs), which emerges through epidermal-dermal signaling and planar polarized morphogenesis. We recently discovered that hair follicle progenitors (placodes) polarize through dramatic and stereotyped rearrangements organized into a counter-rotational pattern of collective cell movements. These movements completely reorganize the placode epithelium along the AP axis – centrally located cells are displaced anteriorly to lead follicle growth, while outer cells are swept posteriorly to become the placode rear. Counter rotational rearrangements require planar cell polarity (PCP) and myosin activities and are driven by polarized junctional remodeling and directed neighbor exchange. Importantly, these cell rearrangements generate not only morphological asymmetry, but also reposition HF progenitors from a radial to planar polarized organization, this establishing cell fate asymmetry. Strikingly, these movements mimic the large- scale cell flows that accompany primitive streak formation in the chick embryo and share features of convergent extension movements that drive axis elongation in gastrulation and neurulation. Based on these findings, we propose that PCP-directed neighbor exchange is a core and versatile mechanism underlying polarized morphogenesis that extends well beyond its established role in convergent extension.

Distinct Signaling Roles for Type I Receptors within a BMP Heterodimer Receptor Complex.

Benjamin Tajer, James Dutko, Mary Mullins University of Pennsylvania, USA

The Bone Morphogenetic Protein (BMP) pathway patterns dorsal-ventral (DV) axial tissues during gastrulation. The zebrafish embryo is an excellent system to investigate the mechanism of BMP signal transduction during DV patterning, as many BMP pathway mutants are available and, unlike in mammals, these mutants survive to show DV patterning defects. When signaling, a dimeric BMP ligand assembles a receptor complex composed of two type I and two type II receptors. Type II receptors phosphorylate and activate type I receptors, which in turn phosphorylate Smad proteins. Phosphorylated Smad then regulates gene expression. This model, however, is overly simplistic as there are two conserved classes of type I receptor, Bmpr1 and Acvr1l, and two conserved classes of type II receptor Bmpr2 and Acvr2, all of which are necessary for early vertebrate embryonic development. Our previous findings demonstrate that BMP2/7 heterodimers are the only ligands that signal in DV patterning. This sufficiency arises from the the unique ability of the heterodimer to integrate both type I receptor classes into the BMP receptor complex, as Bmpr1 preferentially binds the BMP2 ligand, and Acvr1l binds BMP7. We have also found that kinase dead Acvr1l cannot rescue acvr1l deficiency, while, surprisingly, kinase dead Bmpr1 can rescue bmpr1 deficiency. Through truncation experiments we found that, while Bmpr1 kinase function is unnecessary, the Bmpr1 intracellular domain is required for DV patterning. Additional domain swap experiments suggest that the intracellular domains of Bmpr1 and Acvr1l are not interchangeable, and play separate functional roles in BMP signaling. I am now determining the location and nature of the motifs within Bmpr1 and Acvr1l that impart these specific functions through a series of smaller domain swap experiments.

Dynamic patterns of migration of FGF-expressing cells guide Drosophila tracheal branching morphogenesis

Lijuan Du, Sougata Roy University of Maryland, USA

An elegant branched tubular network of epithelial cells constitutes diverse types of animal organs, such as Drosophila trachea, vertebrate lung, kidney, and vasculature. Commonly, a gradient of the secreted growth factors is hypothesized to guide the directed migration of the growing epithelial tubes. To investigate the mechanisms of induction and migration of a branch by signaling proteins, we focused on Drosophila trachea. In Drosophila embryo, one FGF homolog, branchless (bnl), expressed in six different clusters surrounding a tracheal placode, which expresses the Bnl-receptor, breathless (btl), induces branching morphogenesis of the primary branches from the placode. By checking endogenously expressed Bnl:GFP and Btl:Cherry from genomic knock-in alleles, we uncovered that, Bnl:GFP does not form a pre-patterned gradient. Rather, Bnl gradients have diverse and dynamic branch-specific shapes and are receptor- bound. Moreover, we show that the tracheal cells project cytonemes to directly contact the bnl-source and receive Bnl. Therefore, to investigate how a contact- dependent local signaling might mediate Bnl-dependent guided migration in trachea, we developed a highly reliable bnl-specific targeted expression driver, bnl-LexA, using the CRISPR/Cas9 based genome editing to simultaneously mark the bnl-expressing cells along with the trachea. Live imaging of such embryos revealed a unique morphogenetic movement of the bnl-sources followed by a coordinated migration of tracheal branches. We propose that migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branches. In the future, it will be important to investigate how bnl-source might spatiotemporally coordinate its own migration ahead of a growing tracheal branch, and whether the paradigm of coordinated migration of the signal source and recipient cells can be generalized in patterning of all other branched tissues.

Making one nose from two: genetic and morphological mechanisms of facial development.

Heather L. Szabo-Rogers Center for Craniofacial Regeneration, Department of Oral Biology, University of Pittsburgh School of Dental Medicine.

Human orofacial clefting (OFC) is the most common congenital anomaly. OFC has many phenotypic variations from unilateral cleft lip to isolated cleft palate to midfacial clefting. The molecular and physical determinants that contribute to the development of OFCs are complex and range from single gene disorders to complex gene-environment interactions. To model OFC morphogenesis, we have been analyzing the craniofacial phenotypes in the Unicorn mouse line. The Unicorn line is an ENU (N-ethyl-N-nitrosurea) mutagenized mouse line. Unicorn mutants develop a striking midfacial cleft phenotype including two physically independent nostrils that include normal skeletal elements and a duplicated nasal septum. In part the phenotype arises because the organizer of proximal-distal outgrowth, the frontonasal ectodermal zone, is displaced laterally predisposing the outgrowth of two independent nostrils. During normal development, the nasal septum develops through a medial merging event between the paired medial nasal prominences. Prior to the fusion event in Unicorn mutants, we observed increased tight junctions in the medial nasal prominence epithelium. We observe a mild midfacial cleft in Beetlejuice mutants. The Beetlejuice mouse line has missense mutation in Prickle1, therefore, we hypothesize that midline merging events in the craniofacial region require Wnt/planar cell polarity signaling pathway. Our data provides insight into novel pathways and morphological mechanisms for OFC.

Regulatory roles of microRNAs in development

Jia L. Song Department of Biological Sciences, University of Delaware, Newark, DE, USA

The microRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. In early development, cell specification and pattern formation are controlled by cross- regulation of gene regulatory networks (GRN) and signaling pathways. Our overarching hypothesis is that miRNAs perform the critical regulatory function of controlling signaling morphogen gradients and critical regulators to ensure proper development. The goal of our research is to understand how miRNAs integrate GRNs and signaling pathways to drive development. To accomplish this goal, we use the sea urchin model to study complex developmental processes that are shared by a wide range of organisms. The sea urchin model has an exceptionally well-studied GRN and most of its miRNA families consist of a single species, which makes it amenable to unique, powerful functional analysis. To understand the regulatory roles of miRNAs, we have performed single miRNA perturbation analyses, as well as examined miRNA regulation of signaling pathway components. Since miRNAs, GRNs, and signaling pathways are highly conserved in animals, this research advances the field of miRNA research and contributes to the fundamental understanding of early developmental processes.

POSTERS

ID #3884 MicroRNA suppression of Dishevelled results in Wnt pathway associated developmental defects Nina Faye Sampilo1, Nadezda Stepicheva1, Aun Zaidi1, Priya Nigam1, Lingyu Wang2, Wei Wu2, Athula Wikramanayake2 1University of Delaware, USA; 2University of Miami, USA MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3’untranslated region (UTR) of target mRNAs and silence translation. MiRNAs are key regulators of the Wnt signaling pathways which impact cell proliferation, migration, polarity and other developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled (Dsh/Dvl), an important signaling protein upstream of β-catenin. The sea urchin Dvl mRNA isoforms have similar spatial localization in early development, but one isoform has distinct ciliary staining in the larval stage. Using luciferase assays and site-directed mutagenesis, we demonstrated that the different isoforms of Dvl are directly suppressed by miRNAs. By blocking miRNA suppression of all Dvl isoforms using miRNA morpholino antisense oligonucleotides target protectors (miRNA MASO TPs), we observed dose-dependent defects of spicule length, patterning of the primary mesenchyme cells, gut morphology and aberrant cilia length and arrangement. These defects likely result from increased Dvl protein, leading to perturbation of Wnt dependent signaling pathways and additional Dvl-mediated processes. Overall, our results indicate that miRNA suppression of Dvl isoforms plays an important role in ensuring proper early development.

ID #3888 Substrate specific regulation of CDK5 activity by intermediate filaments in developing cortical axons. Christopher Bott University of Virginia, USA The intermediate filaments (IFs) in the brain have distinct temporal and cellular distributions during development, and been implicated in regulating both kinase and phosphatase signaling pathways in neurons and other cell types through a variety of mechanisms. Nestin is a unique IF protein required for Cyclin Dependent Kinase 5 (CDK5) activity and localization during development of the neuromuscular junctions of mice. In neurodevelopment, nestin is traditionally used as a neural stem cell marker, but we have identified a population of cortical neurons that continue to express nestin in their axons transiently during differentiation. The transient expression of nestin in neurons raised the question of what roles neuronal nestin might play, and we hypothesized that nestin may affect CDK5 dependent processes in neurons. We now show that nestin-expressing neurons are more sensitive to semaphorin 3A, an early CDK5 dependent axon repulsive guidance cue, compared to neurons not expressing nestin. Early growth cone sensitivity to semaphorin 3A is thus upstream of CDK5 activation and enhanced by nestin. We evaluated a number of neuronal CDK5 substrates for nestin binding and identified doublecortin (DCX), a microtubule binding/regulating protein important for growth cone guidance, as a novel nestin binding protein. Biochemical experiments suggest that nestin acts a scaffold for active CDK5 and DCX thus promoting phosphorylation of DCX. Phosphorylated DCX no longer promotes microtubule stability, leading to greater sensitivity to semaphorin3a downstream of CDK5 activity. Evaluation of other neuronal IFs reveal that that neurofilament medium, which is expressed later in neuronal maturation, suppresses DCX phosphorylation by CDK5. Expression of nestin and neurofilament medium may represent different maturational states of neurons (early/dynamic and late/stable, respectively), allowing for substrate specific variance of CDK5 activity mediated by temporally expressed scaffolds.

ID #3895 Spop regulates Gli3 activity and Shh signaling in dorsoventral patterning of the mouse spinal cord Hongchen Cai, Aimin Liu The Pennsylvania State University, USA Sonic Hedgehog (Shh) signaling regulates the patterning of ventral spinal cord through the effector Gli family of transcription factors. Previous in vitro studies showed that an E3 ubiquitin ligase containing Speckle-type POZ protein (Spop) targets Gli2 and Gli3 for ubiquitination and degradation, but the role of Spop in Shh signaling and mammalian spinal cord patterning remains unknown. Here, we show that loss of Spop does not alter spinal cord patterning, but it suppresses the loss of floor plate and V3 interneuron phenotype of Gli2 mutants, suggesting a negative role of Spop in Gli3 activator activity, Shh signaling and the specification of ventral cell fates in the spinal cord. This correlates with a moderate increase in the level of Gli3 protein in Spop mutants. Furthermore, loss of Spop restores the maximal Shh pathway activation and ventral cell fate specification in the Gli1;Sufu double mutant spinal cord. Finally, we show that loss of Spop-like does not change the spinal cord patterning in either wild type or Spop mutants, suggesting that it does not compensate for the loss of Spop in Shh signaling and spinal cord patterning. Therefore, our results demonstrate a negative role of Spop in the level and activity of Gli3, Shh signaling and ventral spinal cord patterning.

ID #3899 Nutrient-dependent Development of the Glial Niche Coordinates Neuroblast Exit from Quiescence Xin Yuan University of Virginia, United States Neuroblasts (NBs), the Drosophila neural stem cells, enter quiescence at the end of embryogenesis, and reactivate after freshly hatched larvae consume their first complete meal. In response to food consumption, insulin-like peptides (Dilps) are produced and secreted locally from glia and systemically from insulin producing cells in the brain. Local Dilp signaling activates the highly conserved PI3-kinase growth signaling pathway in NBs, leading to their reactivation. While NBs rely on Dilps to reactivate, it remains unclear whether other cell types within the early larval brain also require Dilp signals, either locally or systemically, for growth. Like NBs, we find that the cortex glia, which ensheath NBs and their newborn progeny, as well as trachea require food consumption and PI3-kinase activation for growth. When PI3-kinase activity is reduced or when dietary nutrients are withdrawn, cortex glia fail to elaborate membrane and ensheath NBs, and tracheae fail to infiltrate the brain hemispheres. Growth and elaboration of both cortex glial membranes and tracheae coincide with NB reactivation from quiescence, but only cortex glial growth is required for NB reactivation. When PI3- kinase is reduced in cortex glia, NBs fail to reactivate in response to food consumption. NBs also fail to reactivate in response to larval feeding when cortex glia are genetically ablated. We conclude that development of the NB glial microenvironment is nutrient- regulated and is required for NB reactivation. Furthermore, we are investigating dynamic changes within the brain tissue architecture that occurs in response to animal feeding and how tissue architecture impacts NB proliferation decisions.

ID #3900 Glial Cell Interactions Across The Motor Exit Point Transition Zone Laura Fontenas, Sarah Kucenas University of Virginia, USA The nervous system is divided into two complementary halves, and their complementarity is uniquely possible through transition zones that enable interactions between the two systems. Motor neurons originate and reside in the ventral-lateral spinal cord while their axons exit the central nervous system (CNS) through motor exit points (MEP) to reach their targets in the periphery. Oligodendrocytes and Schwann cells (SC), which are central and peripheral myelinating glia, respectively, migrate along the same axons and eventually myelinate distinct axonal segments restricted to their own half of the nervous system. However, how this segregation of glia and myelination occurs is not well understood. Previously, our lab showed that CNS-derived peripheral glia, called MEP glia, exit the spinal cord at the MEP, divide and ultimately differentiate into myelinating glia that ensheath spinal motor root axons and segregate oligodendrocytes to the CNS. To determine if MEP glia use the same signaling mechanisms that drive SC migration, proliferation and differentiation we are using in vivo imaging in zebrafish to examine glial-glial interactions across the transition zone during nervous system assembly. Additionally, we have identified new MEP glial markers and are creating both reporter lines and CRISPR mutants to further understand the functional features of these intriguing glial cells and provide novel information about the mechanisms underlying spinal nerve assembly.

ID #3902 Vangl1/2 function in convergence and extension and neural tube closure Maraysa de Oliveira Melo, Emily Noonan, Ann Sutherland University of Virginia, USA Antero-posterior elongation is driven by convergence and extension (CE), a fundamental process for body plan formation. In the mouse embryo, morphogenesis of the early neural tube involves CE, driven by epithelial cell intercalation, as well as bending and closure of the neural plate into a tube, driven by cell shape change and cytoskeletal reorganization. At the genetic and molecular level, components of the planar cell polarity (PCP) pathway have been shown to regulate these polarized cell behaviors. Mutations of the Van Gogh like 2 (Vangl2) gene, lead to mice with a short body axis and open neural tube from the hindbrain to spinal cord regions (craniorachischisis). Previous data from our lab showed that the Vangl2 Loop tail (Lp) mutant, affects cell intercalation efficiency but not polarity of neural plate cells. As the close homolog Vangl1 has a similar localization to Vangl2 in the neural plate, our hypothesis is that Vangl1 may compensate to maintain normal polarity in the absence of Vangl2. In wt embryos, the cell pair separation occurs mostly in the anterior-posterior direction. Using a line carrying knockout alleles of both Vangl genes we find that loss of Vangl1 and 2 (Vangl1gt/gt;Vangl2ko/ko) affects the polarity of cell pair separation, while the knockout for Vangl2 with one copy of Vangl1 (Vangl1gt/+;Vangl2ko/ko) does not. The polarity of intercalation is also lost in embryos lacking Vangl1 and one copy of Vangl2 (Vangl1gt/gt;Vangl2ko/+), and in mutants for Vangl1 only (Vangl1gt/gt;Vangl2+/+), suggesting that Vangl1 is important for polarity and not Vangl2. In absence of Vangl2, the apical area of neural cells is significantly increased and the actin and myosin filament organization is disrupted, suggesting a role of Vangl2 in apical constriction. These data show that the polarity of CE is primarily regulated by Vangl1 while Vangl2 is responsible for regulating the apical changes that drives neural tube bending and closure.

ID #3903 Keeping it together: the role of FGF signaling in embryonic ventral wall closure Michael Boylan National Cancer Institute, USA Omphalocele is a severe ventral wall (VW) defect in which the abdominal contents have herniated through an enlarged umbilical ring during embryonic development. In humans, omphalocele is associated with increased mortality, but the etiology of this condition is poorly understood. We serendipitously observed omphalocele after generating mice in which the genes coding for the FGF8 subfamily (Fgf8, Fgf17 and Fgf18) were inactivated specifically in the primitive streak and emerging mesoderm using TCre. We examined the contribution of each of the three genes to the penetrance of the phenotype, and determined a genetic hierarchy (Fgf18>Fgf8>Fgf17). Omphalocele is thought to be caused by defective myoblast migration from the somites, leading to a failure in VW closure. Fgf8 subfamily null embryos also show impaired myoblast migration, suggesting the defect we observe is analogous to omphalocele in humans. Experiments using different Cre lines show that the Fgf8 subfamily is dispensable in the muscles themselves, and may be required in the presomitic mesoderm (PSM) or somites. The role of Fgf8 in the PSM has been well studied, but any roles of Fgf17 and Fgf18 have not been previously identified. We observed ectopic cell death in the somites of Fgf8 subfamily mutants, which could explain the defects in the ventral muscles. To test whether the cell death observed contributes to the VW defect, we additionally knocked out the pro-apoptotic genes, Bak and Bax. We found that the incidence of omphalocele dropped significantly when apoptosis is suppressed genetically, suggesting that the observed somitic cell death causes omphalocele. We are currently investigating whether the somite specification program is malfunctional; we are also examining the role of FGF receptors 1 and 2 in VW closure. This project will clarify the role of FGF signaling in VW formation, which has received little attention despite its medical relevance. ID #3906 Evolutionary genetics of ovarian arrest in Drosophila melanogaster Priscilla A. Erickson, Daniel Y. Song, Helen M. Stone, Alan O. Bergland University of Virginia, USA Organisms living in temperate environments utilize environmental cues such as light and temperature to make physiological and developmental adjustments that exploit and avoid favorable and unfavorable times of year. When young female Drosophila melanogaster from temperate climates encounter cool, fall-like conditions, they enter a reproductive quiescence (diapause) characterized by an absence of oocyte maturation. However, the genes and pathways underlying this developmental plasticity remain undercharacterized. Using a novel association mapping approach, we are examining the genetic basis and evolutionary history of diapause in D. melanogaster. We generated two outbred populations representing eastern North American clinal diversity by intercrossing 68 inbred lines for 4-5 generations. We exposed hybrid females to different temperatures and phenotyped 2800 flies for ovary development. We have reconstructed full genomes for all phenotyped individuals using low-coverage (~0.5X) Illumina sequencing data, enabling us to perform a genome-wide association study for loci controlling temperature-induced ovarian arrest. Functional studies of candidate genes are ongoing. Further, we have placed the outbred populations in outdoor mesocosms and will use pooled sequencing to assess overwintering survival and the fitness costs and benefits of loci associated with ovarian arrest. We will also examine the frequency of diapause-associated alleles in wild North American populations sampled throughout spring and fall. These integrative experiments will shed light on the molecular mechanisms insects use to alter development in response to seasonal changes and will provide valuable insight into the evolutionary history of this ecologically relevant developmental plasticity.

ID #3907 Regulation of the cell cycle and cell fate by TGFß signaling in larval zebrafish retina Elizabeth Hannifin, Sarah Vogel, Michael Koropsak, Edna Ferreira, Maeve Downey, Katie Brandt, Husni Alasadi, Jenny Lenkowski Goucher College, USA Several studies indicate that the TGFβ signaling pathway plays an important role in regulating proliferation of progenitors and cell differentiation during mammalian retinal development and zebrafish retinal regeneration. Specifically, TGFβ signaling has been shown to promote Müller glial cell fate, inhibit proliferation, and increase differentiation into photoreceptors during rodent retinal development. We are using chemical genetics and genetic approaches in zebrafish to manipulate TGFβ signaling in order to study how the signaling pathway regulates the cell cycle and cell differentiation during retinal development in fish, which has not yet been described. Our preliminary studies of retinal development in zebrafish using fluorescence immunohistochemistry indicate that the ciliary marginal zone where stem cells reside in the mature retina may have fewer proliferating cells and the retina may have fewer Müller glial cells when TGFβ is upregulated. Ongoing experiments are aimed at generating a new transgenic fish in which to study cell cycle dynamics in vivo and to examine cell-cycle exit in the developing retina when TGFβ signaling is misregulated.

ID #3908 Using Zebrafish to better understand the whole Daam Family! Carlee Cunningham, Kaushik Nama, Christopher Cassella, Amulya Surakanti Temple University, USA Wnt signaling is a highly conserved group of pathways that dictate processes such as cell fate and morphogenesis during embryonic development. Canonical Wnt signaling involves the shuttling of β-catenin into the nucleus where it controls expression of target genes, establishing dorsal-ventral patterning, whereas non-canonical signaling works through small GTPases to govern actin polymerization necessary for gastrulation movements. A main player in non-canonical Wnt signaling is Daam1, which has been shown to be essential for convergent extension and neural tube closure in Xenopus laevis. We have been working to understand if the related protein Daam2 also has a role in this pathway. Studies in frog showed that xDaam2 is implicated similarly to xDaam1 in non-canonical Wnt signaling and has comparable knockdown phenotypes. Due to conflicting opinions on the role of Daam2, we are using zebrafish to further study all of the Daams. Zebrafish have a duplicated zDaam1 gene, both of which are expressed like xDaam1: expression is maternal and then persists throughout at least the first 24 hours of development. zDaam2 is not expressed maternally but becomes detectable at early somitogenesis. Whole mount in situ hybridization shows zDaam1a/1b/2 have ubiquitous expression that concentrates in the head region by 24hpf, with zDaam2 expression delayed until somitogenesis. Individual maternal-zygotic mutants for Daam1a, Daam1b, and Daam2 do not display noticeable phenotypes during development or adulthood, and we suggest that this may be due to genetic compensation by the intact zDaam genes. We have been working to establish double and triple mutants to answer this question and are also using morpholinos to affirm any observed knockout phenotypes. The goal of this project is to better our understanding of how the three zDaam genes work together to control morphogenetic development and confirm their role in non-canonical Wnt signaling.

ID #3909 Tubular organ morphogenesis by transcriptional regulation of the translational machinery Rajprasad Loganathan Johns Hopkins University, USA Cell growth is an essential determinant of organ morphogenesis. In particular, tubular organ form is critically dependent on the growth characteristics of constituent cells. We describe the role of a BTB-domain nuclear factor, Ribbon (Rib), during the assembly of the Drosophila embryonic salivary gland (SG), a tubular secretory organ, specialized for high capacity protein synthesis and secretion. Rib functions tissue autonomously to facilitate SG tube elongation. SG cell specification in rib mutant embryos is normal as are other morphological features such as localization of junctional protein markers and epithelial polarity markers. SG cell number in the rib mutants is also comparable to controls. A major morphological characteristic underlying the tube elongation defect in rib mutants is a significant reduction in cell volume, affecting cytoplasmic but not nuclear size. To determine how Rib controls cell growth by transcriptional regulation, we performed SG-specific ChIP-Seq, which revealed that the major target of Rib was the ribosome. Rib also bound genes encoding translation factors and chaperones that are required for protein synthesis. Transcript analysis of a subset of ribosomal protein genes bound by Rib revealed its requirement for their full levels of expression. rib mutant SG cells also showed nucleolar morphological defects along with cytoplasmic accumulation of P-bodies implying compromised ribosome biogenesis and an abundance of untranslated mRNA. Interestingly, Rib immunoprecipitates with the TC- rich sequences embedded at the transcription start site of ribosomal protein genes suggesting that it might function in transcription initiation at these loci. Taken together, transcriptional regulation of the translational machinery by Rib is required for the coupling of SG cell growth with the tubular organ form.

ID #3911 ftz-f1 is necessary to maintain germline stem cell function in Drosophila oogenesis Samantha McDonald, Hanna Berghout, Amelia Blake East Carolina University, USA Stem cells are fundamental for organ formation and function in mammals and insects.For example, tissue resident stem cells serve as the foundation for gamete production in Drosophila melanogaster and many other organisms. Nuclear receptors are prospective mediators of stem cell activity because of their conserved roles in physiology and tissue homeostasis; however the mechanisms by which nuclear receptors regulate stem cell self-renewal and proliferation have yet to be identified.In the mammalian NR5 family, SF-1 and LRH-1 have roles in gonadogenesis and organ formation. In Drosophila, the NR5 family nuclear receptor, ftz-f1, is necessary for embryogenesis and adult morphogenesis following puparation. Ftz-f1 is expressed in several populations of cells in the ovary, including germline stem cells (GSCs), though its function in those cells is unknown. We hypothesize that Ftz-f1 is necessary for GSC regulation. Using clonal analysis through Flippase/ Flippase Recognition Target (Flp/FRT) mediated recombination and tissue specific RNAi, we studied loss of ftz-f1 function in early stages of oogenesis. Tissue specific knockdown of ftz-f1 with the UAS- Gal4 system in the germline showed a decrease in the number of GSCs over time. Moreover, GSCs harboring two copies of the null ftz-f1ex7 mutation were more frequently displaced from the somatic niche, as compared to controls. We are currently assessing the role of ftz-f1 in GSC daughters. We also observed abnormal ovariole phenotypes following loss of ftz-f1 function, including the appearance of punctate cyst nuclei, suggestive of follicle cell or cyst death. Our data implicates ftz-f1 in directing GSC self- renewal in the ovary. Our future experiments will test how ftz-f1 regulates stem cell self- renewal, proliferation, and differentiation.

ID #3915 Combining Fragment Analysis and RFLP to Detect Rare Edited Alleles Chon-Hwa Tsai-Morris, Stephen G. Kaler, Benjamin Feldman NIH/NICHD, USA We are seeking to establish best practices for our core facility to generate and identify zebrafish with precise genome edits using CRISPR/Cas9-based methods. Because precise genome edits are generated at relatively low rates, genomes carrying precise mutations must be identified from a pool in which wild-type genomes, standardly mutagenized genomes (due to NHEJ errors) and genomes with imprecise incorporations of template are more highly represented. This low rate and diversity of outcomes presents challenges in (1) the analysis of injected F0 embryos to determine whether a given strategy shows promise and (2) the identification of germ-line transmitting F0 adults. We will describe the creation and detection of a targeted amino- acid substitution in the atp7a gene using a single-stranded oligonucleotide (ss-ODN) “donor” template. To facilitate detection of precise edits, we modified our standard fluorescent-PCR fragment size analysis1 to detect acquisition of two unique restriction- enzyme sites that we also included in the ss-ODN oligo. Using this method for rate estimation, we detected one in twenty F0 embryos carrying precise edits. In a separate cohort of adult F0s, 0 of 16 showed germ-line transmission, but fin biopsies of an additional 15 sibling F0s identified two candidates carrying precise edits in their soma and one of these showed germ-line transmission.The atp7a mutation we created is a cognate to a human mutation that causes distal motor neuropathy in adolescents and young adults2. Accordingly, we will now proceed to the medical-research phase and goal of this project, which is to characterize the potential disease progression of motor neurons in zebrafish homozygous for this mutation. We will also test the applicability of our strategies to new loci by seeking to generate single amino-acid changes in other genes under investigation at NICHD.

1Carrington et al., PMID 26253739

2Kennerson et al., PMID:20170900

ID #3916 Investigating Roles for Slitrk1 in the Developing Zebrafish Spinal Cord Barbara Lom1, Alishah Lakhani1, Morgan Shannon1, Justin Strickland1, Jennifer Round2 1Davidson College, USA; 2Ursinus College, USA The Slitrks are a novel gene family of six transmembrane proteins with potential to play important roles in the central nervous system due to their structural similarities to the well known Slit and Trk proteins families. Slitrks are known to influence neurite outgrowth, neuronal survival, synapse formation, and behavior. Mutations in Slitrk genes have been associated with neuropsychiatric disorders such as Tourette’s syndrome and OCD spectrum disorders. Slitrk1 is the only member of the Slitrk family that lacks tyrosine phosphorylation sites in the intracellular component of its transmembrane protein. In addition, overexpression of Slitrk1 promotes neurite outgrowth, whereas overexpression of other Slitrks inhibits neurite outgrowth. Thus, Slitrk1 may have distinct roles in neuritogenesis and synaptogenesis. Slitrk1 mRNA is expressed robustly throughout the young zebrafish spinal cord and protein expression localizes to Rohon-Beard (RB) neurons, early and transient mechanosensory neurons that innervate the trunk, sense information from the environment, and mediate initial escape responses. To determine if Slitrk1 plays a role in RB neuron development and function we conducted knockdown experiments by microinjecting antisense morpholino oligonucleotides (AMOs) to inhibit Slitrk1 translation in early zebrafish development. Slitrk1 knockdown did not affect secondary motorneurons but produced small consistent and significant reductions RB neuron numbers at 48 hpf. Thus, this study provides preliminary insights into potential roles of Slitrk1 in vertebrate central nervous system development that may begin as early as neurogenesis and/or neuronal survival.

ID #3917 Transcriptional Control of the Formation of the Left-Right Organizer Jonathan Fillatre, Bernard Thisse, Christine Thisse University of Virginia, USA Formation of mature organs from a totipotent cell, the egg, involves a progressive restriction of lineage potential. This process is achieved through changes in chromatin and transcription factor (TF) networks: genes associated with pluripotency are progressively silenced by DNA methylation, histone modifications and chromatin compaction while key TFs selectively activate the expression of tissue specific genes. Using the differentiation of the Kupffer’s vesicle, a ciliated organ known to be the zebrafish Left-Right Organizer (LRO) as a model system, we investigated how the activity of TFs and epigenetic modifications of the chromatin control organogenesis in vivo. We discovered that at gastrula stage the activity of transcriptional effectors of the Hippo signaling pathway is essential for the formation of the LRO. Indeed analysis of the phenotype of morphants and Cripsr/Cas9 mutants demonstrate that the activity of the TFs (TEAD1a/3a) and of the transcription cofactors that mediate (Yap, Taz) or regulate (Vgll4b, Vgll4l) the transcriptional outcome of the Hippo pathway, is required in the LRO precursors for the formation of the LRO. Using transcriptome analysis, we show that Vgll4l and Yap/Taz are master regulators of the formation of the LRO and control the activity of major signaling pathways (Notch, WNT, Nodal) as well as the expression of TFs involved in ciliogenesis (e.g.: Foxj1a). In addition they regulate, in the LRO precursors the expression of genes coding for writers and readers of DNA methylation marks, providing the first evidence of a spatial and temporal control of DNA methylation by the transcriptional activity of downstream effectors of Hippo signaling. Finally, we found that loss of function (morphants and mutants) of these epigenetic factors also results in LRO differentiation defects. Altogether, we propose that the regulation of DNA methylation by Yap/Taz and Vgll4l is a novel mechanism by which Hippo pathway contributes to organogenesis.

ID #3918 Investigating the formation of spatial and directional patterns in the mammalian epidermis Liliya Leybova Princeton University, USA Organ morphogenesis involves a complex interplay between epithelial-mesenchymal interactions, cell fate induction, and tissue polarity. However, it remains unclear how this interplay coordinates morphogenesis in the developing skin epithelium. The mammalian skin, which is decorated with spatially patterned, globally aligned hair follicles (HFs), is an excellent system to explore how spatial and directional cues coordinate epithelial morphogenesis. HFs develop from multicellular placodes that emerge from the embryonic epidermis in waves of evenly spaced epithelial clusters. Epithelial- mesenchymal crosstalk specifies HF progenitors, while planar cell polarity (PCP) orients the direction of HF growth and the asymmetric positioning of early HF lineages. Recently, we developed methods to perform long-term live imaging of epidermal development to capture the individual and collective cell behaviors that drive polarized morphogenesis of mammalian HFs. We discovered an unanticipated and novel pattern of collective cell movements that generates both morphological and cell fate asymmetry of developing follicles. Shortly after placode formation, HF progenitors undergo dramatic cell rearrangements coordinated in a counter-rotational pattern of cell flow that repositions placode cells within the epithelial plane. To generate the pattern of cell flow, spatial patterning of radial cell fates cooperates with PCP to direct polarized cell neighbor exchanges through myosin-dependent junction disassembly. Using a combination of live imaging, genetic manipulations, and transcriptional profiling, I will investigate how the spatial patterning of distinct placode lineages instructs these collective cell movements. I hypothesize that spatial patterning establishes two radially- arranged lineages that differ in motility, contractility, and cell-cell adhesion properties, and that these differences are essential for the generation of counter-rotational cell flow.

ID #3920 Comparative analysis of the response to Notch signaling perturbation across Xenopus species during embryonic development Mark Pownall, Ronald Cutler, Margaret Saha College of William and Mary, USA Embryonic development is a robust process during which embryos must respond and compensate for changes in order to achieve consistent patterning; however there are still questions about the limits and mechanisms of this robustness. Using tetraploid Xenopus laevis as a model, we have shown that embryos respond to perturbations of the highly-conserved Notch signaling pathway in a compensatory manner. RNA-seq analysis of Notch perturbed X. laevis embryos revealed that homeologs respond differentially to this perturbation, suggesting that the polyploidy of X. laevis may contribute to the compensatory abilities. To address this question, we have perturbed Notch signaling in X. borealis, a tetraploid species that is closely related to X. laevis, and characterized the response over time. Similarly to X. laevis, a compensatory response is seen in X. borealis, but embryos appear morphologically deformed throughout development, suggesting that X. borealis embryos may be more severely affected by this perturbation. RNA-seq was performed on Notch perturbed X. borealis embryos to quantitatively assess the response over time and compare changes in homeolog expression in X. laevis and X. borealis embryos following Notch perturbation. A de novo assembly of the X. borealis transcriptome was generated for further analysis. These data have revealed differences in the response to Notch perturbation between X. borealis and X. laevis.

ID #3921 Modeling Rare Diseases in Caernorhabditis elegans Ben Nebenfuehr NIDDK/NIH, USA Humans harbor approximately 7,000 rare diseases, an estimated 80% of which are monogenic. A rare disease is defined as affecting less that one in 1,500 people. Combined, these rare diseases affect nearly 1 in 10 Americans (25 to 30 million people), and treatments only exist for around 5% of these diseases. Thanks to the advent of whole genome sequencing, the gene(s) responsible for many rare diseases have become known, opening the door for more comprehensive studies.

Caenorhabditis elegans share >40% of their 20,000 genes with humans, have a short generation time, and are amenable to genetic modification, allowing us to study human disease-genes through their worm orthologs.

Mutations in the potassium ion channel KvLQT1 are implicit in the rare, autosomal dominant Long-QT Syndrome 1 (LQT1). The alpha subunit of this channel, KCNQ1, is evolutionarily conserved between humans and the nematode C. elegans (KQT-3, 87.9% amino acid identity). Regions necessary for voltage sensing and potassium trafficking have even higher similarity, suggesting a conserved cellular role between the two distinct organisms.

Timothy Syndrome (TS) is a rare autosomal dominant disease characterized by repolarization delays between heart pumps, defects in heart development, cutaneous syndactyly, autism spectrum disorders, and other developmental malformations. This syndrome is linked to a specific missense mutant in the calcium channel CACNA1C. The worm ortholog of CACNA1C is egl-19 (84.2% identity) and existing mutant alleles display various phenotypes ranging from hyper-contracted and constitutively egg-laying to flaccid and egg-laying defective.

We have generated several patient alleles in kqt-3 and egl-19 and will present our findings.

ID #3922 Understanding germline sex determination in Drosophila: The cis- and trans- regulation of Sex lethal Raghav Goyal, Ellen Baxter, Mark Van Doren Johns Hopkins University, USA In Drosophila, Sex lethal (Sxl) is necessary and sufficient for female identity in both somatic cells and the germline. However, while Sxl has been well studied in the soma, neither its activation nor its targets in the germline are understood. In both the germline and soma, the presence of two X chromosomes leads to Sxl activation. Interestingly, we find that the nature of this regulation at both the cis and trans levels is different in the germline from the soma. Our promoter analysis reveals that different cis sequences are required for regulation of Sxl’s female-specific promoter (SxlPE) in the germline. Further, while SxlPE is activated during embryonic stages in somatic cells, we only observe activation in the female germline during the late first instar larval stage. We are also identifying trans-acting factors that regulate Sxl in the germline. Previous work has shown that the combination of X chromosome “counting” transcription factors that activates Sxl in the soma does not do so in the germline. However, we found that one of these factors, sisterless A (sisA), is important for Sxl activation in the germline. Germline knockdown of sisA results in an ovarian tumor phenotype and germ cell loss, similar to knockdown germline Sxl. Strikingly, Sxl expression is lowered in these ovaries and germline loss is rescued by expression of a Sxl cDNA. Using RNA-FISH, we have also found that sisA is expressed in the germline around the time of Sxl activation. Put together, our data suggests that sisA lies upstream of Sxl in the germline primary sex- determination pathway. Through this work we aim to understand how intrinsic sex determination is regulated in the germline, and how sexual identity of the germline interacts with sex-specific somatic development to control proper gametogenesis.

ID #3924 The limb development program is restricted to the mesenchymal compartment in salamanders Sruthi Purushothaman, Ashley W Seifert University of Kentucky, USA Limb organogenesis is an ideal model to investigate how cellular and molecular networks have developed during tetrapod evolution. Comprehensive studies in chicken and mouse embryos have led to an integrative limb development model for vertebrates. Urodeles, however, appear to deviate from key parts of this model. For instance, they undergo pre-axial skeletal specification and do not develop an AER. Expression analysis of key limb development genes has not been extensively studied and where it has been, results from the literature are conflicting. The present study explores the axolotl limb with regards to spatiotemporal expression of key developmental genes that set up the anterior-posterior and dorsal-ventral axes. Whole mount in-situ hybridizations (WISH) were done on developing axolotl limb buds at 6 morphologically defined stages. Sectioned limbs were used to define cellular expression in ectodermal and mesenchymal compartments along with regionalization along the anterior-posterior and dorsal-ventral axes. We also used real time PCR to validate the observed spatial expression patterns in the limbs. Almost all genes examined were restricted to the mesenchyme in developing limbs, including Fgf ligands normally expressed in the mouse and chicken AER. Shh expression, which is normally restricted to the zone of polarizing activity in the posterior margin of tetrapod limbs, exhibited bipolar expression in an anterior and posterior zone. We also tested the role of mesenchymally restricted Fgf8 during limb outgrowth using the Fgf-receptor inhibitor SU5402 and found the resultant limb phenotype closely resembled Msx2cre;Fgf8 knockout mice. While our findings support conservation of a common genetic limb program in tetrapods, they also demonstrate that this molecular machinery has shifted in Urodeles such that it resides predominantly in the limb mesenchyme.

ID #3925 Modeling NGLY1 Deficiency in C. elegans Carina Graham National Institutes of Health, USA NGLY1 Deficiency is an autosomal recessive disorder characterized by global developmental delays, neurological abnormalities, alacrima, hypotonia, auditory neuropathies, liver fibrosis, and hyperkinetic movement disorders. Since its discovery in 2012, fewer than 60 patients have been diagnosed worldwide. The nematode C. elegans represents an opportunity to model such ultra-rare diseases due to its easy husbandry and high orthology with the human genome.To model NGLY1 Deficiency, the orthologous nematode gene png-1 was altered via the CRISPR-Cas9 DNA editing system. Specific single nucleotide mutations, carried by real human patients, were replicated in png-1. Phenotypes were characterized via physiological, chemical, and genetic screens. It was found that png-1 mutants display a hypersensitivity to proteasomal stress. When proteasome activity is inhibited, either by clinical drug treatment or by RNAi of proteasomal subunits, png-1 mutant worms display larval arrest or lethality not seen in wild-type worms. Subsequently, mutant worms were subjected to random chemical mutagenesis, and proteasomal inhibition assays were repeated on survivors. Lines of worms were established from individuals who showed suppression of the larval arrest phenotype. These worms theoretically carry mutations in genes that represent potential therapeutic targets for human patients.

ID #3926 Determining the developmental roles of the folic acid metabolism gene mel- 32/Shmt in Caenorhabditis elegans Jessica Sullivan-Brown West Chester University of Pennsylvania, United States Neural tube defects are common and serious birth defects in which the brain and/or spinal cord are exposed outside the body. Supplementation of foods with folic acid, an essential vitamin, is associated with decreased risk of neural tube defects; however, the mechanisms by which folic acid influence neural tube defect risk is unclear. Our research seeks to identify the basic cellular roles of known folic acid metabolism genes during morphogenesis using the roundworm Caenorhabditis elegans (C. elegans) as a simplemodel system. Using RNA interference, we showed that mel-32, a homolog of the mammalian folic acid metabolism gene Serine hydroxymethyltransferase (Shmt), is required for embryonic development, supporting previous research characterizing the null mutation. We used live imaging to analyze defects in embryonic development when the mel-32/Shmt gene is depleted. Our data provides evidence that defects in mel- 32/Shmt result in a doubling or tripling of cell cycle lengths during early cell divisions. However, the order of cell divisions, as determined by lineage analysis, is unchanged compared to wild type embryos. Because glycine and thymidylate (dTMP) levels are predicted to decrease in mel-32/Shmt depleted embryos, we performed supplementation experiments to determine if embryonic lethality could be reduced. Our results suggest that supplementation of thymidine but not glycine decreased embryonic lethality in mel-32/Shmt knockdown worms. We propose that mel-32/Shmt has essential roles in cell cycle progression in C. elegans, potentially through affecting cellular levels of dTMP. ID #3928 Investigating the role of tailing in microRNA degradation in C. elegans Katie Prothro, Katherine McJunkin National Institute of Health, USA MicroRNAs are short noncoding RNAs that regulate gene expression by blocking translation and/or destabilizing target mRNAs. Although extensive research has been done on the biogenesis pathway of microRNAs, the mechanisms of microRNA turnover and decay have yet to be characterized. Previous studies have shown a correlation between microRNA decay and an increase in the addition of non-templated nucleotides to the 3’ ends of microRNAs, a process known as tailing. In order to further investigate this correlation, we will induce miRNA decay in C. elegans by inactivating the microRNA biogenesis pathway. MicroRNAs are transcribed as long precursors (pri-miRNAs) that then undergo multiple cleavage events, one of which is carried out by the enzyme Pasha. By using a Pasha-temperature-sensitive strain, we are able to conditionally inactivate Pasha by upshifting temperature. At the higher, restrictive temperature, new, mature microRNAs are not synthesized. Using small RNA deep sequencing, we will be able to investigate the proportion of tailed microRNAs before inactivation of biogenesis and at multiple time points following inactivation, during which miRNAs will be decayed by endogenous mechanisms. To determine whether tailing is causal to microRNA decay, we will attempt to disrupt tailing by knocking down candidate tailing enzymes. If RNAi against a tailing enzyme alters the rate of decay, it would suggest that tailing is a mechanism capable or either promoting or preventing the turnover of miRNA. Altogether, we hope to gain better insight into the turnover of miRNA and its possible relationship to the post-transcriptional modification of tailing.

ID #3931 microRNA-31 regulation of Eve expression in the early sea urchin embryo Chelsea Lee University of Delaware, United States Proper regulation of microRNAs plays vital roles in the embryogenesis of animals. MicroRNAs are small non-coding RNAs that suppress translation and reduce stability of target mRNAs in animal cells. microRNA-31 (miR-31) has been found to play a role in cancer, bone formation, and lymphatic development. Using the sea urchin as a model organism, we investigate how miR-31 regulates skeletogenic cells in developing sea urchin embryos. Previously, we found that knockdown (KD) of miR-31 resulted in defects in the patterning and function of the skeletogenic cells and skeletal formation. Also, perturbation of miR-31 resulted in aberrant Vegf3 signaling which is critical for the patterning of skeletogenic cells and skeletal formation. This study tests the hypothesis that miR-31 indirectly suppresses Vegf3 through its upstream regulators. We found that miR-31 directly suppresses Eve. Importantly, removing miR-31 suppression of Eve in vivo resulted in similar ectopic Vegf3 expression as in miR-31 KD embryos, indicating that miR-31 regulates Vegf3 through suppression of Eve. Further, removing miR-31 regulation of Eve is sufficient to cause skeletogenic defects. We have identified the molecular mechanism of how miR-31 regulates Vegf signaling that impacts skeletogenesis. In addition, we created additional RNA in situ probes to further test the involvement of Wnt signaling in this regulatory mechanism. Overall, our results indicate that miR-31 integrates gene regulatory network and signaling pathways to ensure proper development.

ID #3932 Analyzing the Structural and Functional Changes of TOP-2 variants in C. elegans Tommy Wilmoth, Aimee Jaramillo-Lambert University of Delaware, United States During meiosis, cells undergo one round of DNA replication followed by two rounds of cell division to form haploid gametes. In meiosis I, homologous chromosomes segregate while in meiosis two, the sister chromatids segregate before cytokinesis to form haploid cells. Topoisomerase 2 is an enzyme that helps to facilitate DNA disentanglements. This enzyme has been studied extensively in mitosis but not in meiosis. The proper function of top-2 in C. elegans has been shown to be necessary for proper chromosomal segregation in meiosis I. Previously, we demonstrated that a temperature sensitive mutant, top-2(it7), causes chromosomes to fail to segregate in spermatogenesis when incubated at 24°C. The top-2(it7) mutation changes an Arginine at amino acid 828 to Cysteine. This mutation lies within an a/b fold of TOP-2 called the tower domain that is located within the catalytic domain, a zone that has been proposed to interact with DNA. We hypothesize that the Arginine at position 828 is particularly important for the structure and function of the tower domain. Recently, we have characterized an Arg828àAla change, which has shown high embryonic viability but slight segregation defects. An Arg828à Trp change results in sterility due to germline absence. We are also investigating other domains of TOP-2. The c-terminal domain, which is not highly conserved among Topoisomerase 2 homologs, is thought to be important for regulation of TOP-2 through protein-protein interactions and post- translational modifications. The c-terminus of C. elegans TOP-2 contains 12 phosphorylation (Zielinska et al., 2009) and 2 possible SUMOylation sites. We generated a deletion of the c-terminus of C. elegans, removing 11 of the 12 phosphorylation sites in the c-terminus and 2 possible SUMOylation sites. We discovered that removing the c-terminus results in sterility, due to the absence of a germline. Currently, we are continuing to analyze structural and functional changes in TOP-2.

ID #3933 Eyeless uncouples neuroblast proliferation from dietary nutrients in Drosophila Conor Sipe, Sarah Siegrist University of Virginia, United States Neurons in the Drosophila brain are generated from asymmetric cell divisions of neural stem cells, known as neuroblasts (NBs). Most NBs enter quiescence at the end of embryogenesis coincident with declining maternal nutrient stores; upon larval feeding, these nutrient-sensitive NBs reenter the cell cycle and begin another round of proliferation that continues until early pupal stages. In contrast, a small subset of central brain NBs, the mushroom body (MB) NBs, never enter quiescence and divide continuously throughout development regardless of nutrient intake. Both subtypes reside in close proximity to one another and share a common macroenvironment, suggesting that quiescence versus proliferation decisions are regulated in a cell-intrinsic manner. We have demonstrated that Eyeless (Ey), a transcription factor predominantly expressed in MB NBs, is required for nutrient-independent NB proliferation. When Ey is knocked down, MB NBs exit the cell cycle in response to dietary nutrient withdrawal; conversely, when Ey is ectopically expressed in all NBs, some non-MB NBs continue to divide independent of dietary nutrient conditions. Therefore, Ey is a cell-intrinsic factor both necessary and sufficient to uncouple NB proliferation from dietary nutrient intake. We are currently investigating if conventional cell growth pathways are required for Ey to exert this function. We find that the transcription factor Myc is also required for nutrient-independent proliferation of MB NBs, whereas PI3-kinase signaling is not. Myc expression in MB NBs with reduced Ey can rescue their ability to proliferate during dietary nutrient withdrawal. Since Myc protein and transcript levels are not altered in Ey RNAi MB NBs, we propose that Ey and Myc act in parallel pathways to control MB NB nutrient-independent proliferation. Our work highlights an important role for lineage- specific factors in regulating proliferation decisions in response to nutrient availability in a cell-autonomous manner.

ID #3934 Investigation of Embryonic Plasticity Through Anterior-Posterior Perturbations and Gene Cloning Chloe Naman Whitney, Nana Ya Amoh, Phil Breno, Sonali Dabhi, Amy Forehand, Victor Li, Regan Sindelar, Lulu Zhang, Margaret Saha Department of Biology, College of William and Mary, United States While all cells are able to compensate for genetic and environmental perturbations they experience, embryonic cells display a particularly profound level of plasticity. However despite its importance, little is known about the molecular genetic basis of this plasticity, in spite of its wide application to regenerative medicine and other basic biological processes. In order to investigate this plasticity we performed a series of experiments in which embryonic tissue along the anterior-posterior axis of Xenopus laevis gastrulae were perturbed in three ways. In rotation embryos the presumptive neural axis was removed and rotated 180 degrees, reversing the anterior-posterior axis. In transplant embryo perturbation the tissue was removed and replaced with the corresponding tissue of donor embryos in the original configuration of the tissue. In autologous perturbation, embryo tissue was removed and replaced in its original position to determine which genetic changes were due only to perturbation as opposed to those additionally affected by cellular relocation. RNA sequencing was used to determine which genes were all differentially expressed (upregulated) in the perturbed embryos. These included genes such as osgin-1, an oxidative stress-induced growth inhibitor for which perturbation results in cell apoptosis; dtx4, a ubiquitin ligase gene regulating the Notch sequence; h1fx and HDAC7, both histone family members; psme3, a proteasome activator subunit; complement factor B, a trypsin gene involved in macular degeneration; complement component 9, a growth cone guidance gene; and brambleberry-like, likely involved in karyomere fusion. While additional genes were also differentially expressed, our current project involves cloning and characterizing these genes in order to examine their expression patterns with the goal of better understanding at a genetic level how X. laevis embryos are able to adapt to physical perturbations. ID #3935 Importance of injury induced senescence during regeneration in Acomys compared to fibrotic repair in Mus Sandeep Saxena, Ashley W. Seifert University of Kentucky, USA During epimorphic regeneration, blastemal cells re-enter the cell cycle and proliferate to form new tissue. Cell cycle re-entry or arrest is at least partly regulated by cellular senescence which negatively impacts the proliferative pool of cells during regeneration. What remains unclear is whether cells in regenerating systems possess an increased propensity to proliferate and are refractory to signals that induce senescence. To test this idea we used spiny mice (Acomys), a mammalian taxa that exhibit enhanced regenerative ability and are able to replace skin and musculoskeletal tissue. Using an ear hole injury, Acomys show resident cell activation, cell cycle progression and proliferation in contrast to Mus musculus (Mus), who heal identical injuries via fibrotic repair and exhibit limited cell cycle progression. Culturing primary ear pinna fibroblasts in vitro we found that Acomys cells proliferated longer in culture with significantly more cell population doublings (PDs) compared to Mus cells before these cells senesced. Where Mus cells experienced crisis in ~40 days, Acomys cells proliferated for 90 days in culture. This difference was further magnified under physiologically relevant oxygen (3%) where Acomys cells proliferated for ~140 days. We also analyzed replicative capacity in another regenerating (Oryctolagus cuniculus-rabbit) and non-regenerating (Rattus rattus) mammal. Surprisingly, fibroblasts from both species showed no signs of senescence after 150 days. To ascertain if Acomys cells are more resistant to oxidative or inflammatory-mediated stress we exposed these cells to H2O2 and LPS at sub-lethal doses that induce senescence in Mus. Remarkably, Acomys fibroblast exhibited normal proliferation and resisted senescence as determined by SA-ßgal staining. Together, our data suggests that resident cells in regenerating species possess higher intrinsic proliferative capacity and a higher threshold to resist stress-induced senescence.

Funding: NIH

ID #3936 MicroRNA Regulation of Mesodermal Cell Fate in Early Sea Urchin Development Kalin Konrad University of Delaware, USA microRNA-124 (miR-124) is a highly conserved, brain enriched microRNA (miRNA). The objective of this project is to investigate how miR-124 regulates mesodermal cell specification. The purple sea urchin, Strongylocentrotus purpuratus, will be used to examine the function of miR-124 in regulating the Delta/Notch signaling pathway in early development. Sea urchins express all major gene families as in humans and have abundant sheds that result in transparent embryos. The sea urchin Delta/Notch signaling pathway specifies mesodermally-derived blastoceolar and pigment cells that perform immune functions. We bioinformatically identified two or more potential miR- 124 binding sites in the 3'UTRs of Notch, Ese (blastocoelar cell transcription factor), and Gcm (pigment cell transcription factor). We hypothesize that miR-124 regulates mesodermal cell fates by suppressing Delta/Notch signaling pathway and its downstream targets. To test the direct regulation of miR-124 on these genes, we cloned their 3'UTRs downstream of Renilla luciferase reporter constructs. For Notch, one functional miR-124 regulatory site was identified. To test the impact of miR-124 suppression of Notch, we block miR-124 suppression of Notch with Target Protector Morpholino (TP MASO) that is designed to be complementary to the validated Notch miR-124 seed and flanking sequences. We are in the process of assessing the effect of miR-124 suppression of Notch on mesodermal cell fate changes using blastocoelar and pigment cell markers in whole mount in situ hybridization experiments. We discovered that miR-124 inhibition caused ectopic misexpression of Ese. We will test the molecular mechanism of how miR-124 alters Ese expression. Results from this study will identify the regulatory mechanism of miR-124 in mesodermal cell fate determination.

ID #3937 Contact-mediated long-range dispersion shapes a self-generating FGF morphogen gradient during branching morphogenesis of Drosophila trachea Lijuan Du, Alex Sohr, Sougata Roy University of Mayland, USA Concentration gradients of morphogens specify positional information to pattern tissue morphologies. How the positional information is established with reproducibility and precision is one of the central questions in tissue patterning. The traditional view of cell- cell communication considers that the secreted signaling proteins diffuse in the extracellular space to generate concentration gradients. Here, using endogenously expressed FGF:GFP and FGFR:Cherry from genomic knock-in alleles, we uncovered a cytoneme-mediated self-generating mechanism for a long-range receptor-bound FGF gradient in the Drosophila larval air-sac-primordium (ASP), a wing-disc-associated tracheal branch. A restricted group of wing disc cells expresses FGF to induce growth and migration of ASP during 3rd instar larval development. We showed that the ASP cells extend FGFR:Cherry-containing cytonemes to contact the wing-disc fgf-source to directly receive FGF:GFP. FGF:GFP moves together with FGFR:Cherry along the surface of cytonemes that extend from the ASP to the disc and forms a long-range receptor-bound gradient that adopts recipient ASP-specific contours. The number of cytonemes the ASP cells extend is proportional to the FGF:GFP amount they receive, which gradually reduces with increasing distance from the fgf-source, producing a receptor-bound ASP-specific gradient contour. FGF acts as a morphogen, inducing concentration-dependent activation of four genes in different zones of the recipient ASP epithelium. Pointed-P1, a high-threshold target, and Cut, a low-threshold target, antagonize each other and differentially feedback-regulate the number of FGFR- containing cytonemes, thereby creating regions with higher-to-lower numbers of FGF- receiving cytonemes. Therefore, a self-sustaining spatial asymmetry of cytoneme- mediated dispersion through signaling-feedback sculpts gradient contours, adopting precise tissue-specific morphologies.

ID #3939 Novel BMP-Smad1/5 interactions in a zebrafish model of FOP Robyn Allen, Eileen Shore, Mary Mullins University of Pennsylvania, USA The developing zebrafish is patterned along its dorsoventral (DV) axis by a gradient of BMP-Phospho-Smad1/5 (pSmad1/5) signaling in a process that is conserved from insects to humans. Essential to this signaling process is the type I BMP receptor, Acvr1, which forms a tetramer with other BMP receptors to phosphorylate Smad1/5 in response to binding BMP ligand. In the disease Fibrodysplasia ossificans progressiva (FOP) a single nucleotide substitution in human Acvr1 (617G>A, R206H), leads to over- activation of the BMP-pSmad 1/5 signaling pathway, perturbing skeletal development and causing heterotopic ossification in adulthood. To investigate the still uncertain mechanism by which FOP-Acvr1 enhances BMP signaling activity, we used zebrafish embryonic DV patterning. Perturbations to BMP-pSmad1/5 signaling directly correlate to a series of distinct dose-dependent patterning phenotypes in the zebrafish embryo. Misexpression of hAcvr1-R206H, as well as several variant FOP-Acvr1 mutations, causes increased pSmad1/5 signaling and ventralization of zebrafish embryos. Recent studies suggest that FOP-Acvr1 may have altered ligand responsiveness compared to WT Acvr1. We confirmed that BMP ligand enhances pSmad1/5 signaling through hAcvr1-R206H. Surprisingly, Activin A, a ligand that normally binds Acvr1b and signals through pSmad2/3, was also confirmed to enhance pSmad1/5 signaling by hAcvr1- R206H. Further, we found that BmpR1, a type I receptor normally required for pSmad1/5 signaling and DV patterning in the zebrafish, is not required for pSmad1/5 over-activation by hAcvr1-R206H or G328R. These data suggest that the mechanism by which FOP-Acvr1 signals is not bound by the same receptor and ligand partner requirements as WT-Acvr1. Further studies of the mechanism by which FOP-Acvr1 over-activates BMP signaling will provide unique insight into how this fundamental cell signaling pathway functions in development.

ID #3940 Identifying Molecular Mediators of OPC Spacing Maria Ali University of Virginia, United States Oligodendrocytes myelinate central nervous system (CNS) axons in order to facilitate rapid signal transduction and provide protection from the surrounding environment. They are derived from oligodendrocyte progenitor cells (OPCs), which migrate throughout the developing CNS and become evenly distributed. While most OPCs mature into oligodendrocytes, a population of OPCs remains evenly distributed in the CNS throughout adulthood. How OPCs disperse through the CNS to myelinate axons is unknown, despite this being a crucial step in nervous system development. In both developing and mature nervous systems, OPCs are highly motile cells with dynamic membrane processes that change direction and retract their processes upon contact with neighboring OPCs. This phenomenon, known as contact-mediated repulsion (CMR), influences the local migratory behavior of OPCs and could facilitate the rapid dispersal of OPCs in the CNS. To identify novel mediators of OPC spacing and CMR, I conducted an unbiased drug screen using 430 kinase inhibitors and screened for altered OPC numbers and spacing in the dorsal spinal cord of zebrafish larvae. In conducting this screen, I identified numerous compounds that either reduce OPCs numbers or alter OPC spacing. Using in vivo imaging in combination with identified compounds will reveal novel mechanisms of OPC developmental spacing that could provide insight into OPC dysfunction in disease.

ID #3941 The Control of Germline Stem Cell Sexual Identity in Drosophila melanogaster Pradeep Bhaskar1, Sheryl Southard2, Kelly Baxter1, Mark Van Doren1 1Johns Hopkins University, USA; 2Chang Gung University, Taiwan Sexual dimorphism is common throughout the animal kingdom, with males and females exhibiting phenotypic characters specific for their sex. While a great deal is known about the establishment of sexual identity in somatic cells, this process is much less well understood in the germline. Germline sexual identity is critical for sex-specific development of germline stem cells and production of sperm vs. eggs. Thus, it is an essential aspect of animal sexual reproduction and human fertility. Germ cells depend on both signals from the somatic gonad as well as their own sex chromosome genotype to determine their sex. Therefore, when the “sex” the germline fails to match the “sex” of the soma, germline development is severely disrupted. How somatic signals and germ cell intrinsic cues act together to regulate germline sex determination is a key question about which little is known in any organism. We have previously identified the JAK/STAT pathway as a key male determining signal from the soma to the germline in the embryo. Further, the RNA-binding protein Sex-Lethal (SXL) and the chromatin factor PHF7 have been identified as key components promoting female vs. male germline identity, respectively. We find that the JAK/STAT pathway continues to regulate male identity in adult germline stem cells. It does this, in part, by acting as a direct transcriptional regulator of the male identity factor Phf7. Our data also indicate that PHF7 is also regulated at the post-transcriptional level to ensure its male-specific expression. Further, female germline stem cells must inhibit JAK/STAT signaling in order to preserve their female identity. This is mediated through SXL which is expressed in female germline stem cells and blocks JAK/STAT pathway activity. Together, this work provides a framework for how the germline’s own sex chromosome constitution combines with important signals from the soma to regulate germline stem cell sexual identity.

ID #3942 Multipotent Neural Crest Cells Phagocytose Apoptotic Debris in the CNS and PNS Yunlu Zhu, Samantha Crowley, Sarah Kucenas University of Virginia, USA During neural development, 50% of proliferating cells undergo programmed cell death to eliminate superfluous neuronal cells in both the central nervous system (CNS) and the peripheral nervous system (PNS). Therefore, a rapid, efficient phagocytic response is crucial for the clearance of the vast amount of cellular debris in order to prevent the stimulation of inflammation. However, in the trunk of developing embryos, neurogenesis happens prior to the infiltration of early yolk-sac-derived professional phagocytes. How are apoptotic cells removed from the trunk spinal cord region in early neurogenesis remains largely unknown. Neural crest cells (NCCs) are a multipotent cell lineage that give rise to a wide variety of cells from PNS neurons and glia to skeletal tissue. Here, using live imaging in zebrafish, we demonstrate that migratory NCCs respond rapidly to dying cells and phagocytose cellular debris around the neural tube. During their migration, peripheral NCCs even have the ability to enter the CNS through motor exit points and clear debris in the ventral spinal cord. Live imaging of novel transgenic lines revealed that dynamic lytic vesicles formed after neural crest engulfment are phosphatidylinositol 3-monophosphate-positive and lamp1-positive phagolysosomes. In addition, blocking the detection of ATP by a purinergic receptor inhibitor significantly decreased the recruitment of NCCs towards cellular debris. Our findings indicate that neural crest phagocytosis is mechanistically similar to that of professional phagocytes. Furthermore, we show that phagocytosis-related pathways are down-regulated in NCCs at later developmental stages, which leads us to hypothesize that macrophages cooperate with NCCs and become the major scavenger in the PNS after their emergence. Taken together, our study reveal a novel role of NCCs in phagocytosis of debris during embryonic neurogenesis.

ID #3943 Analyzing ALG-2 mediated mir-35 family regulation and the mir-35 family role in sex determination in C. elegans Lars Benner, Katherine McJunkin NIDDK, USA The mir-35 family of microRNAs containing mir-35-42 is essential for C. elegans embryogenesis. The mir-35 family is strictly regulated throughout development as it is expressed during early embryogenesis and is abruptly decayed at the onset of the first larval stage. Here we present two projects examining different aspects of mir-35 regulation and biology.

First, we want to understand how mir-35 loading impacts its regulation. Since miRNAs are less likely to decay when loaded onto an Argonaute protein, by examining the expression levels and turnover mechanism of the Argonaute protein in which the mir-35 family is preferentially loaded, ALG-2, we can better understand mir-35 regulation. To test this, we utilized CRISPR-Cas9 to fuse a photoswitchable fluorescent protein to ALG-2 expressed from its genomic locus. Subsequent photoconversion allows pulse- labeling and measurement of ALG-2 turnover throughout development. By tagging ALG- 2 at its known locus, we keep endogenous ALG-2 levels while clarifying the mechanism of Argonaute degradation via knockdown of specific catabolic pathways.

The second project examines the mir-35 family’s interaction with the germline sex determination pathway. The mir-35 family is involved in preventing premature male gene expression during embryogenesis. A surprising aspect of mir-35 sex determination biology is that feminizing the germline in a mir-35-41 mutant background causes skewed sex ratios favoring female progeny. We are examining the molecular basis of this male preferential death, investigating the role that male development and the sex chromosome karyotype have on lethality. By feminizing the germline and soma of mir- 35-41 mutant worms, we can understand the extent that these two factors play in male death and thus gain further insight into the mir-35-41 role in the sex determination pathway. Overall, our project seeks to gain understanding into the regulation and biology of this crucial embryonic miRNA family. ID #3944 Glutamine metabolism regulates proliferation and lineage specification in mesenchymal stem cells. Yilin Yu, Leyao Shen, Deepika Sharma, Courtney Karner Duke University School of Medicine, USA Mesenchymal stem cells (MSC) are critical for bone homeostasis by providing osteoblasts throughout life. With age and in certain conditions, MSC are incorrectly specified or depleted, resulting in reduced osteoblast formation, decreased bone mass, and diminished bone regeneration in response to injury. Little is known about the intrinsic mechanisms regulating MSC proliferation or specification. Cellular metabolism is emerging as a critical regulatory node during differentiation. MSC increase glutamine consumption during osteoblast differentiation but the physiological requirements of glutamine metabolism in MSC are unknown. Glutaminase (GLS) catalyzes the rate- limiting first step in glutamine metabolism, the deamination of it to form glutamate. Here, we demonstrate GLS is required for the proliferation and specification of MSCs. Genetic deletion of a conditional Gls allele (Glsfl/fl) in MSCs using either Prx1Cre or LeprCre significantly reduces bone mass and increases marrow adiposity in vivo and limits bone regeneration in a nonstabilized fracture model. Decreased bone mass and regeneration is due to reduced osteoblast numbers and diminished bone formation. Colony forming unit assays demonstrate that osteoblast depletion is due to a significant reduction of MSC proliferation as well as altered MSC specification favoring the adipocyte lineage. Conversely, Gls deletion in specified osteoblasts using Sp7Cre, resulted in reduced bone mass due to diminished bone formation with no effect on MSCs or osteoblast numbers. Our data illustrate a biphasic role for GLS and glutamine metabolism in MSC. First, GLS is necessary for MSC proliferation and osteoblast specification. Then, GLS stimulates bone forming activity in specified osteoblasts. Collectively, these data indicate stimulating GLS activity may provide a valuable therapeutic approach to expand MSCs in aged individuals and enhance osteoblast specification and activity to regenerate bone in human bone diseases.

ID #3946 Investigating combinatorial transcriptional control of ecdysone response gene E74 during cyst development in the Drosophila ovary. Lindsay Davenport, Elizabeth Ables East Carolina University, United States Oogenesis is an essential process by which an egg develops from undifferentiated cells in the ovary. This process has been widely studied; however, many of the molecular mechanisms that regulate oocyte development and growth remain unclear. During oogenesis, germ cells are surrounded by somatic cells to form follicles, which develop into a mature oocyte. Steroid hormones largely drive this process; in Drosophila the predominant steroid hormone is ecdysone. Ecdysone signaling is necessary for seemingly divergent processes and responses are cell-type specific. As ecdysone receptors are ubiquitous in the ovary, it is unclear how ecdysone promotes specific cellular responses to promote follicle growth and survival. We hypothesize that Ecdysone Receptor works with other transcription factors to regulate target genes in a cell and stage-specific manner. To test this hypothesis, we focus on the regulation of an ecdysone target gene, Ecdysone-induced protein at 74EF (E74), induced in germline cysts during follicle formation. We used enhancer mapping to investigate what regions of the E74 gene locus are sufficient to drive expression in the ovary. We identified two intronic regions that are sufficient for reporter expression; both are bound by the chromatin binding factor Trithorax-Like (GAGA factor or Trl). We postulate that Trl binds E74 enhancer regions to specifically promote cyst development in response to ecdysone signaling. To investigate this, we will determine whether Trl promotes E74 expression in early follicles. We are also currently investigating whether Trl promotes follicle development and whether the Trl binding sites in the E74 locus are critical for E74 function. Understanding the mechanisms that underlie the processes of oogenesis can give us valuable insights into female reproduction.

ID #3947 Convergent Thickening; a newly characterized morphogenic machine that generates force for blastopore closure in Xenopus laevis David Shook1, Jason Wen2, Doug DeSimone1, Rudi Winklbauer2, Ray Keller1 1University of Virginia, USA; 2University of Toronto, Canada Gastrulation in amphibians involves the internalization of the lower portion of the early embryo, which includes the prospective endoderm and mesoderm through the blastopore, thereby shaping the basic body plan. These movements are driven by forces generated by the embryonic cells and integrated at the tissue level as “morphogenic machines” that give rise to embryonic shape change and re- organization. Here we describe our recent characterization of one such machine, Convergent Thickening (CT), including its pattern and timing, morphogenic movements, cellular mechanism, and biomechanics. CT is expressed by cells in the involuting marginal zone (IMZ) laying around the blastopore, from the earliest stages of gastrulation, until cells roll around the blastopore lip and are internalized (“involute”), at which point they transition to different cell behaviors. Explants of the IMZ in culture converge and thicken to a greater extent and more rapidly than do explants of non- involuting tissue, and generate tensile force along their long, mediolateral axis. In the intact embryo, CT generates tensile force around the blastopore, driving its closure. Our evidence suggests that CT is driven by a change in the interfacial tension between the presumptive mesodermal cells lying in the IMZ and the overlying superficial epithelial layer. This is supported by observations of tissue behavior, measurements of tissue surface tension, and measurements of the force of thickening. CT is clearly distinct from the other morphogenic machine that operates within the cells beginning in the IMZ, Convergent Extension (CE), which also generates tension around the blastopore, but after involution. CT continues to operate in embryos that do not express CE, and depends on different patterning and effector molecules. CT appears to be a highly conserved machine across the anurans and different species vary in their dependence on it to close their blastopore.

ID #3948 N-terminal and central domains of APC function to regulate branch number, length and angle in developing optic axonal arbors in vivo Taegun Jin1, Gregory Peng1, Esther Wu1, Shrey Mendiratta2, Tamira Elul1 1Touro Univ-California, USA; 2University of California Berkeley, USA During formation of neuronal circuits, axons navigate long distances to reach their target locations in the brain. When axons arrive at their target tissues, in many cases, they extend collateral branches and/or terminal arbors that serve to increase the number of synaptic connections they make with target neurons. Here, we investigated how Adenomatous Polyposis Coli (APC) regulates terminal arborization of optic axons in living Xenopus laevis tadpoles. The N-terminal and central domains of APC that regulate the microtubule cytoskeleton and stability of b-catenin in the Wnt pathway, were co-expressed with GFP in individual optic axons, after which their terminal arbors were imaged in tectal midbrains of intact tadpoles. Our data show that the APCNTERM and APCb-cat domains both decreased the mean number, and increased the mean length, of branches in optic axonal arborsrelative to control arbors in vivo. Additional analysis demonstrated that expression of the APCNTERM domain increased the average bifurcation angle of branching in optic axonal arbors. However, the APCb-cat domain did not significantly affect the mean branch angle of arbors in tecta of living tadpoles. These data suggest that APC N-terminal and central domains both modulate number and mean length of branches optic axonal arbors in a compensatory manner, and also define a specific function for the N-terminal domain of APC in regulating branch angle in optic axonal arbors in vivo. Our findings establish novel mechanisms for the multifunctional protein APC in shaping terminal arbors in the visual circuit of the developing vertebrate brain.

ID #3949 Uncovering a Spatiotemporal Profile of FGF Action in Early X. Laevis Development Timothy McMullen University of Virginia, USA Many studies show that FGF signaling serves a critical role in the induction of somitic mesoderm and notochord in early development and that inhibition or absence of wild- type FGFR in X. laevis inhibits the induction of somites and notochord. Others have demonstrated the existence of FGF8 gradients in chick and mouse embryos which may contribute to the so-called segmentation clock. However, the spatiotemporal aspects of FGF function in early Xenopus development remain poorly characterized. Our work uses varying concentrations of the FGFR tyrosine kinase inhibitor SU5402, applied at specific stages through early development, to compile a more complete spatiotemporal profile of the role of FGF in somite and notochord induction. This investigation employs immunofluorescence microscopy to assay morphological perturbations which result from FGFR inhibition in early X. laevis development. Our preliminary results show that SU5402 blocks mesodermal induction most significantly when administered between stages 8 and 9.5, indicating crucial FGF action during this period. Additionally, inhibition at earlier stages of the stage 8-16 interval correlates with increasingly truncated notochord and reduced somite patterning along anteroposterior axis with downstream effects on neurulation and blastopore closure. In aggregate, these findings provide a strong foundation for the further spatiotemporal characterization of FGF activity in Xenopus development. We will also discuss current progress in our study, including the development of optogenetic controls of FGFR function as well as the use of halo-tagged FGF to study the spatiotemporal dynamics of specific FGF ligands.

ID #3950 Mechanism of 5'-tyrosyl-DNA phosphodiesterase (tdpt-1) Mediated Suppression of DNA Topoisomerase 2 (top-2) during meiosis in C. elegans Nirajan Bhandari1, Harold E. Smith2, Andy Golden2, Aimee Jaramillo-Lambert1 1Department of Biological Sciences, University of Delaware, Newark DE 19716, USA; 2National Institute of Diabetes and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA Meiosis is a highly coordinated event involving a single cycle of DNA replication followed by two cycles of chromosome segregation. DNA Topoisomerase II (TOP-2) is required to relieve the topological stress associated with unwinding of DNA during replication, recombination and sister chromatid segregation. In addition, TOP-2 has been also found to maintain chromosome structure. The role of TOP-2 has been studied well in mitosis but is not very well defined in meiosis. Our lab has recently characterized a genetic allele of top-2 gene [top-2(it7)] in the worm Caenorhabditis elegans. Top-2- (it7) is a temperature sensitive (ts) allele with a role in male meiosis or spermatogenesis. Previously, we demonstrated that top-2-(it7ts) sperm that develop at restrictive temperature of 240C have chromosome segregation defects at anaphase I of meiosis, which results in embryonic lethality after fertilization. To identify the genetic pathway in which top-2 functions during meiosis, we performed a genetic suppressor screen on the top-2-(it7ts) worms. We identified 12 suppressors of top-2-(it7ts) embryonic lethality. Through whole genome sequencing we found that 6 of the suppressors were different point mutations in the gene 5'-tyrosyl-DNA phosphodiesterase (tdpt-1). tdpt-1 is an ortholog of human tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 is involved in the removal of trapped Top-2-DNA complexes. Currently, our lab is focused on elucidating the mechanism of tdpt-1 mutant- mediated suppression of top-2-(it7ts) embryonic lethality. We have found that the tdpt-1 suppressors rescue the embryonic lethality of top-2-(it7ts) to near wild-type levels and also ameliorate the chromosome segregation defects of top-2-(it7ts) worms.For future studies, we plan to validate the rescue of top-2 (it7ts), perform TDPT-1 localization study with respect to TOP-2 during meiosis, and investigate TOP-2 as a potential binding partner of TDPT-1.

ID #3951 The neutral amino acid transporter Slc38a2/SNAT2 is necessary for endochondral ossification in mice Leyao Shen1,2, Yilin Yu1, Courtney Karner1,2 1Department of Orthopaedic Surgery, Duke University, USA; 2Department of Cell Biology, Duke University, USA Skeletal development is dependent upon the coordinated differentiation and activities of both chondrocytes and osteoblasts. During endochondral ossification, mesenchymal progenitors first differentiate into chondrocytes that secrete the cartilaginous template of the developing bone. Bone-forming osteoblasts then differentiate and secrete bone matrix to replace the cartilage template. We have recently identified glutamine metabolism as a critical regulator of osteoblast differentiation and bone formation. However, little is known about how glutamine uptake is regulated during osteoblast differentiation. Here we have identified the sodium-coupled neutral amino acid transporter 2 (SNAT2, encoded by Slc38a2) as a critical regulator of chondrocyte and osteoblast differentiation during endochondral ossification. Expression analyses demonstrate Slc38a2 is highly expressed in both chondrocytes and osteoblasts during embryonic and post-natal skeletal development. Radiolabeled uptake assays highlighted a significant increase in both glutamine and methyl-amino-isobutyric acid (MeAIB, a substrate of SNAT2) consumption during differentiation. Targeting Slc38a2 using CRISPR/Cas9 in primary calvarial osteoblasts significantly diminished both glutamine and MeAIB uptake and prevented osteoblast differentiation in vitro. Mice homozygous for a null allele of Slc38a2 (Slc38a2-/-) were dead at birth. Slc38a2-/- mutant mice were characterized by delayed chondrocyte hypertrophy as early as e14.5 and persistent cartilage matrix in the marrow space at birth. Delayed osteoblast differentiation and reduced bone formation was also evident at e14.5. Collectively, our data indicate that both chondrocyte hypertrophy and osteoblast differentiation require Slc38a2/SNAT2 activity. Future studies will be necessary to elucidate the role of Slc38a2 in both chondrocytes and osteoblasts in vivo.

ID #3952 The Role of Cytokinetic Abscission in Neural Progenitor Cell Fate Decisions Katrina McNeely, Noelle Dwyer University of Virginia, United States Neural progenitor cells (NPCs) of the embryonic cerebral cortex undergo polarized cell division within the ventricular zone to produce the neurons that form the cortex. Cytokinesis, last step of cell division, regulates the separation of two daughter cells through cleavage furrowing and establishment of a microtubule structure (midbody). The midbody is severed to separate the two daughter cells. Recently, cytokinesis has been implicated in cell fate determination, but the specialized mechanisms in the developing brain remain unclear. We hypothesize that temporal and structural changes in cytokinesis of embryonic NPCs may lead to changes in cell fate. We previously reported a mouse mutant with microcephaly resulting from a mutation to Kinesin-6 family member Kif20b, a midbody protein. The Kif20b mutant embryos have smaller, thinner cortices and reduced neurogenesis. Also, mutant NPC midbodies are wider and misoriented, suggesting defects in late cytokinesis. We have shown the loss of Kif20b in HeLa cells causes delayed furrowing and dysregulated abscission. Here, to explore the role of Kif20b and cytokinesis dynamics in NPC cell division we used an in vitro dissociated cortical NPC culture and live imaging of cortical slab explants. In NPC cultures, we found mutant NPCs at E11 had increased neuronal daughters at the expense of progenitor daughters. However, at E15 a similar number of neuronal daughters are produced from control and mutant NPCs. Interestingly, at E11 daughter pairs were more likely to have an associated midbody remnant than at E15. We also developed a method for live imaging of abscission in cortical explants to observe the temporal dynamics of abscission. Currently, we are using these in vitro and in vivo systems to study the detailed cytoskeletal structure and temporal dynamics of normal and abnormal cytokinesis in cerebral cortex development. Our results support the idea that abscission dynamics influence daughter fates during NPC divisions.

ID #3953 failure to launch is essential for glial development Andrew Latimer, Sarah Kucenas University of Virginia, USA Establishing a functional nervous system requires spatial and temporal coordination of cell specification, proliferation and migration within and between its central and peripheral halves. The development and efficient functioning of both halves depends upon diverse types of glia. In the spinal cord, migratory and proliferative oligodendrocyte progenitor cells (OPCs) arise from the pMN domain and undergo several steps of maturation before becoming myelinating oligodendrocytes. In the peripheral nervous system, motor axons are myelinated by neural crest-derived Schwann cells. Motor nerves are in turn ensheathed by the perineurium formed by perineurial glia, which are born in the ventral spinal cord and migrate into the periphery via motor exit points. Perineurial glial association with motor nerves is required for proper nerve formation and Schwann cell myelination. failure to launch (ftl) was identified in a zebrafish mutagenesis screen for genes that regulate perineurial glial development and results in their absence along motor nerves in mutant embryos. ftl mutants appear to develop normally until around 48 hours post-fertilization (hpf), when we observe brain and spinal cord apoptosis, abnormal floorplate morphology and absence of migrating perineural glia and OPCs. Interestingly, spinal cord radial glia are selectively lost at 48hpf, leading us to hypothesize that OPCs normally require the presence of these cells and their fibers to migrate out of the pMN domain. We have sequenced the genomes of ftl mutants in order to determine the affected gene and are currently interrogating potential candidates.

ID #3954 Steroid hormone regulation of distinct RNA-binding proteins promotes GSC self- renewal Danielle S. Finger, Vivian Holt, Elizabeth T. Ables East Carolina University, United States Reproductive capacity in many organisms is maintained by the activity of germline stem cells (GSCs), which maintain an undifferentiated fate while creating daughters that will differentiate. A complex network influences stem cell fate, including local signaling and long-range endocrine signals. The molecular mechanisms by which hormone signaling is integrated to control cell fate is largely unknown. To elucidate these mechanisms, we study the role of ecdysone, a Drosophila melanogaster steroid hormone, in the control of GSC function. Female GSCs are directly regulated by the steroid hormone ecdysone, which is structurally and functionally similar to estrogen. Using a reverse genetic screen, we identified Heterogeneous nuclear ribonucleoprotein at 27C (Hrb27C) as a putative target of ecdysone signaling. Hrb27C is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins, which function to bind mRNAs and regulate their splicing, maturation, and localization. We tested the hypothesis that specific hnRNPs function downstream of ecdysone signaling to promote GSC self-renewal. We identified four hnRNPs, squid (sqd), hephaestus (heph), Hrb27C, and Hrb87F whose expression was decreased in ecdysone mutants, suggesting that they are targets of ecdysone in the ovary. Loss-of-function analyses confirmed that Hrb27C, sqd, and heph are required in GSCs for self-renewal. Our data suggest that GSC loss in the absence of Hrb27C, sqd, and heph is due, at least in part, to deregulation of BMP signaling, which represses differentiation in GSCs. Our data supports the model that specific hnRNPs help maintain stem cell fate in response to ecdysone signaling by stabilizing the expression of BMP signaling transcripts. Given the similarity between Drosophila and human steroid hormone signaling our study will help clarify how stem cell activity is tethered to physiological changes and provide insight into the molecules critical for this response.

ID #3955 Interrogating the regulation and function of the mir-35 family of microRNAs Bridget Donnelly1,2, Kenneth Murfitt3,4, Eric Miska3, Katherine McJunkin1 1NIH, USA; 2Johns Hopkins University, USA; 3University of Cambridge, UK; 4Novartis, UK The mir-35 family of microRNAs is developmentally regulated in Caenorhabditis elegans and is maternally contributed as well as zygotically expressed in early embryos. Expression of the mir-35 family is essential for viability and the regulation of mir-35 family is tightly regulated at various stages during C. elegans development; mir-35 family is sharply decayed at the end of embryogenesis. Moreover, the mir-35 family is haploinsufficient for the regulation of sex determination indicating that the embryonic steady state abundance is tightly controlled. While suppressors of mir-35 family lethality have not been identified, two loss of function mutants, nhl-2 and sup-26, have been found to suppress the sex determination (pseudomale) phenotype, suggesting that this phenotype may be used as a readout for a genetic screen. We propose to conduct a forward genetic screen using a strain that is sensitized to changes in mir-35 family abundance. We will mutagenize these worms and isolate suppressors of the phenotype. We expect worms that have a suppressed pseudomale phenotype will have mutations in proteins that either modulate the abundance of mir-35 family or affect other downstream sex determination genes. We are also interested in how mir-35 family expression is limited to oocyte and embryo stages of development. A screen of >1000 RNA binding proteins identified a single factor, pumilio homolog protein PUF-9, as the only factor that modulates the temporal expression pattern of mir-35; PUF-9 siRNA- treated C. elegans exhibit mir-35 perdurance beyond the embryo stage. In order to understand the mechanism of PUF-9-mediated mir-35 decay, we aim to identify cofactors that bind PUF-9. We are also interrogating whether the mechanism of mir-35 family turnover is sequence specific or non-sequence specific. Overall, we aim to elucidate the regulation of mir-35 family abundance in the early development of C. elegans and the role of the mir-35 family in sex determination.

ID #3956 Retinoic Acid receptors in hindbrain and spinal cord cell specification Jonathan Huang1, Eve Sutton1, Jessica Cothern2, Gabriel Prado3, Isaac Skromne1 1University of Richmond, USA; 2University of Oregon, USA; 3University of Florida, USA Tissue alignment during embryogenesis is controlled by cell communication signals. Our previous work has shown that Retinoic acid (RA) from the mesoderm is an essential signal to align the hindbrain and spinal cord to occipital and cervical somites. While RA represses spinal cord specification genes in the hindbrain, RA does not do this in the prospective spinal cord territory. A survey of the literature has revealed differential transcription of RA receptors in the neural tube, raising the possibility that different RA receptors mediate different RA functions in the hindbrain and spinal cord. To elucidate the function of each receptor in cell specification, we used reagents to inhibit each receptor individually. First, we characterized the effect that different isoform- specific inhibitors of RA receptors have on hindbrain and spinal cord cell specification. Then, we showed that different inhibitors against the same RA receptor isoform cause similar defects. Finally, we showed that inhibition of RARα or RARγ cause different hindbrain and spinal cord developmental defects. Together, our results suggest that different RA receptors regulate different RA functions in the posterior hindbrain and anterior spinal cord.

ID #3957 Loss of Kinesin Kif20b Enhances Axon Extension on L1-CAM Substrate Sara Martin, Katrina McNeely University of Virginia, United States Kinesin-6 family member, Kif20b, is a microtubule motor with a known role in microtubule bundling and neuron morphogenesis. We have previously reported that the loss of Kif20b causes microcephaly. When neurons from brains of mutant embryos are grown in vitro on poly-L-lysine (PLL) theyhave decreased neuron polarization. Interestingly, neurons that are able to polarize have wider, shorter axons that retract more often. We hypothesize that these phenotypes are due to less densely packed and more dynamic microtubules along with localization changes of Shootin1, which has been implicated in polarization and axon outgrowth. Shootin1 connects the actin cytoskeleton to the cell adhesion molecule L1 and has been shown to interact with Kif20b in the brain. Therefore, we predicted that when grown on the L1 substrate, Kif20b-/- neurons would have altered Shootin1 localization and would maintain the shorter axon phenotype. We tested the first part of this hypothesis by measuring the intensity of Shootin1 in the axons of neurons grown on both PLL and L1. We found that Shootin1was decreased in the axonal growth cones of Kif20b-/- neurons on either substrate. But surprisingly, Kif20b-/- neurons plated on L1 grew significantly longer axons than controls. We tested this interesting overgrowth phenotype by growing Kif20b-/- neurons on a different substrate that is known to increase axon outgrowth. Laminin, an important protein for cell attachment and differentiation as well as cell shape, was the perfect candidate. Interestingly, on laminin, Kif20-/- neurons were not significantly longer than controls. Therefore, the increased axon length phenotype is unique to Kif20b-/- neurons on L1. Currently, we are investigating how Kif20b regulates neurite outgrowth through a previously uncharacterized interaction with L1.

ID #3958 Role of calcium activity during early neural development Sudip Paudel1, Atiqur Rahman1, Eileen Ablondi2, Morgan Sehdev2, Peter Kemper1, Margaret Saha1 1College of William and Mary, USA; 2Harvard Medical School, United States Calcium plays a major role in virtually every physiological process as a ubiquitous signaling molecule. The molecular-cellular details of calcium activity in the mature nervous system are well studied, but far less is known regarding calcium activity during the early stages of neural development. Although previous work has demonstrated that the calcium activity plays a crucial role during embryonic nervous system development, we know relatively little about the regularity, the molecular genetic basis, and the pattern of this activity. Using an in vitro system, we have shown that differentiated neurons, in comparison with neuronal progenitor cells, exhibited more high amplitude spikes with longer duration. However, ultimately, it is essential to examine the spatial and temporal patterns of calcium activity in vivoat a single cell level during early embryonic neural development.Therefore, using a genetically encoded calcium marker (GCaMP), we imaged calcium activity of neural plate of whole mount Xenopus laevis embryos at a single cell resolution and subjected these embryos to in situ hybridization to correlate calcium activity with a molecular phenotype. In order to analyze data, we devised a method to compare calcium activity across different embryos by projecting images onto an embryo grid based on Sox2 and neural beta tubulin (NBT) expression to align the spatial location of cells. Our preliminary analysis suggests that a stripe of cells correlating with NBT exhibited more predictable and persistent calcium dynamics with a higher number of high amplitude spikes that have longer duration. These findings underscore the need for further detailed analysis to understand the importance of calcium activity during early neural development.

ID #3959 Creation of a Database of C. Elegans Orthologs for Human Rare Disease Genes Isabella Zafra Martinez1,2, Andy Golden1,2 1National Institutes of Health, United States; 2National Institute of Diabetes and Digestive and Kidney Diseases, United States There are approximately 7,000 known rare diseases in humans, with about 80% of these being monogenic. When combined, nearly 10% of the United States population has a rare disease. Unfortunately, treatments have been successfully developed for less than 5% of these rare diseases. Fortunately, whole-genome and whole-exome sequencing has allowed identification of disease-associated alleles for many of these rare diseases. The nematode Caenorhabditis elegans has 20,000 protein-coding genes, with nearly 40% of these being estimated to have human orthologs. This, along with its physiological simplicity and short life cycle, makes C. elegans a great model organism to study genes associated with human disease. Here we present the creation of a database in collaboration with the NIH’s Undiagnosed Disease Program (UDP) and WormBase with the goal of indicating which monogenic disease genes identified by the UDP have C. elegans orthologs. In this database, we indicate which of these genes in the worm have the corresponding amino acid that is mutated in the disease condition. The ultimate goal of this project is to merge our database with WormBase to highlight C. elegans genes that researchers can consider generating the missense allele associated with a human disease to study its biology and the underlying genetic mechanisms. Because of the high degree of gene conservation across species, such databases in other model organisms should stimulate similar research projects to better understand the function of these genes.

ID #3961 Determining the function and regulation of polymers of nucleotide biosynthetic enzymes during Drosophila oogenesis Jacqueline Simonet1, Sajitha Anthony2, Alana O'Reilly1, Jeffrey Peterson1 1Fox Chase Cancer Center, USA; 2Drexel University College of Medicine, USA CTP synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) are two rate-limiting enzymes in nucleotide biosynthesis. They have been found to polymerize into filaments under conditions of nucleotide depletion or elevated demand for nucleotides in many different species and cell types. For example, these enzymes polymerize in nutrient-starved mammalian cell lines and CTPS polymerizes during normal Drosophila oogenesis, where germ cells undergo rapid cycles of endoreplication and rRNA synthesis. Our lab and others have demonstrated that CTPS assemblies are present during Drosophila egg development and we are using this model system to understand their function and regulation. We also recently reported that, IMPDH filament assembly has no effect on its biosynthetic activity, either as a purified protein or cultured cells. By contrast, others have found that filament assembly of CTPS can either inhibit or enhance activity of the enzyme, depending on the species. We hypothesize that assembly of these enzymes into filaments may regulate some other aspect of their biological function unrelated to their catalytic activity. We are currently examining this question in different ways for each of these two enzymes. For IMPDH, I am utilizing transgenic flies expressing human IMPDH2 constructs that either inhibit or promote filament assembly without altering enzyme activity to functionally rescue mutants of the Drosophila IMPDH gene. Human IMPDH rescues the impaired fertility of Drosophila IMPDH mutant flies equally whether it can form filaments or not, which means that its ability to form filaments is not necessary for it role in Drosophila egg development. For CTPS we are screening genes involved in nucleotide biosynthesis, endoreplication, cell proliferation, and growth to find regulators of CTPS filament formation during Drosophila oogenesis in order to understand how CTPS assembly and disassembly is regulated and what is its biological function during oogenesis.

ID #3962 The neuromodulator adenosine regulates oligodendrocyte migration at motor exit point transition zones Taylor Welsh, Melanie Piller, Laura Fontenas, Sarah Kucenas University of Virginia, United States During development, OPCs migrate extensively throughout the spinal cord, but their migration is restricted at transition zones (TZ). At these specialized locations, unique glial cells in both zebrafish and mice are at least partially responsible for preventing peripheral OPC migration, but the mechanisms of this regulation are not understood. In order to elucidate the signals that mediate OPC segregation at motor exit point (MEP) TZs, we performed an unbiased small molecule screen. Using chemical screening and in vivo imaging, we discovered that inhibition of A2a adenosine receptors (AR) causes ectopic OPC migration out of the spinal cord. In our studies, we provide in vivo evidence that endogenous neuromodulation by adenosine regulates OPC migration along motor axons, specifically at the MEP TZ. This work opens exciting possibilities for understanding how OPCs reach their final destinations during development and identifies mechanisms that could promote their migration in disease.

ID #3963 Characterizing Sox21-protein interactions and their subsequent functions in the coordination of neurogenesis in Xenopus laevis Dillon Damuth Georgetown University, USA While transcription factors (TFs) primarily serve to alter transcription, some require a partner protein(s) to efficiently affect the transcriptional state of target genes. Since transcriptional regulation is a key factor of embryogenesis, it can be assumed these TF interactions may help coordinate development. However, the means by which partner proteins serve to guide the function or specificity of a transcription factor is not well understood. Investigating how these interactions contribute to cell lineage coordination will contribute fundamental information not only applicable to embryogenesis, but also to future research aiming to guide pluripotent cells toward a specific fate.

The Sox family of TFs offers promising subjects to study how protein interactions contribute toward cell fate, since they are an example of TFs requiring protein interactions. Although it is known that Sox TFs require a partner protein, little is known regarding the interactomes of each Sox protein, and even less known about the functional significance of these interactions. Sox21, a SoxB2 protein, offers a particularly unique opportunity to study not only how function is influenced by partner proteins, but also how those interactions contribute to cell fate within the process of neurogenesis.

This project is focused toward two interactions, Sox21-Sox2 and Sox21-Ngn2, in effort to bridge the knowledge gap of how protein-protein interactions may serve to coordinate the specification and differentiation of neurons. Previous data suggests Sox21 is necessary to maintain neural progenitor cells in a sox2+ proliferative state, while low levels are also necessary to promote neural maturation. It is hypothesized that Sox21 is capable of having seemingly contradictory functions due to differential interactions with partner proteins across stages of development. In studying these interactions, we aim to test how these partners may differentially guide transcriptional specificity of Sox21.

ID #3964 MATER matters - breaking the cortical actin barrier during exocytosis in mouse eggs Edgar Vogt, Keizo Tokuhiro, Jurrien Dean National Institutes of Health, NIDDK, USA Regulated exocytosis is a fundamental cellular event for delivering cargo molecules to the cell surface for specific physiological tasks including neurotransmission, immune response, and reproduction. Post-fertilization exocytosis of egg cortical granules releases ovastacin, a metalloendopeptidase that cleaves ZP2 in the extracellular zona pellucida to prevent polyspermy. How cortical granules traffick to the plasma membrane during exocytosis in mouse eggs remains unclear. Here, we use high- and super- resolution imaging to observe exocytosis at single granule resolution in transgenic mouse eggs expressing fluorescently tagged ovastacinmCherry, a unique marker of cortical granules. We characterize the association of the motor protein non-muscle myosin IIA with cortical granules, wherein they become docked at the plasma membrane during exocytosis after clearance of cortical actin. We also implicate the maternal-effect gene Mater in cortical granule trafficking. Eggs lacking MATER fail to tether cortical granules at the egg's cortex. Significantly more sperm penetrate the zona matrix of MaterNull eggs owing to impaired cortical actin clearance, delayed exocytosis and cleavage of ZP2. Thus, MATER regulates cortical granule exocytosis in mouse eggs to ensure an effective post-fertilization block to sperm binding to the zona pellucida.

ID #3965 A network of microRNAs and RNA binding proteins acts maternally to regulate sex determination in the C. elegans embryo. Katherine McJunkin National Institutes of Health, NIDDK Intramural Research Program, USA Although many roles of microRNAs in differentiated tissues have been described, relatively few microRNAs are known to function in early embryonic development. In particular, little is understood about the function of microRNAs expressed prior to the maternal to zygotic transition, when post-transcriptional control of gene expression is widespread. Seeking to elucidate the function of microRNAs in early development, I have focused on the mir-35-41 microRNA cluster in C. elegans, which is expressed maternally and in embryos, and is essential for embryonic development and fecundity.

Here I show that the mir-35-41 microRNA cluster regulates sex determination, preventing aberrant activation of male-specific gene expression in hermaphrodite embryos. Two predicted mir-35-41 target genes are required for the sex determination phenotypes of mir-35-41 mutant embryos, suggesting that they act downstream of mir- 35-41. These target genes, sup-26 and nhl-2, both encode RNA binding proteins, thus delineating multiple new layers of post-transcriptional regulation of the sex determination pathway.

Most players in the sex determination pathway are regulated zygotically, after the inheritance of both sex chromosomes. In contrast, the maternal load of mir-35-41 is largely responsible for regulating sex determination. Because of this maternal contribution to an inherently zygotic process, I propose that mirs-35-41 act as a developmental timer, ensuring a period of naïveté in early embryos, and preventing premature decision-making in sex determination and possibly other developmental processes.

Using CRISPR/Cas-9 to manipulate the endogenous mir-35-41 seed match in the nhl-2 3’ UTR, I observe that repression of nhl-2 by mir-35-41 is not only required for proper sex determination but also for viability, showing that a single microRNA target site can be essential. Our work thus also makes progress towards understanding the essentiality of this maternally-contributed microRNA family.

ID #3966 The role of planar polarity and cell geometry in epidermal stem cell self-renewal Kimberly Box, Bradley Joyce, Danelle Devenport Princeton University, USA The ability to control cell fate through oriented cell division is imperative for the proper development of many organs, such as the stratified epidermis. Basal stem cells of the epidermis can divide in two ways: 1) perpendicularly to the epithelial plane to produce one basal and one suprabasal daughter that goes on to differentiate, and 2) parallel to the epithelial plane, which generates two basal daughter cells and expands the stem cell pool. While mechanisms leading to perpendicular divisions in the murine epidermis are known, those orienting planar divisions have not been explored. Late in embryonic skin development, apical-basal polarity factors align the spindle to promote perpendicular divisions. Thus, we hypothesized that parallel cell divisions might be controlled by planar cell polarity (PCP), which relies on a set of cortical “core” transmembrane components that are asymmetrically localized along the epithelial plane. In agreement with this hypothesis, Vangl2Lp/Lp mutant embryos exhibit increased perpendicular, asymmetric divisions at the expense of planar, symmetric divisions. This defect was not due to increased proliferation rates, nor to mislocalization of cortical spindle anchoring proteins like LGN. Rather, we link the reduction in planar divisions to alterations in cell geometry and cell packing, but these alterations are indirectly caused by the neural tube defects characteristic of PCP mutants. We demonstrate that early in epidermal stratification, there is a close relationship between cell density, interphase cell height:width ratio, and mitotic spindle orientation. Moreover, failure of the epidermis to close over the neural tube in PCP mutants leads to cell crowding and a lower frequency of planar cell divisions. We propose a model in which basal epidermal cells utilize cell packing and shape, rather than cortical PCP cues, to inform planar division orientation.

ID #3967 Ca2+/calmodulin-dependent protein kinase type II (CaMK-II) is required for hematopoietic stem cell specification Camden Kurtz, Robert Tombes, Sarah Rothschild Virginia Commonwealth University, USA Ca2+/Calmodulin-dependent protein kinase type II (CaMK-II) is a Serine/Threonine protein kinase that is activated by Ca2+ and Calmodulin to phosphorylate substrates involved in myriad developmental processes. This project implicates CaMK-II in specification of HSCs, and zebrafish provide an ideal embryonic model to study hematopoiesis. Zebrafish genetic manipulation was achieved through: incubation in chemical inhibitors; injection of notochord-targeted WT and DN CaMK-II constructs with Transposase; and injection of camk2g1 translation-blocking morpholino antisense oligonucleotide (MO). Whole-mount in situ hybridization (WISH) and immunolocalization on zebrafish embryos allowed visualization of key HSC markers and pathway components that implicated CaMK-II in the specification of HSCs. CaMK-II is a negative regulator of shh expression during HSC specification, but CaMK-II does not influence Shh during its well-documented role in vasculogenesis. CaMK-II appears to affect the spatial distribution of Shh protein, which accumulates near the notochord source and differentially affects expression of Shh target genes based on their distance from the notochord. This project also identifies the specific timing requirement for CaMK-II during HSC specification, as inhibition of CaMK-II consistently reduces HSC specification, but only if administered before 18hpf. CaMK-II also downregulates ezh2 in the DA during the time of HSC specification, and the Ezh2 inhibition rescues the loss of HSCs, suggesting that CaMK-II regulates the secretion of Shh from the notochord to epigenetically regulate expression of key HSC specification genes in the DA through EZH2 methyltransferase.

ID #3968 Development of the pectoral fin vasculature in zebrafish embryos Scott Paulissen, Daniel Castranova, Brant Weinstein NIH/NICHD, United States A properly functioning circulatory system plays a critical role in human health, and understanding how blood vessels assemble is important to develop treatments for cardiovascular disease. Using super resolution microscopy, we describe the initial assembly of the vasculature supplying the pectoral fins late during the second day of zebrafish development. The pectoral fins are analogous structures to the forelimbs of mammals. The superficial location of the pectoral fin and its developing vessels make it ideal for observing and studying the events of vascular development including sprouting, anastomosis, lumenization, and the cellular rearrangements associated with these processes. The formation of the pectoral artery occurs via a stereotyped process that involves abrupt linkage to the axial vasculature to initiate blood flow throughout the vessel. This attachment leads to very rapid changes in pectoral artery morphology compared to analogous processes occurring in the intersegmental vessels of the trunk, for example. We will present some of our latest image data illuminating the mechanisms of pectoral artery growth and tubulogenesis.

ID #3969 Calcium signals act through Histone Deacetylases to mediate pronephric kidney morphogenesis Sarah Rothschild, Hunter Lee, Sarah Ingram, Robert Tombes Virginia Commonwealth University, United States Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common heritable diseases in the world, characterized by mutations in PKD1 and PKD2. PKD2, a non-selective cation channel with a high selectivity for Ca2+, has been shown to be necessary for Ca2+/calmodulin-dependent protein kinase type 2 (CaMK-II) activation, enabling kidney morphogenesis. In order to further elucidate how CaMK-II influences kidney development, the interaction between CaMK-II and class II HDACs (HDAC 4, 5, 6) during zebrafish kidney development was investigated using pharmacological inhibition and antisense morpholino oligonucleotides. Although all three class II HDACs are expressed during early development, only HDAC5 morphants exhibited anterior cyst development and defects in kidney cell migration and pronephric duct convolution, similar to PKD2 and CaMK-II morphants. HDAC4 morphants did not exhibit kidney defects. However, suppression of HDAC4 was able to partially rescue kidney convolution in CaMK-II mutants, while suppression of HDAC5 exacerbated these defects. CaMK-II suppression also caused HDAC4 to translocate to the nucleus but did not affect the localization of HDAC5. This data supports a model in which CaMK-II retains HDAC4 in the nucleus, enabling kidney morphogenesis. Further support for this model will be investigated in two ways. First, by observing the sub-cellular localization of HDAC4 and activated-CaMK-II during kidney development. Second, by mutating known CaMK-II phosphorylation sites on HDAC4 and HDAC5 to create nuclear targeted and cytosolic targeted constructs which will be used to evaluate their effects on kidney cell migration and pronephric duct convolution. This will not only provide new information into the role of CaMK-II and HDACs in kidney development, but will also identify potential therapeutic targets for the treatment of ADPKD.

ID #3970 Histone-directed transcriptional co-regulator Brd2 is necessary for proper formation of several organ-systems in vertebrate development Hee Jae Song, Alexandra Quatrello, Angela DiBenedetto Villanova University, USA Brd2 is a member of the bromodomain-extraterminal domain (BET) family of transcriptional co-regulators, which form histone-directed recruitment scaffolds for the assembly of chromatin modification complexes at promoters, thus controlling access of target genes to the transcriptional machinary. Brd2 is involved in the control of proliferation in adult mammalian tissues and in neuronal apoptosis during early vertebrate development. Its role in later development, when organ-systems other than the nervous system are being formed is, however, unknown. Previous studies in zebrafish have shown that complete brd2a knockout by Crispr-Cas9 is embryonic lethal, while brd2a knockdown by antisense morpholino results in reduced hindbrain and central nervous system abnormalities associated with elevated levels of cell death. Since neither of these methods allow for temporal control of Brd2 deficiency, they are unable to reveal any later functions of Brd2 that may exist. In this study, we use a specific Brd2 BET small molecule inhibitor, BIC1, to target Brd2 activity at different stages of development in zebrafish, and find that later Brd2 deficiency results in abnormalities in the brain, trunk, and circulatory and excretory systems. BIC1 was administered at 3, 10, and 24 hpf, and fish were assayed at 24 hpf and 48 hpf for characteristic morphology. Early 3 hpf inhibition showed abnormalities in brain, trunk, and circulation when assayed at 24 hpf and 48 hpf, compared to wild-type. Treatment at 10 hpf showed pronephric duct abnormalities at 24 hpf assay, with recovery at 48 hpf assay. Late 24 hpf treatment showed circulatory and heart abnormalities at 48 hpf assay. Thus, Brd2 function is necessary both for early central nervous system development, and for later circulatory and excretory system formation. These findings expand our understanding of the developmental role of Brd2, revealing its involvement in the proper formation of multiple organ-systems in vertebrates.

ID #3971 Elucidation of the function of miR-31 during early development Carolyn Remsburg1, Nadezda Stepicheva2, Jia Song1 1University of Delaware, USA; 2University of Pittsburgh School of Medicine, USA MicroRNAs (miRNAs) are short, non-coding RNAs that act as post-transcriptional regulators by repressing translation of their target mRNAs. miRNAs have been shown to play important roles in many cellular processes, including cell differentiation, migration and cell cycle. The goal of this project is to evaluate the global function of miR-31 during early development, using the sea urchin embryo as a model. Sea urchins have approximately 50 miRNAs, with the majority of them having a single member in each family, making the sea urchin a tractable model to examine single miRNA function. We previously identified that miR-31 is required for proper development and function of skeletogenic cells. In order to identify additional miR-31 target genes, we injected biotinylated miR-31 to pull down its bound targets. RNA-sequencing was performed to identify differentially pulled down transcripts between control and injected embryos in the blastula stage. Our analysis indicated that several potential miR-31 targeted genes encode proteins that interact with actin. Using whole mount in situ hybridization, we determined the temporal and spatial expression of potential miR-31 target genes, including Fascin, Nr1m3, Kirrel2L_6 and DUSP16. To test the direct suppression of these targets by miR-31, 3' untranslated regions (3'UTR) of these genes are cloned downstream of the luciferase reporter construct. We will use dual luciferase assays to compare the luciferase readouts in 3'UTRs with wild type or mutated miR-31 target sites. The impact of miR-31 regulation of these targets during development will be examined by using morpholino antisense oligonucleotides (MASO) to block miR-31 target sites. We will also examine the function of some of these miR-31 target genes with MASO knockdown and CRISPR/Cas9 mediated knockout approaches. Through identification of the targets of miR-31, our results contribute to a comprehensive understanding of the function of microRNAs in embryonic development.

ID #3972 Studying the origin and function of novel brain vascular-associated cells Marina Venero Galanternik, Ryan D. Gober, Daniel Castranova, Brant M. Weinstein NICHD, NIH, USA The meninges are an external enveloping connective tissue that encases the brain, producing cerebrospinal fluid, acting as a cushion against trauma, nourishing the brain via nutrient circulation, and removing waste. Despite its importance, the cell types present in the meninges and the function and embryonic origins of this tissue are still not well understood. We describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and highly unusual scavenger behavior, these cells appear to be the zebrafish equivalent of mammalian “Fluorescent Granular Perithelial cells” (FGPs), macrophage-like cells about which very little is known and that likely play important roles in brain function and in a variety of CNS pathologies. Using RNA-seq of FACS- sorted FGPs and single-cell profiling of cranial cells, we show that despite their macrophage-like morphology and their perivascular location these cells are molecularly most similar to lymphatic endothelial cells, and lineage tracing and time-lapse imaging demonstrate that these unusual cells transdifferentiate from endothelial cells lining blood vessels of the optic choroid vascular plexus deep inside the brain, from where they migrate to the brain surface. Using forward-genetic screening of transgenic zebrafish for ENU-induced recessive mutations, we recently identified a mutant that appears to be specifically deficient in FGPs, providing us a genetic model that we are using to further explore the likely very important functional role of these cells. Our findings thus far provide the first report of a non-vessel forming, perivascular cell population in the brain that emerges by transdifferentiation from vascular endothelium.

ID #3973 Calcium Signaling in Acute Lymphoblastic Leukemia/Lymphoma Sarah Rothschild1, Wilson Clements2 1Virginia Commonwealth Univ, USA; 2St. Jude Children's Research Hospital, USA Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, where 3000 children under the age of 20 are diagnosed each year. 15% of patients have T cell ALL while 85% are diagnosed with B cell ALL. Different chromosal translocations and genetic lesions have been identified in patients with ALL. Misregulation of calcium signaling has also been observed in patients with leukemia. The multifunctional Ca2+/calmodulin-dependent protein kinase type 2, CaMK-II, is one such protein that is incorrectly activated, leading to disease progression. To determine the role of CaMK-II in ALL, we generated a stable line of zebrafish expressing EGFP tagged constitutively active (T287D, CA) CaMK-II in lymphocytes. Although expression of CA CaMK-II alone did not generate leukemia, the expression of CA CaMK-II in p53 mutant fish caused B cell acute lymphoblastic leukemia/lymphoma to develop as early as four months of age. Immature lymphoblasts are visible throughout the kidney marrow, the zebrafish adult site of hematopoeisis, and spleen. We seek to understand the underlying molecular mechanism of leukemia progression in this model to identify novel targets for therapeutic intervention.

ID #3974 Perturbations to the Notch Signaling Pathway Induce Differential Variability in Gene Expression in the Developing Allotetraploid Frog Xenopus laevis Ronald Cutler, Caroline Golino, Andrew Hallaren, Margaret Saha College of William and Mary, United States The Notch pathway is a juxtacrine signaling pathway involved in determination of neural cell fate during embryonic development among vertebrates. This pathway was perturbed in developing Xenopus laevis embryos by injecting a genetic construct into one cell at the two-cell stage that either impedes or overexpresses Notch signaling, disrupting neural patterning. Embryos show a compensatory response to this perturbation and largely recover by the swimming tadpole stage. We have performed RNA-sequencing to investigate the transcriptional response to Notch signaling perturbations and identify genes involved in compensation from this perturbation. In addition to customary differential expression (DE) analysis, we examined differential variability between groups (Ran & Daye, 2017) at three time points (neural tube, tailbud, swimming tadpole), which compares the consistency of gene expression across biological replicates. Notch pathway impedance resulted in less than 20 significant DE genes at all time points, however many significant differentially variable genes were observed, with a peak of 512 differentially variable genes at the neural tube stage. Notch pathway upregulation resulted in over 100 significant DE genes, in addition to a peak of 546 differentially variable genes at the neural tube stage in the upregulated condition. Interestingly, no genes were shared among DE and differentially variable genes in the impedance conditions, while an average of 3.8% of these genes were shared in the upregulated conditions. Significantly enriched differentially variable gene ontology (GO) terms at the neural tube stage include calcium-mediated signaling in the impedance condition and regulation of cell morphogenesis involved in differentiation in the upregulated condition. Discordance between DE and differentially variable gene sets and GO enrichments suggest the importance of differentially variable genes in Notch signaling perturbation and compensation from it.

ID #3975 Mechanical Signals Drive Airway Smooth Muscle Differentiation in the Embryonic Mouse Lung Jacob Jaslove1,2, Celeste Nelson1 1Princeton University, USA; 2Rutgers Robert Wood Johnson Medical School, USA Lung disease in newborns causes significant morbidity and mortality worldwide. Progress in treating these disorders will require an understanding of how the body creates the complex branched architecture of the lung. During development, the airways are comprised of a tube of epithelial cells that are wrapped by a layer of involuntary muscle tissue known as airway smooth muscle (ASM). We recently found that ASM plays an essential role in development by mechanically inducing bifurcation of the airway epithelium. Formation of ASM, in turn, appears to be controlled by mechanical forces. This led us to ask whether mesenchymal cells from the developing lung will differentiate into ASM when they are physically stretched, and to ask what genes and proteins allow the cells to respond to stretch. We constructed a mechanical stretch device to investigate these questions, and found that MLg mouse lung mesenchymal cells upregulate the smooth muscle markers α-smooth muscle actin and transgelin when stretched in culture. To distinguish between the different smooth muscle cell types in the developing lung, we validated the expression of the different isoforms of smoothelin as markers for ASM and vascular smooth muscle. Using muscle to shape developing organs is a simple and powerful tool with which the embryo assembles complex structures. Understanding how mechanical forces regulate the formation of smooth muscle may reveal new targets for treating lung diseases that result from mechanical disturbances or may enable tissue engineers to use smooth muscle to create self-assembling organs in the lab.

ID #3976 Molecular regulation of vascular smooth muscle cell recruitment to arteries during development Amber Stratman, Brant Weinstein NIH, NICHD, USA The preferential recruitment of vascular smooth muscle cells (vSMCs) to arteries versus veins during early development is a well-described phenomenon that has traditionally been attributed to higher levels of blood flow rates and of shear stress through the arterial vasculature. Although the preferential recruitment of smooth muscle to arteries has been appreciated for many centuries, little is known about the molecular pathways responsible for this preference. Here, we show that the cxcl12 ligand and its receptor cxcr4 are both expressed on embryonic arteries during stages of vSMC acquisition. Using zebrafish genetic mutants, RNA/DNA over expression studies, and in vitro mechanistic analysis in primary human cell lines, we find that cxcl12/cxcr4 signaling within arterial endothelial cells leads to increased pdgf-bb ligand production, thus resulting in increased vSMC recruitment to arteries. Shortly after the onset of blood flow, expression of klf2a, a well-characterized blood flow–regulated gene that negatively regulates cxcr4 expression, transiently becomes heavily polarized to veins. This inhibits vSMC recruitment to veins by limiting expression of cxcr4 and pdgf-bb to the arterial vasculature. Together, our findings illuminate an early developmental molecular signaling axis that is regulated by blood flow and drives preferential recruitment of smooth muscle to the arterial vasculature.

ID #3977 Does Sonic Hedgehog specify digit identity directly as a morphogen, or indirectly through relay signals? Jianjian Zhu National Cancer Institute-Frederick, USA Sonic Hedgehog (Shh) controls both digit morphology and number during limb development. Shh knockout (null) limbs form a single digit-like structure. How Shh regulates digit identity still remains unsettled. Classic models propose that accumulated Shh signals (higher concentration or longer duration) is required to specify posterior digits arising from Shh descendants. However, those Shh descendants become refractory to Shh over time. By timed genetic deletion of Shh in mouse limbs, we proposed a biphasic model that Shh specifies digit progenitors in the early transient phase, and sustained activity regulates the number of digits that form. To validate our model, we have deleted the pro-apoptotic genes Bax and Bak to completely block apoptosis in Shh conditional mutants with tamoxifen-activated Cre. When Shh is deleted very shortly after initiation of its expression, 100% of Shh conditional mutants with one functional Bax allele (with apoptosis) have a Shh-null phenotype. However, when there is no Bax/Bak function (apoptosis blocked), 50% of Shh conditional mutants form up to 5 digits with completely normal morphology, indicating an early transient pulse of Shh specifies progenitors of all the digits. Interestingly, labeling of progenitors responding to this early pulse of Shh (Gli1CreER+) shows that only digits 4 and 5 respond directly to Shh, while anterior digits (d1-d3) do not. To study if downstream relay signals play a role in specifying anterior digits, enforced Shh signaling in posterior digits (Shh descendants)in Shh null embryos is adopted, and rescues the normal digit 1, the anterior-most non-Shh descendant digit. These data support our model that an early transient Shh pulse suffices to determine the fate choice and final morphology of digit progenitors. During this process, posterior digits (d4-d5) respond to Shh directly, while anterior digits (d1-d3) are specified indirectly, rather through relay signals secreted from Shh responsive cells.

ID #3978 Wnt/ß-catenin regulates an ancient signaling network during zebrafish scale development Andrew Aman1, Alexis Fullbright2, David Parichy1 1University of Virginia, USA; 2University of Utah, USA Understanding how patterning influences cell behaviors to generate three dimensional morphologies is a central goal of developmental biology. Additionally, comparing these regulatory mechanisms among morphologically diverse tissues allows for rigorous testing of evolutionary hypotheses. Zebrafish skin is endowed with a coat of precisely patterned bony scales. We use in-toto live imaging during scale development and manipulations of cell signaling activity to elucidate core features of scale patterning and morphogenesis. These analyses show that scale development requires the concerted activity of Wnt/β-catenin, Ectodysplasin (Eda) and Fibroblast growth factor (Fgf) signaling. This regulatory module coordinates Hedgehog (HH) dependent collective cell migration during epidermal invagination, a cell behavior not previously implicated in skin appendage morphogenesis. Our analyses demonstrate the utility of zebrafish scale development as a tractable system in which to elucidate mechanisms of developmental patterning and morphogenesis, and suggest a single, ancient origin of skin appendage patterning mechanisms in vertebrates.

ID #3979 FGF activity is a robustness factor for clock oscillations in somite segmentation Matthew Anderson1, Ryoichiro Kageyama2, Mark Lewandoski1 1National Cancer Institute, USA; 2Kyoto University, Japan Many essential embryological processes must be robustly buffered against parametric variability to ensure normal development. A case in point is the oscillatory expression pattern of the transcriptional repressor, Hes7, in the presomitic mesoderm (PSM). Such Hes7 oscillations are at the heart of a molecular PSM clock that regulates the proper segmentation of the paraxial mesoderm. This segmentation is essential for the regular formation of somites along the vertebrate axis, which develop into the segmented vertebral column and its associated muscle, tendons, and dermis. Precise control of Hes7 is imperative for somite formation as even subtle mutations, such as removing a single intron in Hes7 that only disrupt mRNA halflife dynamics, result in an altered vertebral column. We have previously shown that Fgf4 and Fgf8 comprise a classical “wavefront” activity that keeps PSM cells undifferentiated. Somite segmentation occurs when cells in a certain clock phase leave the wavefront. Here, we show that Fgf4 is needed for proper clock oscillations. Inactivation of Fgf4 in the PSM results in both diminished Hes7 levels and altered Hes7 oscillations, leading to cervical and thoracic vertebral defects, These defects are more extreme when one copy of Fgf8 is additionally removed and Hes7 oscillations are rescued when Fgf8 is overexpressed. However, all markers of wavefront activity are normal in these mutants. Therefore, we conclude that clock oscillations require an FGF signal that is genetically separable from the wavefront requirement. We hypothesize that this FGF role in clock oscillations is permissive and provides a robustness factor for somite segmentation, buffering the system to changes in Hes7 expression dynamics.This idea predicts that removing one Hes7 copy, which in a wildtype background causes only low penetrance scoliosis, in our Fgf4 mutants will cause a synergistic defect with extreme vertebral defects; we are testing this now.

ID #3980 Detection of bone mineralization in larvae and adult zebrafish using Carbon nanodots Esmail Miyanji, Lesly Corado-Santiago, Bryle Barrameda, Isaac Skromne University of Richmond, USA Bone weakness and deformity arise from abnormal growth, development, remodeling, or disease. Towards developing new strategies to mitigate bone-related health problems, we are investigating the bone-binding properties of Carbon nanodots (C-dots) in larvae and adult zebrafish, as a method for the targeted delivery of drugs to bones. Our published work shows that C-dots are non-toxic to larvae, and that they bind with high affinity and specificity to mineralized bones: manipulations that increase or decrease calcium mineralization, respectively increase or decrease C-dots deposition in bones. These bone-binding properties are not affected by the derivatization of the C- dots with amine, fluorescein or biotin groups. Preliminary research shows that C-dots can also bind to exposed bones after fin amputation. Further work will determine the bone-binding properties of C-dots to adult bones during remodeling, wound healing and regeneration.

ID #3981 Investigation of top-2(it7ude6) in Suppressing Embryonic Lethal Mutation, top- 2(it7), in C. elegans Dina Collins1, Harold E. Smith2, Aimee Jaramillo-Lambert1 1Department of Biological Sciences, University of Delaware, United States; 2National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States Meiosis is a unique form of cell division that results in haploid cells that contain half the number of chromosomes of the parent cell. Topoisomerase II (TOP-2) is an enzyme that promotes chromosomal integrity in mitosis by resolving topological problems in double-stranded DNA. Studies have demonstrated that TOP-2 also plays a role in meiosis, but the exact mechanisms are unknown. Previously, we identified a mutation in C. elegans TOP-2, top-2(it7), which may be the missing element in connecting the role of TOP-2 in meiotic events. The top-2(it7) mutation is a temperature sensitive embryonic lethal mutation. At the restrictive temperature (24°C), ~99.9% of top-2(it7) embryos die. To identify putative genes that interact with top-2 during meiosis, we performed a genetic suppressor screen and found 12 suppressors of top-2(it7) embryonic lethality. Out of the 12 suppressors, my studies focus on Suppressor 19. Interestingly, Suppressor 19 (allele designation ude6) is a top-2 intragenic mutation that substitutes aspartate 809 with an asparagine in the topoisomerase domain of the protein. We aim to determine how top-2(ude6) is causing the suppression of the mutant phenotype of top-2(it7). First, to confirm that top-2(ude6) D809N is directly involved in suppressing the top-2(it7) R828C mutation, we are using CRISPR-Cas9 genome editing to recreate top-2(ude6) in the top-2(it7) strain. Secondly, top-2(it7ude6) will be “rescued” by using CRISPR/Cas9 genome editing to change the mutant asparagine at amino acid 809 back to the wild-type aspartate. For future experiments, we will conduct a thorough analysis of the germline via gonad dissection and DAPI staining techniques. Additionally, the top-2(ude6) D809N mutation will be recreated in wild-type animals to investigate if the mutation has its own novel phenotype. Ultimately, we expect these studies to advance our understanding of how the structure of the TOP-2 protein contributes to its roles in chromosome segregation.

ID #3982 The Genetic Basis of Natural Pigment Pattern Diversity in Danio kyathit Braedan M. McCluskey1, Susumu Uji2, John H. Postlethwait3, David M. Parichy1 1Department of Biology and Department of Cell Biology, University of Virginia, Charlottesville, VA, United States; 2Fisheries Research and Education Agency, Yokohama, Kanagawa, Japan; 3Institute of Neuroscience, University of Oregon, Eugene, OR, United States Vertebrates display a striking diversity of pigment patterns, even between closely related species. To understand the evolution of pigment pattern, we are combining the knowledge of the genetic, developmental, and cellular basis of stripe formation in zebrafish (Danio rerio) with natural variation in the closely related species, Danio kyathit, which has both spotted and striped morphs. Although many Danio hybrids are sterile, these two morphs produce fertile hybrid offspring allowing for traditional genetic mapping approaches to understand their phenotypic differences. To understand the genetic basis of natural pigment pattern variation, we phenotyped 648 individuals from two mapping crosses. Using Restriction site Associated DNA sequencing (RAD-seq) for a subset of these individuals, we genotyped over 30,000 loci, constructed a genetic map, and identified quantitative trait loci. Our genetic map cross revealed segregation distortion across several chromosomes inherited from the hybrid parent, strongly suggesting that either meiotic drive or hybrid incompatibilities are pushing the spotted and striped morphs of Danio kyathit to become distinct species. Our QTL analysis identified a locus associated with multiple phenotypes: pigment pattern (as measured by several metrics), standard length and body depth. The QTL contains several promising genes known to affect pigmentation and body size in zebrafish mutants. Comparing gene order between zebrafish and more distantly related species, we find that this QTL includes a genomic region that underwent a series of large chromosomal inversions during the diversification of the Danio genus. Combining the genetic and genomic resources available for zebrafish with the study of natural variation across species, we have uncovered a chromosomal hotspot for genomic and phenotypic evolution.

ID #3983 Rab35 is Essential in Sea Urchin Gastrulation Michael Testa, Carissa McKinney, Jia Song University of Delaware, United States Rab35 is a small GTPase conserved in all metazoans. It mediates membrane protein trafficking between the plasma membrane and the early endosomes at the cell surface. Little is known about the physiological role of Rab35 in development. This study examines the function of Rab35 in development. Previous studies indicated that Rab35 is involved in fundamental cellular roles, such as migration, apical-basal polarity, and cell division, which are all important processes in embryonic development. We hypothesize that Rab35 is involved in regulating cellular morphogenesis. To test this hypothesis, we use the sea urchin as an experimental model, because they shed a large number of gametes, undergo external fertilization, have transparent embryos, have a relatively short, early developmental period, and have one of the best defined endomesodermal gene regulatory networks. To test the function of Rab35, we use Rab35 morpholino antisense oligonucleotide (MASO) to knockdown Rab35. In addition, we utilized site directed mutagenesis to mutate the Rab35 coding sequence into a constituently active form (Q67L) and dominant negative form (S22N).The resulting mRNA of these Rab35 constructs is microinjected into newly fertilized zygotes, and embryos are collected at various developmental time points to examine Rab35 perturbation effects. Preliminary results indicate that Rab35 MASO-treated embryos displayed a range of defective gastrulation phenotypes, ranging from a lack of invaginated gut, to exogastrulation. Importantly, these gastrulation defects are rescued with either the sea urchin Rab35 or the human Rab35, indicating the specificity of the Rab35 knockdown. This study reveals the critical function Rab35 in regulating gastrulation in development.

ID #3984 The RNA-binding protein ETR-1 plays an important role in male reproduction Ruby Boateng1, Kristina Ramirez1, Andy Golden, Ph.D2, Anna Allen, Ph.D1 1Howard University, USA; 2NIDDK/NIH, USA The ELAV-Type RNA-binding protein, ETR-1, is one of two CELF-family RNA-binding proteins that exist in C. elegans. Our studies have shown that ETR-1 depletion via RNAi results in defects in muscle development, a reduced reproductive capacity and increased number of germ cells undergoing apoptosis in C. elegans hermaphrodites. We will show here that ETR-1 also plays a significant role in male reproduction. Spermatogenesis genes in C elegans are typically resistant to RNAi, therefore most researchers study spermatogenesis defects using mutant alleles. Only few genes such as sp-3/4 genes have been shown to result in a penetrant male sterility phenotype when depleted via RNAi in an enhanced sensitivity to RNAi worm strain [rrf-3(pk1426)] (Chu et al., 2006). We will show that RNAi depletion of ETR-1 in rrf-3 worms also results in a strong male fertility defect. Males depleted of etr-1 via RNAi are 64% less fertile that control(RNAi) animals. Males depleted of ETR-1 have deformed tails, prematurely activated spermatids similar to spe-47(hc198) mutants, and delayed activation of spermatids. Some of the spermatids have "rod-like" and "spike-like" projections, similar to phenotypes observed in spe-15(ok153) spermatids, and suggests defects in Major Sperm Protein polymerization and sorting of cellular components respectively. As we begin to elucidate how ETR-1 might be functioning in C. elegans, including during spermatogenesis, we performed RIP-seq to identify the mRNA targets of ETR-1. Initial analysis of our RIP-seq data indicates an enrichment of calcium binding and Major Sperm Protein genes, both of which are involved in sperm activation. The mRNA targets of ETR-1 in conjunction with the phenotypes will enable us to deduce the mechanism of ETR-1 in spermatogenesis. ID #3985 Activating mutations in FGFR leads to a competitive advantage in Drosophila germline stem cells Kathy Le Johns Hopkins School of Medicine, USA The Paternal Age Effect describes how older men are more likely to have children with genetic disorders than younger men. This is attributed to an age-related accumulation of sperm with spontaneous mutations that are thought to originate in germline (sperm- producing) stem cells (GSCs). Stem cells with advantageous mutations can out- compete other stem cells; however, competition between GSCs is still poorly understood. My previous research showed that overexpressing breathless (btl), an activated form of the FGF receptor, in Drosophila causes GSC competition. This suggests a relationship between GSC competition and the Paternal Age Effect because the human homolog of btl is known to cause Apert’s Syndrome, a Paternal Age Effect congenital disorder. Therefore, the goal of my project is to effectively model stem cell competition in the Drosophila melanogaster testis and to find specific gene mutations that cause GSC competition that could be associated with the Paternal Age Effect. Additionally, characterizing the advantages that are conferred by these mutations will allow for a greater understanding of the mechanisms behind stem cell competition. Further uncovering the mutations and mechanisms that underlie the Paternal Age Effect disorders will be a prerequisite for finding ways to prevent mutation accumulation and genetic defects.

ID #3986 Ecdysone regulates epithelial barrier maturation in wing imaginal discs. Danielle DaCrema, Ryunosuke Yano, Rajan Bhandari, Adrian Halme Department of Cell Biology, University of Virginia School of Medicine, USA Drosophila imaginal discs (larval precursors to adult organs) lose regenerative capacity late in larval development in response to high circulating levels of the steroid hormone ecdysone. To better understand the changes ecdysone produces in the imaginal discs, we examined a key function of epithelia: the epithelial barrier. The epithelial barrier is formed by septate junctions in invertebrates and is a semi-permeable diffused barrier that separates the lumen of organs from the insect hemolymph. We modified an epithelial barrier-permeability assay to measure changes in the epithelial barrier of wing imaginal discs. Using this assay, we observed that the epithelial barrier of wing imaginal discs is permeable early in the third larval instar, but becomes impermeable on a time- scale that correlates with the ecdysone increases. Although early discs are permeable, they are less permeable than discs with non-functional barriers indicating that this is a maturation of barrier function not the establishment of the epithelial barrier within the imaginal disc. We demonstrated that increased ecdysone induces barrier maturation, and that limiting ecdysone signaling through local expression of dominant negative ecdysone receptors prevents barrier maturation. To determine how septate junctions change during epithelial barrier maturation, we examined the localization of several septate junction components before and after barrier maturation. Coracle, a Drosophila homolog of protein 4.1 and a core component of the septate junctions, is diffusely localized along the lateral cell membrane prior to epithelial barrier maturation. Following barrier maturation, Coracle becomes tightly localized to the septate junctions. Once Coracle is tightly localized at the junctions, it becomes necessary for the function of the barrier. We are currently examining whether ecdysone regulates the maturation of the epithelial barrier by regulating the localization of Coracle to the septate junctions.

ID #3987 Interpretation of the BMP Morphogen Gradient Patterning the Dorsal-Ventral Axis of the Zebrafish Embryo Hannah Greenfeld University of Pennsylvania, USA Bone Morphogenetic Protein (BMP) acts as a morphogen to pattern the dorsal-ventral (DV) axis in all vertebrates. In zebrafish, a gradient of BMP signaling activity across the embryo forms during gastrulation, where the highest level of BMP signaling is found ventrally and the lowest levels dorsally. BMP signaling specifies ventral cell fates, whereas dorsal tissue specification requires suppression of BMP signaling. However, it is unknown how cells along the DV axis interpret and translate distinct levels of BMP signaling into differential gene activation to specify cell fate. We are investigating how the BMP activity gradient is interpreted to generate discrete gene expression domains that specify distinct cell types. We have identified genes that are directly regulated by BMP signaling by performing RNA-seq on bmp7-/- embryos treated with a translation inhibitor and rescued with BMP2/7 protein injection. To assay how the BMP gradient is interpreted transcriptionally, we use a quantitative immunofluorescence assay of nuclear phosphorylated Smad5 (pSmad5), the transcriptional effector of BMP signaling. By measuring the spatial relationships between the nuclear pSmad5 signaling activity gradient and the boundaries of target gene expression, we have identified the threshold levels of pSmad5 that are required to induce expression of target genes. We have identified multiple genes with expression boundaries that correlate with distinct levels of pSmad5. To test if the differential pSmad5 levels act as thresholds for the induction of different genes, we examined the change in gene expression domain size in mutants that effect the shape of the BMP signaling gradient across the embryo. We found that a discrete threshold level of pSmad5 establishes the expression pattern of the target gene, foxi1. This work suggests that cells’ interpret distinct levels of pSmad5 to activate the expression of target genes.

ID #3989 Identifying ETR-1 as a novel player in the engulfment of physiological germline apoptotic cells Sofiat Atoba, Ruby Boateng, Anna Allen Howard University, United States ETR-1 is a conserved ELAV-type RNA-binding protein that plays a role in muscle development and has recently been identified by our lab as functioning during engulfment of germline apoptotic cells. etr-1(RNAi) animals exhibit a reduced brood size and an increase in physiological germline apoptotic cells. Co-depletion via RNAi of ETR-1 and CED-1, an engulfment gene, rescued the phenotypes associated with ETR- 1 depletion. This suggested that ETR-1 might be playing a role in engulfment through either one of the three established engulfment pathways or a novel engulfment pathway. The goal of our research is to investigate where ETR-1 functions during physiological germline apoptosis to ensure normal germ line function. We obtained a deletion allele etr-1(tm6221) that is homozygous lethal and balanced it over egl- 26(ku228). Characterization of etr-1(tm6221)/ egl-26(ku228) animals showed a reduced brood size compared to wild-type animals and an increase in germline apoptotic corpses, supporting our etr-1(RNAi) data. Conducting similar analysis of six engulfment mutants from two of the three established engulfment pathways, confirms the published increase in germline apoptotic corpses and for the first time we report the percentage of fertility decrease associated with each of these mutants. We generated double mutants of etr-1(tm6221) and various available engulfment mutants and are analyzing their fertility and quantifying the number of germline apoptotic cells using Differential Interference Contrast microscopy. If etr-1 and an engulfment gene are in different engulfment pathways, we should observe an increase in apoptotic cells in the double mutants compared to the single mutants. If the two genes act in the same engulfment pathway, we anticipate there would be no change or a suppression in the number of apoptotic cells compared to the single mutants. These studies will provide us with a deeper understanding of the mechanism of engulfment of germline apoptotic corpses.

ID #3990 Ecdysone limits wing imaginal disc regeneration through Broad Z1 Faith Karanja, Sara Weintraub, Subhashuri Sahu, Adrian Halme University of Virginia, USA As tissues develop, their regenerative capacity is often diminished. For instance, Drosophila melanogaster imaginal discs lose regenerative capacity at the end of larval development. The timing of loss of regenerative capacity coincides with the rapid increase in systemic levels of the steroid hormone ecdysone, a key coordinator of Drosophila developmental progression. We have showed that increasing systemic ecdysone levels by feeding larvae ecdysone is sufficient to limit regeneration in their imaginal tissues. Based on these data, we hypothesized that ecdysone signaling promotes changes in the imaginal disc epithelium that interfere with activation of regeneration pathways. We find that in the wing discs the expression of broad splice variant Z1 (br-Z1), an early transcriptional target of the pre-pupal ecdysone peak, begins at the same time that the discs lose regenerative capacity and inversely correlates with the regenerative capacity of the wing imaginal disc. When regeneration competent imaginal discs are damaged, Br-Z1 expression is delayed, which is consistent with the extension of the regenerative period in these larvae. In contrast, feeding larvae ecdysone produces premature Br-Z1 expression. By overexpressing Br- Z1 early in the imaginal disc development, we determined that Br-Z1 cell-autonomously suppress the expression of Wingless (Wg) and Dilp8, which are necessary for regeneration. Inhibiting broad expression in late third instar imaginal discs using broad RNAi allows tissues to activate regenerative pathways past the regeneration restriction point. We are currently exploring the mechanism through which BrZ1 limits regeneration and whether ecdysone signaling also functions to shape the regenerative response and duration through differential expression of broad splice variants. Our findings would provide insight into how endocrine signals act on tissues to regulate their regenerative competence. ID #3992 Regulation of the Migration and Function of Skeletogenic Cells Alexander George, Tyler McCann, Jia Song University of Delaware, United States The non-canonical Wnt/Calcium signaling (ncWnt/Ca2+) pathway has been shown to regulate cell motility and epithelial to mesenchymal transition, implicating its role in various developmental processes, such as gastrulation in zebrafish and proper organ formation in mice. We use the sea urchin Primary Mesenchyme Cells (PMCs), the skeletogenic cells in developing sea urchin embryos, to examine directed cell migration. The working hypothesis is that PMC migration and function are regulated by the ncWnt/Ca2+ signaling pathway. We have shown that disruption of elements downstream of the ncWnt/Ca2+ pathway using pharmaceutical drugs resulted in embryos with decreased skeletal length and altered PMC migration patterns. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) produced further developmental defects, including aberrantly enlarged cells and improper migration of the multipotent cells. Activation of PKC resulted in an anterior shift and decreased spatial expression domain of Eve transcripts, and a posterior shift of the spatial expression domain of Nodal. Eve is a key transcription factor that indirectly regulates PMC migration and function and Nodal establishes the embryonic dorsal-ventral axis. Interestingly, PMA treated embryos and microRNA-1 (miR-1) knockdown embryos exhibited similar skeletal phenotypes. MicroRNAs (miRNAs) are short, non-coding RNAs that bind to the 3’UTR of target mRNA transcripts to repress translation. MiR-1 is identified to bind to PKC and CDC42 of the ncWnt/Ca2+ signaling cascade, indicating a potential role in regulating the ncWnt/Ca2+ signaling pathway. We will investigate the direct regulation of miR-1 of PKC and CDC42 with dual luciferase assays and site-directed mutagenesis of the potential miR-1 binding sites. This study identifies the molecular mechanisms of how the ncWnt/Ca2+ pathway impacts the directed migration and function of PMCs and contributes to a better understanding of cell motility in the context of development.

ID #3993 Analysis of Upstream Regulatory Regions in Differentially Regulated Homeologs in Xenopus Laevis Samantha Phillips, Ronald Cutler, Mark Pownall, Margaret Saha College of William and Mary, United States of America The binding of trans-acting factors to the cis-regulatory regions of genes is an essential aspect of transcriptional regulation. Comparative analysis of the variation within upstream regions of a gene can reveal key regulatory regions. A recent allotetraploidy event in the model organism, Xenopus laevis, caused a whole genome duplication that provides an opportunity to study the effects of polyploidy in a vertebrate model system. Temporal expression patterns show differentially expressed homeologs, yet how this expression correlates with differences in upstream regions remains unclear. Here we focus on the Tweety (TtyH) gene, a chloride channel that was identified from an RNA- Seq experiment perturbing the Notch signaling pathway that resulted in homeolog specific expression in the S version of TtyH1. To explain overall expression pattern differences between the TtyH homeologs during Notch perturbation and normal embryonic development, we compared the 5KB and 10KB upstream regions of TtyH1 and the TtyH3 homeologs. Alignments between each homeolog pair are concentrated near the transcription start site. We performed a de novo motif enrichment on the upstream regions to generate hypotheses about transcription factors that potentially cause differences in expression. On the TtyH1 5KB upstream region, the L homeolog has a cluster of motifs associated with the sox1 transcription factor. Additionally, the TtyH1 10KB region contains a greater amount of motifs in the L homeolog than the S version. The L homeolog of the TtyH3 5KB upstream region contains 7 additional de novo motifs than the S homeolog where this trend is maintained when analysis is extended to the 10KB region. These results, paired with data on TtyH expression from the egg to swimming tadpole stages for X. laevis, suggest neofunctionalization. We intend to continue our investigation of upstream regions of homeologous genes that are differentially expressed in response to Notch signaling.

ID #3994 Insights into the role of pax-3 and the gene regulatory network that specifies hypodermal cell fates in C. elegans Margarita Correa-Mendez, David M. Eisenmann University of Maryland Baltimore County, USA The formation of the C. elegans skin (the hypodermis) is an excellent model to study how general categories of cell types acquire a specific cell fate. Early events in C. elegans embryogenesis induce hypodermal precursor cells to differentiate into either dorsal, lateral, or ventral hypodermal cells. Our work suggests that the specification of the three major hypodermal cell fates may involve cross-regulation between important transcription factors, many of which have homologs in humans and other model organisms. Previous work from our lab showed that the Paired-box transcription factor PAX-3 is involved in specifying the ventral (P cell) hypodermal fate during embryogenesis. We have shown that PAX-3 acts in the ventral hypodermis by repressing the expression of genes required to adopt a lateral hypodermal (seam cell) fate, specifically the GATA factor egl-18 and ceh-16/engrailed. In addition to our work on PAX-3, other authors have shown the involvement of REF-2, VAB-15 and HLH-3 in the specification of the ventral hypodermis. Our data from reporter expression analysis as well as RNAi experiments suggests that PAX-3 acts earlier during embryogenesis, upstream of these additional transcription factors. Finally, we have found through a yeast one-hybrid approach, that the T-box factor TBX-8 binds a cis-regulatory region of the pax-3 promoter. Combinatorial RNAi of tbx-8 and its redundant factor tbx-9 caused ectopic expression of pax-3 and other ventral reporters in the dorsal hypodermis. Our results indicate that the T-box factors TBX-8 and TBX-9 act in the dorsal hypodermis by repressing, either directly or indirectly, the expression of genes required to specify the ventral hypodermal fate. With our current data and ongoing experiments, we aim to extend the knowledge on the regulatory interactions that control these cell fate decisions earlier during development.

ID #3995 Utilizing the Drosophila tracheal system as a model for seamless tube formation in an undergraduate research course Jodi Schottenfeld-Roames Princeton University, United States Transformation of epithelial cells into complex functional organs is a critical step in development. Organs such as the lungs and kidney are composed of multicellular tubes (two or more cells attached to one other by junctions), whereas a large percentage of capillaries in the vertebrate vascular system are composed of seamless tubes (single cells that lack junctions). Although the morphogenetic events that help generate a multicellular tube can vary, it is clear how cells joined together by epithelial junctions create a membrane-bounded lumen; what is not obvious is how a single cell hollows out to create an internal lumenal membrane de novo. The Drosophila tracheal system is composed of a network of tubes that forms by specialized tip cells leading the migration of new branches and mediating their interconnection. Some of these tip cells become “terminal cells” that go on to form branched seamless tubes. Using terminal cells as a model, I have uncovered a number of cytoskeletal and membrane trafficking regulators that help orchestrate this seamless cell-to-tube transformation. In previous work, I showed that the dynein motor complex is one major contributor to de novo lumen formation, however the dynein-associated cargo required to generate a seamless tube has been largely uncharacterized. Candidate RNAi screens to identify factors that might crosstalk with the dynein motor complex to promote apical membrane formation have served as the foundation for independent undergraduate research projects, as well as a 60-person undergraduate research course. Using this approach, we have uncovered several genes including, Syntaxin1A, Syntaxin5, and Syntaxin18 that act at distinct steps in the secretory pathway and have important inputs into both seamless tubulogenesis and branch formation in terminal cells. Efforts to determine the role of these proteins, as well as other secretory pathway components during terminal cell morphogenesis are currently underway.

ID #3996 A Common Genetic Etiology for 2,3 syndactyly and Maladaptive Behaviors Audrey Chang1, Sara Pirzada2, Siddharth Srivastava3, Nirmal Singh4, Marco A Grados1 1Johns Hopkins University, Baltimore, MD, USA; 2Shaikh Khalifa bin Zayed Al Nahyan Medical and Dental College, Lahore, Pakistan; 3Harvard University, Boston, MA, USA; 4All India Institute of Medical Sciences, New Delhi, India

Background. Cornelia de Lange Syndrome (CdLS), a rare congenital genetic disorder characterized by dysregulation in cohesin and transcription, results in CNS dysgenesis and limb malformations. Previous reports associate 2,3 syndactyly, a common feature in CdLS, with autism features, hyperactivity, and mood liability/self-injury. An exploration of common gene sets for 2,3 syndactyly and maladaptive behaviors is conducted.

Methods. A search for genes implicated in 2,3 syndactyly was conducted. Identified gene sets were catalogued for CNS expression and Gene Ontology (GO) pathways using GeneCards and the Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. To test for overrepresented pathways, the Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System reported probabilistic overrepresented pathways.

Results. Gene sets etiologically related to 2,3 syndactyly are associated with the 3p21.31 locus. Within this locus, 127 genes were catalogued for CNS expression and KEGG pathway representation. The major genes were RHOA, GNAI2, PLXNB1, SEMA3B, DAG1, NPRL2, LAMB2, PRKAR2A, PTH1R, and SEMA3F and the main KEGG pathways were locomotion, signal transduction, response to stimulus, and cell communication. The apoptosis and cell death pathways were among overrepresented pathways. 3p21.31 genes in this pathway are CCR1, CCR5, CCR2, CCR9, CXCR6, and CCR3. Interestingly, these genes are also involved in neuroplasticity, learning and memory and the apoptosis pathway (2,3 syndactyly).

Conclusions. 3p21.31 genes play a causative role in 2,3 syndactyly. Within this locus, gene sets with overrepresented pathways include the CCRx genes, which are involved in immune signaling (chemokines). However, they are also overrepresented in the apoptosis pathway (2,3 syndactyly) as well as suppression of cortical plasticity, learning and memory (maladaptive behaviors), plausibly providing the etiological link between 2,3 syndactyly and maladaptive behaviors.

ID #3997 Altering Levels of the Histone Demethylase Gene, Jarid1C, Leads to Changes in Stem Cell Traits Theresa Geiman, John Tehan, Patrick Frick Loyola University Maryland, USA Jarid1C is a histone demethylase enzyme that removes the acvite epigenetic mark of histone H3K4 tri-methylation from chromatin. It is an important gene in mammalian development since many mutations of Jarid1C have been found in human with X-linked Intellectual Disability (XLID). Epigenetic modifier proteins such as Jarid1C are important for controlling gene expression through generation of heterochromatin or euchromatin at specific chromosome loci. Stem cell characteristics are closely linked to the precise control of gene expression in cells at any given time, with incorrect gene expression possibly leading to severe developmental defects or developmental arrest. We have altered levels of the Jarid1C gene in an in vitro mouse model of stem cell differentiation and evaluated the stem cell traits such as stem cell gene expression, cloning, soft agar, and alkaline phosphatase activity. We have also used a stem cell reporter system and seen changes in drug resistance under the control of an important stem cell gene promoter. Better understanding of the function of Jarid1C in stem cells by altering expression levels provides valuable information aout the mechanisms and genes that control the transition from a stem cell to a differentiated cell state in mammalian cells.

ID #3999 The role of p53 in mediating Kif20b mutant neuron morphology defects Madison Hecht University of Virginia, United States Some of our brain’s most powerful and innate processes occur in the cerebral cortex, including spatial perception, abstract reasoning, and judgement. To perform such functions, proper growth of the cortex requires precise divisions of neuroepithelial stem cells (NSCs), which first expand the stem cell pool and then produce post-mitotic neurons through symmetric and asymmetric divisions. When divisions are disrupted, conditions such as microcephaly, or small brain, can occur. Our lab discovered a novel mouse model for microcephaly, caused by mutation of the Kinesin-6 family member Kif20b, a plus-end directed kinesin motor protein known for its involvement in cytokinesis. Exciting new experiments demonstrate that p53, a cell cycle regulator, plays a pivotal role in Kif20b-/- microcephaly. By crossing the p53-/- and Kif20b-/- mouse lines, we discovered that p53 co-deletion rescues the apoptosis and decreased cortical size in Kif20b-/- mice.

Equally important to proper cortical functioning is the formation of correct neuron morphology. Specifically, embryonic cortical neurons must polarize by growing dendrites and axons to receive and transmit signals respectively. In Kif20b mutant cultures with wild-type p53, we found decreased numbers of neurons, defects in neuron polarization and increased DNA damage in neurons compared to controls. Interestingly, in Kif20b;p53 double mutant cultures, we found no differences in the number, polarization and DNA damage of cortical neurons. Additionally, because of Kif20b’s role in cytokinesis we looked for differences in the formation of binucleate stem cells and neurons, as cells that fail cytokinesis can undergo furrow regression. However, neither the single Kif20b mutant nor Kif20b;p53 double mutant NSC cultures showed differences in the number of binucleate cells formed compared to controls. Our data suggests that defects in neuron polarization and DNA damage in Kif20b-/- neurons may occur due to p53 activation.

ID #4000 Lhx1 role in regulation of neuromesodermal progenitors cell fate NIKOLAOS P. MANDALOS Cancer and Developmental Biology Laboratory, CCR, NCI-Frederick, Frederick, MD, US The primitive streak (PS) is generated from epiblast stem cells in the egg cylinder, at early embryonic development and it establishes the spatiotemporal formation of germ layers and bilateral symmetry. At the stage of anterior primitive streak (PS) patterning, a bipotent cell population arises, called neuromesodermal progenitors (NMPs), that contribute to the development of spinal cord and paraxial mesoderm in the mouse embryo. Recent studies propose that the molecular mechanisms that control the generation and differentiation of NMPs may differ from the ones acting at the posterior primitive streak and anterior neural plate. In an effort to study this distinguished embryonic cell population, an in vitro cell model of NMPs has been established from embryonic stem cells (ESCs) and epiblast derived stem cells (EpiSCs), upon induction of Wnt signaling. In vitro derived NMPs co-express the mesodermal and neural markers, Brachyury (T/Bra) and Sox2 respectively, mimicking the cells in the epiblast associated with the primitive streak. We have performed microarray screening of in vitro derived NMPs to better define molecular markers that govern the differentiation of EpiSCs towards NMPs, revealing the upregulated expression of Lhx1 transcription factor. EpiSCs in which Lhx1 expression has been blocked with siRNA fail to form NMPs, even after the activation of Wnt signaling at precise time point. These findings suggest that the expression of Lhx1 is essential for the transition of EpiSCs towards NMPs. We hypothesize that Lhx1 controls the cell fate of EpiSCs into NMPs by blocking the expression of the homeodomain-containing transcription factor Otx2, a well characterized epiblast cell marker. Lhx1 overexpression in EpiSCs will further reveal its role in epiblast pluripotency loss and NMP and PS formation, as NMPs could prove to be a cell type useful for tissue engineering and regenerative medicine applications.

ID #4002 Going Green: Illuminating Schwann Cell Behaviors and Contributions in Regenerating Axolotl Limbs. Sarah Elayne R. Hryniewicz, Andrew V. Marinich, Karen Crawford St. Mary's College of Maryland, USA The purpose of this study was to take advantage of the green fluorescent protein (GFP) axolotl to visualize Schwann cell activity and contribution to stump and regenerated limb elements in Ambystoma mexicanum. To accomplish this, large segments of GFP upper arm brachial nerve axons along with their supporting glial tissues were grafted alongside intact nerves in comparable regions within sibling white hosts. Grafts were allowed to heal for a week or more and then host animals were amputated at either the mid upper arm, through the grafted GFP positive tissue, or more distally through the mid-lower arm. Our rationale was that the first scenario would allow for visualization of GFP positive Schwann cells as they contributed to the blastema and regenerating limb, while the second would explore whether the presence of a distal level amputation wound and regenerating blastema might stimulate Schwann cell outgrowth and/or migration. Our results revealed a propensity for grafted Schwann cells to not only migrate in both intact and regenerating limbs, but also proliferate and myelinate the neurons that innervate those new limbs. Because of these findings, we supplemented our study to explore whether the grafted Schwann cells could respond to nerve growth factor. To do this, Affigel Blue beads soaked in mouse nerve growth factor (NGF, 1µg/ml) were inserted into either intact or regenerating sibling white hosts that had previously received GFP nerve grafts. Beads were inserted in a variety of orthogonal positions to explore possible axial differences in Schwann cell response to the presence of NGF. Beads soaked in phosphate buffered saline were used as control. These experiments are currently underway. Additional examples from our laboratory highlighting the use of GFP donor to white host siblings to explore questions of pattern formation and graft stem cell potency will be included.

ID #4004 From GAD Regulation to Synthetic Biology Grace Solini, Alexander Chalpin, Margaret Saha College of William and Mary, United States GABAergic inhibitory neurotransmission is a necessary aspect of a functioning vertebrate nervous system. However, the transcriptional mechanisms underlying GABAergic neuronal regulation remains largely uncharacterized. Using the allotetraploid model Xenopus laevis, we examined the transcriptional regulation of glutamic acid decarboxylase (GAD67), which catalyzes the rate-limiting step in GABA synthesis. Putative cis-regulatory elements were identified by in silico comparison of upstream regions between Xenopus GAD67 and syntenic vertebrae homologs. These results were utilized to clone EGFP constructs for functional transgenic experiments, which identified presumptive regulatory regions upstream of the GAD67 coding sequence. We also characterized variation among different wild caught individuals and are now using motif enrichments to identify hypothetical transcription factor profiles for both homeologs. This will allow us to form conjectures regarding the transcriptional regulation of differential GAD67 homeolog expression, and eventually piece together the complex regulatory mechanisms of overall GAD67 expression. In addition, we are also pursuing the design of a synthetic morphogen gradient capable of relaying spatial information to cells through a concentration gradient of signaling molecule. We will create two cell populations, a sender population flanked against a receiver population, where induced sender cells drive production of signaling molecule VGEF at a given rate. This will form a concentration gradient of VGEF along the receiver cell population, which through a VGEF-MESA receptor will induce different responses in receiver cells depending upon the concentration of signaling molecule. This will result in the creation of different cell populations (marked by different responses) based upon the spatial location of different cells, effectively mimicking the remarkable feature of cell-directed spatial organization seen in morphogen gradients.

ID #4008 Abscission defects in neural stem cells are associated with p53-dependent microcephaly Jessica Little, Noelle Dwyer University of Virginia, USA Proliferation and cell death pathways are tightly regulated in neuroepithelial stem cells (NSCs) in order to produce a cerebral cortex of normal size and function. In a novel mouse model for microcephaly discovered by our lab, the mutation of the kinesin Kif20b results in reduced brain size. In cell lines, Kif20b regulates the last step of cytokinesis, abscission, during which a structure called the midbody mediates the last separation between two cells. NSCs undergo a polarized form of cytokinesis within the neuroepithelium that is poorly understood. Kif20b localizes to midbodies in NSCs, and interestingly, we found midbody abnormalities in NSCs along with increased apoptosis in Kif20b-/- cortex. Our hypothesis is that the loss of Kif20b causes impaired abscission that results in apoptosis in a subset of NSCs, depleting the progenitor pool and impairing neurogenesis.

To test our model for the etiology of Kif20b-/- microcephaly, we attempted to inhibit apoptosis by crossing the Kif20b mutant mouse to a knockout for the pro-apoptotic Trp53 (gene encoding the tumor suppressor p53). We found that p53 deletion rescues apoptosis in Kif20b-/- mice. Strikingly, inhibition of apoptosis through p53 deletion is able to fully restore cortical thickness and dramatically improves lifespan in Kif20b mutants. However, previously observed midbody abnormalities are not rescued in Kif20b;p53 double mutant mice and new midbody defects are revealed, suggesting that abscission defects are upstream of p53 activation. This data is the first indication that defects in cytokinetic abscission in NSCs could activate a p53-dependent apoptotic pathway to cause microcephaly.

ID #4009 Urban Immersion: A Staircase of Discovery in Developmental Biology Research Alana O’Reilly Fox Chase Cancer Center, USA Urban Immersion has dual goals: 1) to recruit and retain under-represented students in cancer research and 2) to address experimentally how a critical social determinant of health, diet, impacts cancer pathways of relevance to under-represented minority communities. The program is built on underlying principles of citizen science, where members of the public contribute valuable ideas and data to ongoing research projects. Urban Immersion combines a series of successful pilot programs into a comprehensive citizen science training model to promote initial research literacy for thousands of students representing communities that are dramatically under- represented in STEM research careers. Completion of each step of the program provides iterative learning, preparing students for any career involving problem solving or data collection. Students who complete all levels of the program are well-prepared for college STEM majors, having the research cultural currency, experience, and inclusion in the scientific community to succeed. Parallel, integrated programs for students and teachers provide a feedback loop of training, enabling participation of hundreds of students in authentic research. This low-cost, high throughput approach where HS students provide data for cancer research projects will reduce the time required to identify diet-derived compounds with biological activity against cancer pathways in a relevant model system (Drosophila melanogaster). The program is nearly infinitely scalable, as any signaling pathway or genetic mutation that is conserved between fruit flies and humans can be screened against millions of compounds derived from diet. This new approach to scientific discovery engages high school students in research while identifying new diet-based methods for improving health. ID #4042 Testing the R1 peptide on kidney development in zebrafish Tejas Mahadevan Padmanabhan, Vishal Keshari, Basma Adeeb, Elon Denio, Elizabeth Stackhouse, Michael Belko, Cuong Diep Indiana University of Pennsylvania, United States Almost 10% of American adults suffer from some sort of chronic kidney disease and current therapies for end-stage kidney disease are not ideal. Unlike humans, zebrafish can regenerate new kidney tissues after injury using stem cells that express the lhx1a transcriptional activator. lhx1a is also expressed in stem cells during kidney development in zebrafish and other organisms, including humans. Mutations in lhx1a result in kidney agenesis, indicating that its function is essential for kidney formation. To understand the molecular function of lhx1a, we carried out a genetic selection for interacting proteins. We found that lhx1a interacted with itself, suggesting homodimerization of lhx1a. Deleting the LIM domain of lhx1a enhanced dimerization. Others have shown that deleting the LIM domain of lhx1a also activated the protein, converting into an active transcriptional activator. Therefore, we hypothesize that the LIM domain negatively regulates homodimerization and activation of lhx1a, and thus activation of lhx1a requires dimerization. To determine whether lhx1a dimerization is physiologically relevant in vivo, we performed a different genetic selection to identify small random peptides that bind to lhx1a. We rationalized that such peptides binding to lhx1a would affect its dimerization and function (negatively or positively). Our pilot selection resulted in one peptide (called R1) that bound to lhx1a. Next, we will inject one-cell embryos with mRNA encoding the R1 peptide, which is fused to an internal loop of EGFP for stabilization and proper presentation. If the R1 peptide binds to lhx1a and alters its dimerization and/or function, then we anticipate getting a kidney defect such as edema and misexpression of kidney genes. If confirmed to affect kidney development, the R1 peptide could serve as a tool to dissect the molecular mechanism of lhx1a in regulating kidney stem cells during development and regeneration.

ID #4055 Intronic enhancers impact exon usage across the genome of the developing human brain Christine Charvet, Paul Soloway Cornell University, USA It is well known that enhancers play a crucial role in regulating expression of target genes but whether enhancers play a role in generating isoforms is less clear. A large number of enhancers are intronic and these intragenic enhancers may have a cis- regulatory impact on isoforms. We identified putative intronic enhancers profiled from a combination of histone modification patterns and DNA accessibility annotations. We use RNA seq data to assess whether intronic enhancers differentially impact the expression of exons upstream and downstream to enhancers across the genome of human fetal brains and we validate our results across cell lines (i.e., leukemia, embryonic stem, lymphoblastoid cells). RNA seq data were obtained from previously generated data as part of the ENCODE project and from the Allen Brain Institute. We found that exon expression upstream to intronic enhancers is significantly reduced relative to those that are downstream in the human fetal brain and across cell lines. Intronic enhancers also have a lasting impact on exon usage over the course of development. Thus, enhancers may generate phenotypic diversity by regulating gene expression as well as isoforms.

ID #4070 Cellular analysis of ear pinna development in murid rodents Shishir Biswas, Corin Hacker, Ashley Seifert University of Kentucky, USA Models of epimorphic regeneration, such as zebrafish fins and salamander limbs, have long been studied through the lens of development. Deconstructing how specific cell lineages contribute to regenerated tissue requires an understanding of cell lineages during organ formation. African spiny mice (Acomys cahirinus) have emerged as a bonafide model of epimorphic regeneration as they are capable of re-growing excised body skin and regenerating musculoskeletal tissue in the ear pinna while closely related murids such as mice heal identical injuries via fibrosis. Since most mammals have external ear pinnae, our 4 mm ear punch assay is particularly useful for assaying regenerative ability across species. Not surprisingly, our understanding of ear pinna development is almost non-existent. The few studies that have characterized ear pinna development have focused specifically on embryonic time points and neglect postnatal development. This study provides a cellular characterization of late gestational and postnatal ear pinna development in Acomys and Mus. Our analysis demonstrates that ear pinna development is largely similar in these species, allowing us to use Mus as a general developmental model. Previous studies have suggested that the entire mesenchyme of the pinna is neural crest derived. Using Wnt1-Cre;ROSAmT/mG reporter mice to lineage trace neural crest we found that muscle is not of neural crest origin. Finally, we used the naturally occurring short ears mouse mutant which have a mutation in Bmp5 to investigate how Bmp-signaling regulates normal pinna development. Although short ear mice have truncated ear pinnae, cellular analysis shows normal tissue architecture with regions devoid of elastic cartilage that is replaced with adipose tissue. Interestingly, Oil Red O lipid and Fabp4 adipocyte staining of heterozygous and null mice suggest that Bmp5 may play a role in differentiation of an adipochondrocyte precursor.

Funding: UK Biology, NSF and OISE (IOS-1353713)

ID #4086 Arabidopsis TSO1 and MYB3R1 form a regulatory module to coordinate cell proliferation with differentiation in shoot and root Zhongchi Liu, Wanpeng Wang Univ. of Maryland, USA Fundamental to plant and animal development is the regulated balance between cell proliferation and differentiation, a process intimately tied with cell cycle regulation. In Arabidopsis, mutations in TSO1, whose animalhomolog is LIN54, resulted in severe developmental abnormalities both in shoot and root including shoot meristem fasciation and reduced root meristematic zone. The molecular mechanism that could explain the tso1 mutant phenotype is unknown. Through a genetic screen, we identified 32 suppressors that map to the MYB3R1 gene encoding a conserved cell cycle regulator. Further analysis indicates that TSO1 transcriptionally represses MYB3R1, and the ectopic MYB3R1 activity mediates the tso1 mutant phenotype. Since animal homologs of TSO1 and MYB3R1 are components of a cell cycle regulatory complex, the DREAM complex, we tested and showed that TSO1 and MYB3R1 co-immunoprecipitated in tobacco leaf cells. Our work reveals a conserved cell cycle regulatory module, consisting of the TSO1 and MYB3R1, for proper plant development.

ID #4087 Assessment of mCherry-fused ephrinB as a live imaging tool Jaeho Yoon1, Hee-Jun Cho1, Laura Knapik1, Moonsup Lee1, Jian Sun1, Julien Homble1, Olivia Fritz2, Ira Daar1, Yoo-Seok Hwang1 1DSTL / CDBL/ CCR/ NCI/ NIH, USA; 2University of Pittsburgh/ School of Pharmacy, USA Although there are hundreds of papers describing ephrinBs’ signaling, the scientific community lacks any reliable live imaging tool for these proteins. There are a number of technical issues which may thwart such efforts for devising one, including the existence of multiple protein cleavages and complex processing of these proteins, which may confound the interpretation of the fluorescent signal in terms of actual peptide identity or membrane-topological location. In addition, modifications of the protein may disrupt the appropriate scaffolding activity and affect the ability to signal in a bi-directional manner.

Here, we attempted to devise a live imaging tool of ephrinB1 and ephrinB2 to visualize the proteins in vivo. We chose 5 positions in the proteins (4 positions in the ectodomain and 1 position in the cytoplasmic domain) according to known cleavage information, and inserted mCherry at these positions. The 5 different constructs for ephrinB1 and 2, were over expressed in Xenopus laevis embryos. At stage 15-19 (neural), embryos were transversely vivisectioned and analyzed by confocal microscopy using time lapse imaging. At the same time, we performed immunoprecipitation of these overexpressed mCherry-fused proteins with known ephrinB1 interacting partners, flotilin and EphB2 receptor in the Xenopus embryonic system.

In addition, we tested phosphorylation of ephrinBs in response to FGF receptor binding. In high-dose overexpression Cyto-B1mCh, shows significant loss of cell-dissociation effect that normally observed with wildtype ephrinB1. In addition, Cyto-B1mCh lost certain degree of tissue separation role of ephrinB1, which was tested in rescue of ephrinB1-knockdown in the ecto-mesoderm boundary of gastrula embryo. In Co-IP analysis with wildtype ephrinB1, Cyto-B1mCh shows only little avidity, representing the attenuation or loss of functional property of ephrinB1 probably was resulted from impaired oligomerization.

ID #4099 TBC1d24-ephrinB2 interaction regulates contact inhibition of locomotion in neural crest cell migration Jaeho Yoon, Ira Daar National Cancer Institute - Frederick, USA Although Eph-ephrin signalling has been implicated in the migration of cranial neural crest (CNC) cells, it is still unclear how ephrinB transduces signals regulating this event. We provide evidence that TBC1d24, a putative Rab35-GTPase activating protein (Rab35 GAP), complexes with ephrinB2 via the scaffold Dishevelled (Dsh), and mediates a signal affecting contact inhibition of locomotion (CIL) in CNC cells. Moreover, we found that in migrating CNC, the interaction between ephrinB2 and TBC1d24 negatively regulates E-cadherin recycling in these cells via Rab35. Upon engagement of the cognate Eph receptor, ephrinB2 is tyrosine phosphorylated, which disrupts the ephrinB2/Dsh/TBC1d24 complex. The dissolution of this complex leads to increasing E-cadherin levels at the plasma membrane, resulting in loss of CIL, and disrupted CNC migration. Our results indicate that TBC1d24 is a critical player in ephrinB2 control of CNC cell migration via CIL.

ID #4116 Understanding the molecular mechanisms of human craniofacial disorders associated with TWIST mutations by studying Caenorhabditis elegans Arwa Alsubait, Ann Corsi Catholic University of America, Washington D.C, USA Twist family members are highly conserved basic helix-loop-helix transcription factors that are important for mesoderm development in all animals.Twist either forms homodimers or heterodimers with E protein homologs. The dimers regulate the expression of downstream target genes by binding an enhancer element called the E- box. In mammals, there are two paralogous genes, TWIST 1 and TWIST 2.Mutations in both genes have been identified in patients with craniofacial disorders. Sweeney-Cox syndrome (SwCoS) and Barber-Say syndrome (BSS) are congenital craniofacial anomalies that arise due to mutations at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1 and TWIST2, respectively. To understand the molecular basis of SwCoS and BSS, we chose Caenorhabditis elegans as a model organism. Caenorhabditis elegans has a single Twist homolog known as HLH-8. HLH-8 is expressed in a subset of mesodermal tissues that include the head mesodermal cell (hmc), enteric muscles (ents), and vulval muscles (vms). We study the Notch ligand gene, arg-1, as an HLH-8 target gene. The 385 bp arg-1 promoter region contains three different E-box DNA sequences (E1, E2, E3) that are sufficient for maintaining the full expression pattern of arg- 1 in the three mesodermal tissues. Transgenic animals with HLH-8 alleles that mimic the mutations found in SwCoS and BSS patients showed arg-1::gfp expression in the hmc and the ents but not in vms. To understand the variability in arg-1 expression, we examined whether the specific sequence of the E-box matters. Our results showed a significant decrease in the hmc arg-1::gfp expression level upon swapping the E-boxes (E3, E2, E3) vs. (E1, E2, E3). Additionally, we found that swapping the E1 sequence with E3 sequence affects vms arg-1::gfp expression in hlh-8 mutants that analogous to the BSS TWIST2 mutants more than SwCoS TWIST1 mutants, suggesting that hlh-8 mutants tolerate the E-box specific sequences differently.

ID #4117 Genetic evidence that Sonic Hedgehog does not act as a morphogen to specify digit identities Jianjian Zhu National Cancer Institute-Frederick, USA Sonic Hedgehog (Shh) signaling from posterior limb bud cells (ZPA) controls both digit identity and number. Shh knockout (null) limbs form a single digit-like structure. Classic models propose that Shh acts as a mophogen (signal concentration or duration sensed) to specify distinct digit identities, with more posterior digits requiring the highest exposure. We previously determined the temporal requirement for Shh, by timed deletion of a conditional Shh (floxed) allele (Shh CKO) with a tamoxifen-dependent Cre. This resulted in digit loss that reflected the normal digit appearance order, rather than a posterior-anterior progression. We proposed a biphasic model that Shh is only required transiently at an early stage to specify digit progenitors (~8hrs), but sustained temporal activity regulates cell survival/expansion enabling all digits to form. To test this model and determine how Shh acts over such a short time window to specify digits, we have now deleted Bax/Bak to determine if restoring cell survival can substitute for late Shh function. When Shh activity is terminated 2-4 hours after onset, 100% of Shh CKOs with normal Bax function have a Shh-null (KO) phenotype. However, if Bax/Bak are deleted, 50% of Shh CKOs form up to 5 digits with completely normal morphology. Genetic lineage tracing of Shh-responding cells shows that only posterior digit progenitors (4,5) have responded directly to Shh in this short time window, even though all 5 digits can be rescued by Bax/Bak removal. To test if downstream relay signals play a role in specifying anterior digits, we have enforced Shh response in “ZPA” cells in Shh null/KO embryos. Our results support a model in which an early transient Shh pulse suffices to specify distinct digit types. During this transient phase, posterior digit progenitors (4,5) respond to Shh directly via short-range autocrine signaling, while anterior digits (1-3) are specified indirectly, via relay signals induced by Shh.

ID #4145 Exploration of Asymmetrical Gene Expression within Ciona Intestinalis Cameron Tumey Swarthmore College, USA The processes underlying left-right asymmetry are highly conserved across a wide range of species. Nodal signaling, H+/K+ ATPase dependent ion flux and ciliary flow are required for lateral asymmetry in both protostome and deuterostome clades yet details regarding how these initial processes lateralize organ morphogenesis remain poorly characterized. We use the invertebrate chordate Ciona intestinalis to investigate asymmetric organ development along the left-right axis. In Ciona, the heart and endoderm are positioned to the right and this asymmetry arises simultaneously. Surprisingly, previous research indicates that Ciona heart asymmetry is dependent on ion flux but does not require Nodal signaling. To understand the link between ion flux and lateralized organ morphogenesis, we have begun to characterize laterally asymmetric gene expression in Ciona embryos. By sequencing RNA in thin sections spanning the left-right axis, we have established a list of 19 candidate genes displaying strongly lateralized expression including orthologs to Cspp1, Gpr161, Taf9, Nup155, Lars2, FoxC, Siah1B, and Prmt9 that are strongly expressed on the right side of the embryo and Tor1B, Sept9, Klhl4, Mef2, Pitx, Nodal, sFzd(Crd), Dnah8, Crkl, Lrrc46, and Kat2a that are strongly expressed on the left side. We have confirmed six of these predicted expression patterns through in-situ hybridization. These studies have revealed that many of the candidate genes are expressed in the trunk lateral cell lineage, a group of mesodermal cells that migrate extensively in the larval head and differentiate into blood and muscle. We have also begun to characterize the dependence of these candidate genes on ion flux using the H+/K+ ATPase inhibitor omeprazole. The further characterization of asymmetrically expressed genes should provide critical insights into the molecular mechanisms driving heart and endoderm asymmetry within Ciona and vertebrate embryos.

ID #4152 Crosstalk between the Wnt and Fgf Pathways in the Formation of Neuromesodermal Progenitors (NMPs) Matthew Koury, Mark Kennedy, Ravindra Chalamalasetty, Robert Garriock, Nikita Salker, Jessica Grisez, Matt Anderson, Sara Thomas, Terry Yamaguchi NCI-Frederick, NIH, Frederick MD 21702, United States The Wnt/b-catenin pathway is a highly conserved pathway that controls embryonic development, often through the regulation of stem cell self-renewal. We are studying how this Wnt pathway regulates the formation of neuromesodermal progenitors (NMPs) during early embryogenesis. NMPs arise in the anterior primitive streak at E7.5, and generate the neural and paraxial mesoderm progenitors that form the spinal cord and the axial skeleton, respectively. Another pathway that is crucial for the formation of NMPs is the Fibroblast Growth Factor (FGF) Pathway. Mutations in genes in either the Wnt or Fgf pathways lead to similar axial truncation phenotypes however it remains unclear how these pathways interact. We propose that the Zinc-finger transcription factors Sp5 and Sp8 could mediate this interaction. Null alleles of Sp5 and Sp8 lead to downregulation of Fgf target gene expression both in vivo and in vitro. Conversely, when Sp5 and Sp8 are overexpressed, Fgf target genes are upregulated. ChIPseq analysis suggests that these proteins might regulate Fgf genes directly as Sp5 and Sp8 bind presumed regulatory elements of Fgf4 and Fgf17. Given that we have previously established that Sp5 and Sp8 are effectors of the Wnt pathway, these results suggest that the Fgf pathway is directly activated by Wnt signals.

ID #4161 Cerebellar folding through Differential Expansion Andrew Lawton1, Tyler Engstrom2, Daniel Rohrbach3, Jonathan Mamou3, Jennifer Schwarz2, Alexandra Joyner1 1Sloan Kettering Institute, United States; 2Syracuse University, United States; 3Riverside Research, United States During human development the cortex and cerebellum fold into intricate and robust patterns increasing the surface area and compartmentalizing the neural circuitry. Models, such as bilayer wrinkling due to differential expansion between the inner and outer layers have been proposed to explain brain folding. Yet the cellular and physical processes that drive folding are unknown. The murine cerebellum is a genetically tractable model to study the development of brain folding. The cerebellum progressively folds, creating 8-10 primary lobules. Growth is primarily driven by proliferation of granule cells within an external granule layer (EGL) on the surface prior to their migration into the inner mass. We found that at the initiation of folding the EGL has uniform cell sizes, shapes, and proliferation throughout its length. The EGL also has a uniform distribution of fibers from specialized glial cells. In addition, preliminary live-imaging of ex-vivo slice cultures showed no regional differences in cell movement mechanics. Therefore the EGL is a uniformly expanding layer. Consistent with bilayer wrinkling models, we found differential expansion between the outer EGL and the inner cortex when folding is initiated. Unlike these models, we found the EGL is stiffer than the underlying cortex, and the EGL thickens such that that its thickness is out-of-phase with its surface height during the initiation of folding. Using an alternative micro-structure model we can capture the initiation of folding driven by differential expansion, and the out-of-phase layer thickness. Our results thus show that the cerebellum uses uniform cell behaviors within the EGL to create a differential expansion between the inner cortex and the outer EGL, which drives folding.

ID #4165 The DEP domain protein LET-99 regulates spindle positioning downstream of multiple polarity cues in C. elegans Lesilee Rose, Malgorzata Liro, Kari Price, Jocelyn Alvarado University of California-Davis, USA Asymmetric divisions produce daughter cells with different fates, and thus are critical for development. During such divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types a cortically localized complex consisting of Gα, GPR, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynein, which exerts pulling forces on astral microtubules to position the spindle. In the C. elegans one-cell embryo, the conserved PAR polarity proteins organize in to anterior and posterior domains that regulate the asymmetry of cortical GPR/LIN-5. A key intermediate in this process is LET-99, a DEPDC1 family protein, which is localized in a banded pattern by the PAR proteins. In contrast, at the 4-cell stage in the EMS cell, spindle alignment depends on partially redundant Wnt and MES-1/SRC-1 signaling pathways initiated from a neighboring cell. The mechanisms connecting cell signaling to the spindle positioning machinery are unknown. Recently, our analysis of temperature sensitive mutants revealed that LIN-5 is required for EMS spindle positioning, potentially in a Gα-independent manner. LET-99 acts in the MES-1/ SRC-1 pathway for EMS spindle positioning. To further elucidate the MES-1/SRC-1 pathway, we tested other candidate genes for a role in EMS spindle orientation. PAR proteins are present in EMS, but are localized in an inner/outer pattern rather than with the spindle axis. We found that PAR-1 may act in the Wnt pathway, but we found no role for PKC-3. In addition, we found that the PAR-1 related kinase, PIG-1, acts in the MES-1/SRC-1 pathway, as does the small G protein Rac. Rac and LET-99 are also involved in furrowing during cytokinesis, and LET-99 can associate with Rac in vitro. We are carrying out additional analyses to determine the relationship between LET-99 and Rac during cytokinesis and EMS spindle orientation.

ID #4179 Par3 integrates planar cell polarity (PCP) signaling to regulate hair bundle morphogenesis Andre Landin Malt, Zachary Dailey, Julia Holbrook Rasmussen, Steven Gercken, Maxwell Madani, Yuqiong Zheng, Xiaowei Lu University of Virginia School of Medicine, United States During development, cochlear hair cells form Vshaped hair bundles(HB) that are uniformly oriented. This process is controlled by the coordinated action of tissue-level PCP signaling, and a hair cell-intrinsic polarity machinery. This machinery is also required for HB morphogenesis through basal body positioning and consists of RacPAK signaling and LGN/Gαi, an evolutionarily conserved complex for mitotic spindle orientation. To further understand how PCP signaling is coupled with HB morphogenesis, we investigated the role of Par3, a PDZ scaffold protein and an evolutionarily conserved regulator of cell polarity, in hair cell. We found that Par3, but not its binding partner Par6, is asymmetrically localized at intercellular junctions in the cochlea during HB morphogenesis. Indeed, analysis of ear specific Par3 knockout (Par3cKO) cochleae revealed defects in both HB orientation and morphogenesis. We first tested whether Par3 promotes asymmetric cortical localization of LGN, like its role in mitotic spindle orientation. To our surprise, asymmetric distribution of LGN was unaffected in Par3cKO hair cells. Instead, levels of PAK activity were decreased in Par3cKO, suggesting a positive role of Par3 in regulating RacPAK signaling. Co-IP experiments confirmed that Par3 forms a complex with Tiam1, a guanine nucleotide exchange factor for Rac, in the ear. Moreover, Tiam1 localization was altered in Par3cKO hair cells. We further demonstrated that Par3 regulates microtubule organization and its stable attachment at the lateral hair cell cortex. Taken together, the results demonstrated a critical function of Par3 in regulating basal body/kinocilium position through the Tiam1-Rac-PAK axis. At the tissue-polarity level, we found that Par3 and the core PCP molecule Vangl2, mutually regulate each other subcellular localization. Thus, we propose that Par3 integrates tissue-level and cell-intrinsic PCP signaling pathways to regulate HB morphogenesis

ID #4181 Essential Role of the channel kinase TRPM7 in Cochlear Morphogenesis Andre Landin Malt1, Maxwell Madani1, Mandar Kulkarni1, Arielle Hogan1, Bimal Desai2, Xiaowei Lu1 1Department of Cell Biology, University of Virginia Health System, USA; 2Department of Pharmacology, University of Virginia Health System, USA Planar cell polarity (PCP) signaling mediates multiple aspects of cochlear development, including elongation of the cochlear duct and the uniform orientation of Vshaped hair bundles atop sensory hair cells. Recent advances have uncovered mult iple pathways for intercellular PCP signaling in the developing cochlea, consisting of the core Frizzled/PCP pathway, the PTK7 and the Nectin- mediated signaling pathways. However, how these intercellular PCP signals are integrated to control hair cell PCP is not understood. To further elucidate mechanisms of PCP signaling during cochlear morphogenesis, we are investigating the function of transient receptor potential-melastatin-like 7 (Trpm7) in mouse inner ear development. Trpm7 is an essential gene, encoding a ubiquitous cation channel with a cytosolic α- kinase domain. In Xenopus, TRPM7 functions synergistically with Frizzled/PCP signaling to control convergent extension movements. Furthermore, published RNA-Seq data show that Trpm7 is expressed in the cochlea, consistent with a function during cochlear morphogenesis. We have generated inner- ear specific Trpm7 knockout mutant (Trpm7cKO) that revealed a cochlear duct significantly shorter with decreased number o f hair cells and disorganized cellular organization, suggesting that Trpm7 is required for cochlear elongation. In contrast to the core PCP mutants, planar polarization of Trpm7cKO hair cells, as indicated by hair bundle orientation and basal body positioning, was largely unaffected. To further understand the cellular patterning defects, we are performing experiments to examine cell proliferation, cell differentiation, cell adhesion and cytoskeletal organization in Trpm7cKO cochleae. We are also interested in determining the specific roles of the ion channel and kinase domains of TRPM7 in cochlear morphogenesis. Details of our analysis will be presented. Taken together, our results reveal an essential role of TRPM7 in cochlear morphogenesis

ID #4215 Evolution in the control of the Gli protein TRA-1 during nematode sex determination Shin-Yi Lin, Yongqquan Shen, Yiqing Guo, Emily Schmidt, Ronald Ellis Rowan University-SOM, USA The majority of Caenorhabditis nematodes are male-female species. However, C. elegans and C. briggsae are both androdioecious, making males and hermaphrodites. In these two species, XX animals develop a female body, but their germlines produce both sperm and oocytes. Spermatogenesis only occurs during late larval stages, yielding a limited cache of sperm. The XX mother can then use these sperm to fertilize her own oocytes during adulthood, although outcrossing with a male is preferred. Thus, the regulation of the sex determination pathway controlling the development of germ cells must have been altered during the evolution of self-fertility. The worm Gli homolog, TRA-1, plays a conserved role in both species, and some of its regulators are conserved as well. Using gene-editing technology, we have created matching sets of mutants affecting similar residues in the C. elegans and C. briggsae homologs of TRA-1 and its regulators. In some cases, the mutants have phenocopied each other, pointing to conserved aspects of TRA-1 function. A missense mutation in the carboxyl terminus of TRA-1 supports the hypothesis that TRA-1, like its Gli homologs in fly and mouse, acts as a transcriptional activator as well as a repressor, and that full-length TRA-1 activator is needed during adulthood to sustain spermatogenesis in males. Other mutations have distinct effects in the two species, highlighting divergent aspects of TRA-1 regulation. First, mutants affecting the regulation of cbr-fem-3 through its 3’UTR show that fem-3 is both necessary and sufficient for sexual development in C. elegans germ cells, but neither necessary nor sufficient in C. briggsae germ cells. Second, a series of cbr-tra-2 mutants that affect two domains of TRA-2—one that governs direct TRA-1/TRA-2 interactions, and another that is involved in FEM-3/TRA-2 interactions— have unique effects. SL is funded by an American Cancer Society Postdoctoral Fellowship.

ID #4338 Efficient genome-editing of wild strawberry genes, vector development, and validation junhui zhou University of Maryland, United States The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR-Cas9 to horticultural plants, especially with regard to germline transmission of targeted mutations. Here we report high efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and Auxin Response Factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6-26 and the wild strawberry U6 promoter FveU6-2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high efficiency genome editing in strawberry. To test germline transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct were analyzed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harboring arf8 homozygous knockout mutations grew considerably faster than wild type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance.

ID #4414 The role of Sox11 during Xenopus laevis nervous system development Pablo Silva-Rodriguez Georgetown University, USA The vertebrate CNS is one of the most complex structures in biology and hence its development is tightly regulated. The transcription factor, Sox11, a member of the large Sox HMG family plays a vital role in this regulation. It has been shown to drive differentiation of neural precursors (NPCs) to neurons in the developing cortex, spinal cord, retina, and also during primary neurogenesis. However, in Xenopus laevis, Sox 11 has also been shown to regulate an earlier step in neural development, neural cell fate specification of ectoderm. Thus, Sox11 has stage-specific functions during neural development, however what regulates these functions is largely unknown.To address this question, we identified Sox11 targets early (blastula) and late (mid- gastrula) during development. To do this we overexpressed Sox11 at the blastula stage (prior to neural specification) and during gastrulation (when neural differentiation begins) and analysed the transcripts with RNA-Seq. We identified three sets of target genes: Those specifically responsive to Sox11 induction during blastula (st. 9), those specifically responsive to Sox11 induction during gastrula (st. 11.5) and those responsive at both stages, supporting the hypothesis that Sox11 dual function is being driven by targeting different downstream genes at different stages. GO analysis shows that the main categories responsive to Sox11 in both stages are CNS/PNS development, axial patterning and sensory system development. We observed a switch to regulation of genes involved in neural differentiation. While only 5% of the target genes were involved in neural differentiation during blastula, percentage increased to 40% during mid gastrula, suggesting Sox11 is targeting neural differentiation genes more actively during gastrulation.

In summary, our data support our initial findings that Sox11 has a stage specific dual function in neural development and this dual function is driven in part by targeting different downstream genes.

ID #4416 Six1 proteins carrying different human Branchio-Oto-Renal (BOR) mutations differentially affect cranial ectoderm patterning and otic vesicle formation Ankita Shah1, Karen Neilson1, Patrick Krohn2, Himani Datta Majumdar1, Sally Moody1 1George Washington University, USA; 2University of Hohenheim, Germany Branchio-oto-renal syndrome (BOR) and Branchio-otic syndrome (BOS), are the second most prevalent hearing-loss birth defects in developed countries. BOS/BOR are characterized by deformities of the external, middle, and inner ear and second branchial arch defects that include fistulas and cysts; renal malformations additionally occur in BOR. Interestingly, there exists considerable variability in the severity, presence and type of abnormalities on the left and right sides of an affected individual and among individuals of the same family carrying the same mutation. While hearing loss and external ear dysmorphologies are the most prominent features of this syndrome, how they arise in the embryo has not yet been addressed. As an autosomal dominant syndrome, BOS patients have one wild-type copy of SIX1 and one mutated allele with a single amino acid substitution. Given the key role of Six1 during early development of the inner ear, the objective of this study was to investigate whether four of the known human SIX1 mutations have different developmental effects on the specification of the embryonic ectoderm into its major components that contribute to syndrome dysmorphologies (epidermis, neural crest, cranial placodes). Using Xenopus, whose ear development and morphology is highly conserved with other tetrapods including mammals, we expressed mutated Six1 proteins and compared their effects on early gene expression in the embryonic ectodermal domains and otic vesicles, and on the morphology of the tadpole inner ear. We found that each has a differential effect on otic development compared to wild-type Six1. These studies have the potential to explain the high degree of phenotypic variability observed in BOS patients and will reveal the consequences of SIX1 mutations.

ID #4417 Disruption of neural circuitry in the mouse model of pediatric dysphagia zahra Motahari, Anastas S Popratiloff, Sally A. Moody, Anthony S. LaMantia George Washington University, USA Pediatric dysphagia, feeding and swallowing difficulty, is a serious complication in 35% to 80% of children with neurodevelopmental disorders. The consequences of pediatric dysphagia include acute choking, and food aspirations leading to nasal, middle ear, and lung infections. Although the causes are unknown, studies in LgDel mouse model of dysphagia suggest the aberrant development of several cranial nerves. Particularly affected is the trigeminal nerve (CNV), which provides sensory innervation to the face and oral cavity as well as motor innervation to the muscles of mastication. Furthermore, in LgDel animals the retinoic acid-mediated anterior-posterior patterning of the hindbrain is disrupted. Since CNV sensory as well as motor/inter neurons arise from distinct rhombomeric locations in the hindbrain, the altered hindbrain patterning suggests that the neural circuits for feeding and swallowing are disrupted. We retrogradely labeled trigeminal sensory and motor axons in living E11.5 embryos using Alexa Fluor 594 biocytin injected into pharyngeal arch 1B. Embryos were incubated at 37° in cell culture medium for one hour, fixed, cleared and prepared for confocal 3D imaging. In wild type animals, both sensory and motor CNV axons project directly toward the correct target, pharyngeal arch 1B. Their pathway is direct, with no indications of wandering or branching. LgDel CNV axons also project to the correct target, However, they branch, misroute, or loop. Interestingly, the CNV phenotype in LgDel can be genetically rescued by diminishing retinoic acid signaling. Thus, 3D imaging in whole E11.5 embryos can resolved compromised axon pathfinding in LgDel animals that might otherwise have been missed using conventional techniques. These results indicate that abnormal individual axon trajectories are at least partially responsible for disrupted construction of a key component of the sensory and motor circuitry that may underlie feeding and swallowing difficulties.

ID #4496 EGFR signaling mediates regeneration after injury in the Drosophila testis stem cell niche Margaret de Cuevas, Leah Greenspan, Erika Matunis Johns Hopkins School of Medicine, USA Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the Drosophila testis stem cell niche, somatic hub cells produce signals that maintain and regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells normally divide only during embryogenesis and are quiescent in adult flies. We previously showed that complete genetic ablation of CySCs causes hub cells to exit quiescence, delaminate from the hub, and transdifferentiate into functional CySCs. Forced expression of cell cycle activators, or knockdown of the cell cycle inhibitor Retinoblastoma-family protein (Rbf), directly in hub cells also causes hub cells to proliferate and transdifferentiate into CySCs. These findings suggest that CySC ablation alters signaling pathways within the niche, triggering hub cells to re-enter the cell cycle and change fate to replace the missing stem cells. To identify these signaling pathways, we knocked down or overexpressed candidate pathway genes in hub cells and screened for loss of hub cell quiescence. We found that the EGFR-MAPK pathway plays a key role in this process. In an otherwise normal testis, activation of EGFR signaling causes hub cells to proliferate and transdifferentiate into CySCs. Moreover, after genetic ablation of CySCs, reduction of EGFR causes a reduction in the number of testes that successfully regain CySCs. These preliminary results suggest that EGFR signaling is necessary and sufficient for promoting hub cell proliferation and transdifferentiation to CySCs. Taken together, our studies reveal that the precise regulation of niche cells, not just the stem cells they support, is important for tissue maintenance and regeneration.

ID #4498 Defective Retinal Ganglion Cell Generation Underlies FOXC1-Attributable Congenital Glaucoma Jurgienne Arizza Umali, Curtis French Discipline of Genetics, Memorial University of Newfoundland, CA Glaucoma is characterized by optic nerve damage and retinal ganglion cell (RGC) death, leading to vision loss. Global estimates show that glaucoma is the leading cause of irreversible blindness, affecting at least 70 million people worldwide. The only known risk factor for the disease is elevated intraocular pressure (IOP), however some patients present with IOP within the reference range, suggesting IOP-independent mechanisms underlying a proportion of disease cases. The forkhead transcription factor, FOXC1, is required for anterior segment eye development and has been linked to glaucoma, however the mechanism by which this occurs isn’t fully understood. Given that homozygous mutation in FOXC1 can cause congenital glaucoma, we hypothesized that FOXC1 mutations cause reduced RGC number through dysregulation of genes involved in RGC genesis and differentiation. We also hypothesize that the zebrafish, a popular model to study ocular development and disease due to its high similarity to the human genome and similar eye structure, is a viable model for FOXC1-dependent glaucoma research. In this study, we quantified and compared RGC numbers and optic nerve sizes during early development in a zebrafish loss of foxc1 function model. Our data demonstrates that complete loss of foxc1 function causes a significant reduction in RGC number and optic nerve size. In situ hybridizations also show that loss of foxc1 function results in a downregulation of atonal homolog 7 (atoh7), a gene involved in RGC genesis and differentiation, which likely accounts for the reduction in RGCs and optic nerve size. Overall, we propose that altered RGC genesis and differentiation as at least part of the mechanism underlying FOXC1-dependent congenital glaucoma, and that the zebrafish serves as a useful model for future studies on the mechanisms underlying this disease. We thank the Glaucoma Research Society of Canada and Memorial University of Newfoundland for research funding and support.

ID #4502 Mitotic kinases choreograph FGF receptor redistribution during asymmetric heart progenitor induction in Ciona. Christina Cota1, Matt Dreier1, William Colgan1, Anna Cha1,2, Brad 1 1Swarthmore College, USA; 2Harvard University, USA Receptor distribution profoundly impacts cellular signal transduction. Cell division requires extensive reorganization of cell membranes that alter the distribution of membrane-bound proteins. As such, mitotic trafficking of signaling components may play a profound, largely uncharacterized role in cell fate specification. Ciona robusta (formerly C. intestinalis Type A) has emerged as an ideal system for investigating in vivo signaling dynamics. In this system, biased mitotic redistribution of Fibroblast Growth Factor (FGF) receptors is required to compartmentalize the response to FGF during differential heart progenitor induction. Here we show that the mitotic kinases; Cyclin dependent kinase 1 (Cdk1) and Aurora kinase direct FGF receptor trafficking during asymmetric heart progenitor induction. Through selective perturbation of these mitotic kinases in pre-cardiac founder cells we have found that, Cyclin- dependent Kinase 1 (CDK1) activity inhibits lysosomal degradation and endosomal recycling of FGF receptor-enriched membranes during mitotic entry while Aurora Kinase is inhibits internalization of FGF receptors throughout mitosis. These results indicate that mitotic progression may play a critical regulatory role during embryonic and stem cell divisions directing receptor redistribution to promote robust responses to signaling during cell fate specification.

ID #4506 Lighting the Way: Can Dermal Stem Cells Form Muscle in Axolotls? Andrew Marinich, Karen Crawford St. Mary's College of Maryland, United States of America Salamanders are well known for their ability to regenerate. Normally, following amputation or injury, an undifferentiated bud of tissue called a blastema forms at the wound site, proliferates, and redifferentiates to restore the missing limb elements. The belief that any limb blastemal cell could contribute to any tissue within the regenerate was examined by grafting mature limb tissues from GFP donors into white host limbs followed by amputation and regeneration (Kragl et al., 2009). In that study, it was conclusively shown that differentiated limb tissues do possess restricted regenerative potentials. For example, muscle or bone cells were shown to contribute only to either muscle or bone, respectively. In contrast , dermal tissue was shown to contribute to dermis, as well as other connective tissues. One lingering question from this study was whether dermal tissue could contribute to muscle. To explore this question, we utilized the accessory limb model (ALM) of regeneration in the axolotl. In our experiments, limb dermal tissue from GFP+ axolotls was grafted to host limb wounds with and without brachial nerve supplementation. Dermal grafts were harvested from the contralateral limb and oriented to maximize limb pattern discontinuities. GFP+ dermal tissue contributions within regenerating or ectopically induced ALM limbs, were followed throughout regeneration with fluorescence microscopy. While GFP+ dermal grafts were only observed to contribute to dermal tissues in nearly all of our experimental animals, in one striking case, dermal tissue was found to contribute extensively to muscle tissue in an ALM ectopic limb. This result suggests that dermal cell potential may be enhanced to include muscle in ALM ectopic limbs. This unexpected result expands our current understanding of dermal tissue potential and suggests that the accessory limb model (ALM) may not be absolutely equivalent to whole limb regeneration in axolotls.

ID #4508 Cell proliferation and nerve regeneration in African Spiny mice Corin Hacker, Shishir Biswas, Ashley Seifert University of Kentucky, USA Regenerating species are widespread across the animal kingdom and include many vertebrates which are capable of appendage regeneration. Most mammals, however, are unable to regenerate tissue in response to injury and instead usually heal via fibrotic repair. Our previous work demonstrating that several species of spiny mouse (Acomys cahirinus,A. kempiand A. percivali) could regenerate skin and large holes made through the ear pinna, shows that some mammals possess enhanced regenerative ability. Building on this work, our lab continues to develop A. cahirinusas a robust mammalian model for studying complex tissue regeneration. Spiny mice completely close 4mm ear punches and regenerate hair follicles, sebaceous glands, elastic cartilage and nerves to restore functional tissue architecture. Conversely, both lab and wild caught mice (Mus musculus) heal identical injuries with scar tissue and form a few disorganized cartilage nodules. In this study we quantified the proliferative index of cells at the injury site during the first 20 days post injury and found that Acomysexhibited a higher rate of proliferating cells than Mus. We next asked if there was a regional bias (i.e., dorsoventral, proximodistal) in proliferative rate and observed a significantly higher number of proliferating cells in the dorsal and distal regions. We hypothesized that the regionalized distribution of proliferation might be associated with regenerating nerve fibers. To test this hypothesis we co-labeled regenerating ear tissue with EdU and ß-III Tubulin or RT97. Indeed, we observed axons re-innervating healing tissue near clusters of proliferating cells. Our results suggest that cell proliferation in spiny mice ear pinna is at least partly dependent on nerves and raises the prospect that suboptimal re-innervation in Mus contributes to fibrotic repair. (Supported by: University of Kentucky, Dept. of Biology, NSF and OISE (IOS-1353713))

ID #4512 Effects of Glyphosate on Motor Development in Embryonic Zebrafish Saad Saleem, Gwendolyn Lewis George Mason University, United States Modern agricultural practices rely heavily on chemical agents such as fertilizers, insecticides and herbicides to combat pests, improve crop yields, and minimize loss of product. One of those chemicals, an herbicide called glyphosate, enjoys widespread use. In fact, it is the most common herbicide in use today and is commercially marketed as Roundup. Glyphosate has been around for nearly half a century, and yet we understand relatively little about its effects on us and other animal species that come in contact with it. The goal of this study is to bridge the research gap on how glyphosate affects motor development in zebrafish in order to provide critical information on the health effects of glyphosate.This is to ensure glyphosate use is safely and effectively regulated. During the study, we looked at the effects of glyphosate on motor development in a zebrafish (Danio rerio) animal model. Overall morphology, motor axon morphology, and motor behaviors were analyzed over the course of this study. The results show significant differences in locomotor activity, overall length, hatching rates, as well as a reduction in overall neuromuscular junction expression.

ID #4519 Phage Development: WC1 and SEA-PHAGES as a Program for Continued Undergraduate Research Monica Bonilla, Ronnie Cutler, Caroline Golino, Margaret Saha College of William and Mary, USA The SEA-PHAGES program serves as an excellent model for seamlessly blending teaching and research, and engaging freshmen in authentic research early in their careers. However, one of the major “problems” facing both SEA-PHAGES students and faculty is how to sustain the desire for continuing research following the excitement and engagement of the freshmen Phage Lab experience. Together with faculty and other students at the College of William and Mary, we developed a program that allows students to continue their phage-related research through a number of different venues. These include: a continuation genomics course; mentored research experiences; and serving as a Teaching Assistant and mentor for Phage Lab and ongoing outreach programs. Participation in these programs has led to the discovery of a novel Rhodococcus phage (WC1) that infects Rhodococcus erythropolis. As part of the continuation and extension of Phage Lab we have performed a latency period experiment and sequenced both the host and the WC1 phage as well as performed RNA-Seq on the WC1 and the host at several time points following infection. By 30 minutes following infection 25% of the reads were from WC1 and at 120 minutes following infection over 80% of the reads were from WC1. In addition to the expected expression of early and late genes, several novel non-coding transcripts were detected early in the infection cycle. In summary, the SEA-PHAGES program has served as an effective model for engaging students in research throughout their entire undergraduate careers.

ID #4522 Conservation of 3 Amino Acids at the C - Terminus of TWIST1 Suggests Similar Structure/Function Role Across Species Michael Gruss Catholic University of America, United States Transcription factors play a critical role in the regulation of gene expression in all life, more importantly do so in a spatiotemporal specific manner during tissue development. TWIST1 is a basic helix-loop-helix subclass of transcription factors that are found in humans and many homologs found in multiple organisms with a high degree of conservation. TWIST1 functions primarily as a transcriptional repressor, but also can function as a transcriptional activator depending on the time, place, and stage of development. In humans, mutations in TWIST1 result in a debilitating craniofacial disease called Saethre-Chotzen syndrome. The aberrant TWIST1 causes crainosynostosis, or the premature fusion of skull sutures into mature bone through osteo-diffentiation pathways, as well as many other complications.

C.elegans hlh-8 gene product is homologous and functionally similar to the human TWIST1 where it is crucial for the development of the mesoderm. Our lab in previous work has shown that the absence of functional HLH-8 results in both a constipated and egg laying defective phenotype in worms. Our current study is to look at the effect of CRISPR/Cas9 induced single point mutations at three specific, evolutionarily conserved amino acids located at the understudied C-terminal domain of TWIST1. This C-terminal domain has been shown to play a critical transactivational role in humans, and two of the three conserved amino acids were linked to Saethre-Chotzen patients. Currently two of the three desired mutants have been successfully created and a variety of phenotypic and genotypic assays are currently under work. Finally, these mutants serve as good candidates for suppressor screen analysis as the alpha helical predicted structure of this domain suggests that a potential binding pocket is formed, suggesting potential protein to protein interaction that could account for TWIST1’s diverse functionality.

ID #4524 Role of N-Myc Downstream Regulated Genes Family in Low Oxygen Adaptation Nguyet Le University of Maryland Baltimore County, USA In the absence of oxygen, the production of ATP decreases dramatically. In humans, the loss of ATP in organs with high metabolic rate, such as the brain, heart and kidney, can lead to cell death. However, under anoxia (0% oxygen), zebrafish embryos enter a hypometabolic state that enables them to conserve cellular energy (ATP) and survive for up to 50 hours. Currently, the molecular mechanisms that initiate and maintain the hypometabolic state in zebrafish are unknown. The knowledge of such mechanisms would be extremely beneficial for therapeutic purposes. In an effort to identify molecules that promote arrest, the Brewster laboratory performed metabolic profiling and found that lactate is one of several metabolites that are up-regulated in embryos exposed to anoxia. Lactate has previously been shown to bind to NDRG3 in hypoxic cancer cells and to promote cell survival, thereby identifying lactate/NDRG interaction as a candidate signal for adaptation to low oxygen. This finding lead us ask whether NDRGs could be activated downstream of lactate binding in the zebrafish embryo to arrest ATP- demanding processes. To address this question, my project was to identify where in the zebrafish embryos are the ndrg family expressed by performing in situ hybridization. Reported here are the expression patterns of several members of the NDRG family.

ID #4525 Conserved and novel role for the Planar Cell Polarity component Vangl2 in shaping the zebrafish anterior neural tube Maraki Negesse, Jonathan Werner University of Maryland Baltimore County, USA Neurulation is the process by which the neural tube, the precursor of the brain and the spinal cord, forms during early development. In the prospective forebrain, neurulation is coupled with eye formation. Fate maps of vertebrate embryos reveal that the prospective forebrain occupies the lateral edges of the eye field. The optic vesicles evaginate as neurulation proceeds and the neural tube is positioned in between the developing eyes. Despite the forebrain regulating many essential physiological functions, little is understood about the cellular and molecular events that shape this brain region. Our laboratory is interested in understanding the cellular dynamics that underlie forebrain morphogenesis, using zebrafish as a model organism. The optical clarity of the zebrafish embryo and its amenability to genetics make it uniquely well suited for studying tissue dynamics. In contrast to more posterior regions of the neural tube, the events that shape the forebrain in this teleost are mostly unknown. Preliminary data from our laboratory indicate that forebrain morphogenesis in zebrafish presents hallmarks of primary neurulation in amniotes, namely medial and lateral hinge point-like structures and neural folds that converge and fuse at the dorsal midline. The medial hinge point (MHP) forms in the superficial layer of the bilayered eye field, which resolves into a single layer via radial intercalation. We report here that disruption of the Planar Cell Polarity (PCP) component Vangl2, previously implicated in MHP in amniotes, is required for the formation of the MHP counterpart in zebrafish. Lastly, we show that radial intercalation of MHP cells is impaired in Vangl2 mutants. Together, these findings highlight conservation of central aspects of primary neurulation in zebrafish and reveal a novel role for the PCP pathway in driving radial intercalation in the developing anterior neural tube.

ID #4527 Identification by suppressor mutation of a novel factor that could cooperate with CeTwist Md Rabby The Catholic University of America, Washington DC, 20064, United States of America TWIST, a basic helix-loop-helix transcription factor plays an essential role in mesoderm development in all animals. CeTwist, or HLH-8, is a homolog of human TWISTin C. elegans. Mutations in human TWIST and CeTwist are linked with many abnormalities in both organisms. HLH-8 directly regulates the expression of its target gene, the Notch ligand arg-1 that is expressed in the head mesodermal cell (HMC), vulval muscles (VMs), and enteric muscles (EMs) (1). When HLH-8 is mutated, worms have defects in VMs and EMs leading to egg-laying difficulties and constipation. In our most recent study, several amino acid substitutions in the DNA binding domain of HLH-8 were made (E29 mutants) (2). All of these strains exhibit different variations of severity of the previous phenotypes mentioned. We predict that mutations can be identified in the C. elegans genome that could alleviate or restore the mutated E29 HLH-8 function. Based on this hypothesis, we performed a suppressor screen on the E29G strain utilizing the mutagen EMS. This strain does not have arg-1::gfp expression in the VMs, but arg- 1::gfp is still expressed in the HMC and the EMs. Our goal is to find a novel factor that may contribute to the tissue-specific expression of arg-1 in the VMs. To find the potential novel factor, we screened the progeny of the mutagenized animals up to the F2 generation looking for restoration of arg-1::gfp expression in the VMs. After screening 132,000 genomes, we have four suppressor candidates. All four strains have WT VM expression of arg-1::gfp but are still egg-laying defective suggesting that they are not revertant mutations. We are currently characterizing these strains further and identifying the mutation causing suppressed phenotype. By discovering this factor, we can learn more about how HLH-8, and potentially human TWIST, regulates transcription.

References:

1. Zhao J., Wang P. and Corsi A.K., (2007) Mech Dev. 124:377-389.

2. Kim S. et.al, (2017), 26: 2118-2132.

ID #4528 SHE-1 controls TRA-2 activity but not TRA-2 protein levels in C. briggsae Jonathan Harbin, Yongquan Shen, Shin-Yi Lin, Ronald Ellis Rowan University-SOM, USA Three species of Caenorhabditis independently evolved the ability to reproduce as self- fertile hermaphrodites. These androdioecious species, C. elegans, C. tropicalis, and C. briggsae, offer ideal subjects for studying the regulation of Gli proteins, since the nematode Gli protein TRA-1 controls sexual fate. The our goal is to understand how TRA-1 works with co-factors to control its targets and to learn how its repressor and activator isoforms are regulated. In Caenorhabditis, the transmembrane protein TRA-2 regulates TRA-1 activity. This Patched-related protein is a receptor for the male sex hormone HER-1. When TRA-2 is active, it releases an intracellular fragment, TRA-2ic. This fragment can interact with both TRA-1 and with FEM-3, a protein that regulates TRA-1 stability. However, little is known about the nature or function of the TRA-2/TRA-1 interaction, although it could provide insight into common activities of all Gli proteins. We are focusing on Cbr-TRA-2, since mutants that eliminate FEM-3 in this species do not prevent hermaphrodite development. To study Cbr-TRA-2 expression we tagged it with an OLLAS epitope tag in the intracellular domain. We detect TRA-2ic::OLLAS of the correct size on a Western blot, and in the nuclei of extruded germ cells using immunohistochemistry. SHE-1 is a novel F- box protein that is needed for self-fertility in C. briggsae. Null mutants of she-1 result in XX animals that produce only oocytes, as well as fertile XO males. Although epistasis tests suggest that SHE-1 controls germ cell fates by regulating Cbr-tra-2, we have not detected change in TRA-2::OLLAS expression levels in feminized she- 1(v35) mutants. However, we have identified a conserved protein, PQN-94, that does play an important role in SHE-1 function. Finally, we have begun tagging Cbr-tra- 2 "mx" mutants that should affect its interaction with TRA-1, so that we can study how altering this interaction changes TRA-2 expression or localization during development.

ID #4529 The function of fem-1 is conserved in the Caenorhabditis tropicalis germline Soumi Joseph, Maria Ivanova, Ronald Ellis Rowan University School of Osteopathic Medicine, USA The nematode genus Caenorhabditis includes both male/female and self-fertile hermaphrodite species. Evolutionary studies have shown that self-fertile hermaphrodites have arisen on three independent occasions in this genus. To understand how the sex-determination pathway was modified during the evolution of hermaphrodites, we have begun a forward genetic screen in C. tropicalis, one of the three self-fertile species. We identified a temperature-sensitive, recessive mutation that transforms both somatic and germline development from male to female. Single nucleotide polymorphism mapping data showed that this mutation is strongly linked to ctr-fem-1. Sequence analysis of the mutant revealed a missense mutation in the last fem-1 codon. This fem mutation shows a strong maternal effect, like that previously observed in C. elegans. The homozygous recessive mutants that lack maternal product develop as complete females, which have a hermaphrodite body structure but do not produce sperm. The homozygous mutants that do have maternal product (provided by a heterozygous mother) show partial feminization: XX animals develop as hermaphrodites but with reduced sperm number, while XO animals exhibit an intersex phenotype with a deformed male tail, protruding vulva and two-armed gonad (like that of hermaphrodites) which contains oocytes in addition to sperm. Although the fem genes are dispensable in the hermaphrodite germline of C. briggsae, a distant relative of C. tropicalis, our study confirms that fem-1 is necessary for spermatogenesis in both sexes of C. tropicalis, just as they are in C. elegans. Thus, a dual role for fem-1 in spermatogenesis is the ancestral trait, which must have been altered during C. briggsae evolution. These results imply that comparisons between C. tropicalis and C. elegans should determine if a requirement for fem-1 activity introduces a developmental bias into the evolution of self-fertility.

ID #4530 Local regulation of radial cell intercalation and re-lumenization by neural fold fusion in Xenopus Grace Anyetei-Anum University of Virginia, USA Grace Patrice Anyetei-Anum, Logan Haley, Seulgi Min, Mark Rimkus, Thao Vu, David Lee, John D.

The development of the neural tube encompasses various morphogenic cell behaviors and interactions. Understanding these processes is of great importance because the causes of neural tube defects (NTDs), including those in humans, remain poorly understood. Significantly, previous work (Davidson and Keller, 1999 Development 126, 4547-4556) described a novel process in which the cells of the multilayered neural plate of Xenopus intercalated radially (radial intercalation) to form a single-layered neural tube, which resulted in convergent extension (CE) to form a longer neural tube immediately after neural fold fusion. They speculated that neural fold fusion is essential for the radial intercalation that further elongates, thins, and reforms the lumen of the neural tube. We tested this hypothesis by mechanically blocking neural fold fusion locally (in an anterior-posteriorly limited region), either by inserting plastic shim-stock or by repeated microsurgery. We assayed for radial cell intercalation with fluorescent labeling of the participating cells, and assayed CE with time-lapse imaging. Current results show multiple layers (lack of radial intercalation) in the regions of blockage versus one layer of cells in closed areas (where radial intercalation occurred), and a decrease of CE in regions of the open neural tube. These results support the hypothesis that radial intercalation and the resulting CE are dependent on neural fold fusion.

ID #4536 Activation of JAK/STAT signaling depends on the vesicle fusion regulator alpha- Snap Afsoon Saadin, Michelle Starz-Gaiano University of Maryland, Baltimore County, USA The well-conserved Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway is critical for normal animal development and immune function, and is implicated in cancer progression and metastasis. To investigate this pathway, we study a set of follicle cells in the Drosophila ovary that switch from epithelial to migratory. Two signaling cells, the polar cells, secrete the cytokine Unpaired (Upd), which activates STAT signaling and specifies nearby cells as the motile border cells. In a mutant screen to identify novel regulators of cell migration, we uncovered a requirement for α-Soluble NSF Attachment Protein (α-Snap) in motile cell specification. α-Snap is known to function in synaptic transmission, membrane fusion, and vesicle trafficking by facilitating association of N-ethylmalemide-Sensitive Factor (NSF) and SNAP Receptors (SNAREs) during vesicle fusion. RNAi-mediated depletion of α- Snap in the follicle cells, including the polar cells, results in egg chambers that lack both polar and border cells. Over-expression of the viral anti-apoptotic gene, p35, in α- Snap depleted egg chambers rescues the polar cells and verifies that the lack of a border cell cluster in mutants is due to impaired signaling, not apoptosis. RNAi and genetic interaction studies indicate that α-Snap cooperates with the target-SNARE Syntaxin 1 and NSF to regulate trafficking of a STAT signaling component. Further genetic analysis provides evidence that α-Snap is specifically required for Upd secretion from the polar cells during oogenesis. Live imaging suggests that changes in intercellular calcium may be linked to this event. These results suggest a new regulatory node – vesicle trafficking – in activating JAK/STAT signaling and induction of cell motility.

ID #4537 Notch signaling is required for termination of neurogenesis during development Chhavi Sood University of Virginia, United States During development, progenitor cells called neural stem cells (NSCs) undergo asymmetric cell division to produce morphologically and functionally diverse neurons and glia which constitute the adult neural circuitry. Integration of stem cell intrinsic mechanisms and environmental cues control the proliferation and elimination decisions of the NSCs. Failure to eliminate at the proper developmental time can lead to over or under production of neurons resulting in human diseases of megalocephaly or microcephaly. Most NSCs in Drosophila (known as neuroblasts, NBs) are eliminated during early pupal development via terminal differentiation, however, what determines the timing of their elimination is currently not well-understood. A targeted RNAi screen was performed in our lab to identify genes required for NB elimination during development. Among the candidate genes tested, members of notch signaling pathway were identified as possible regulators of NB elimination. Activation of notch signaling pathway in the NBs results from its interaction with either delta in the new-born daughter cells or serrate in the glial neighbors. We find that perturbing notch signaling by knocking down notch or either of its ligands results in improper timing of NB elimination. NBs persist ectopically when we knock down notch or its ligand delta, whereas NBs are eliminated prematurely when we constitutively activate notch signaling or knock down its ligand serrate. We propose that during NB elimination, notch signaling is activated differently by delta and serrate which in turn results in activation of different downstream target genes.

ID #4538 Effects of lithium ions on the embryonic development in Xenopus laevis Raymond Keller, Adetokunbo Goncalves, Parabhkiran Kaur University of Virginia, United States A common way to generate dorsoanteriorized Xenopus embryos is acute exposure of the 32-64 cell stage blastula to 0.3 M lithium chloride for 8-12 mins, a treatment that converts the entire marginal zone to an “Organizer”, results in delayed and symmetrical gastrulation, and the formation of progressively more dorsal and anterior tissues of the embryo (Kao and Elinson, 1989 Dev. Biol 132, 81-90). Lithium ions are thought to cause this phenomenon by inhibiting GSK3beta and thus activating the Wnt pathway (Hedgepeth, et al., 1996, Curr. Biol. 6, 1664-1668), a major pathway in embryonic development. Because lithium is used in medicine to treat the manic episodes and depression associated with bipolar disorder (Sproule 1) and in common use in industry, we were interested to see what developmental effects, if any, would occur in chronic exposure to much lower concentrations of lithium. Women being treated with lithium may be pregnant, whether aware or unaware, and the fetus may be affected, thus increasing the relevance of this experiment. Further, it has been shown clinically that higher concentrations of lithium yield greater clinical success in treatment of bipolar disorder than lower concentrations do (Sproule 1). We assayed the effect of lithium chloride on the embryonic development in Xenopus laevis embryos from the early blastula stage onward, from millimolar to micromolar concentrations. Loss of posterior structures, decreased convergent extension of axial and paraxial tissues, failure of blastopore closure, formation of split (double) tails, pigment pattern defects, fin fold defects, edema, and eye and head defects all were observed with decreasing frequency and severity at progressively lower concentrations. However, considerable effects were seen at micromolar concentrations. We conclude that chronic, low levels of lithium ions result in dramatic effects on Xenopus development.