Congress Abstracts - Society for Developmental Biology

viability and morphology of over 200 of these lines have been assessed during organogenesis at embryonic day 14.5 (E14.5). Any
dysmorphology including growth retardation, oedema, craniofacial, skeletal and neural tube defects are recorded and annotated. This
recessive lethality screen has now been extended thanks to a strategic award from the Welcome Trust involving the Deciphering
Mechanisms of Developmental Disease (DMDD) consortium, an innovative and ambitious programme of research involving members
of the UK developmental biology community. This programme includes additional phenotyping tests (encompassing embryonic days
E9.5, E14.5 and E18.5) and will use a combination of comprehensive whole embryo 3D imaging, placental histopathology,
transcriptomics and nervous system functionality assessment in order to identify abnormalities in embryo structure and development.
All data will be made freely available, enabling individual researchers to identify lines relevant to their research and provide valuable
insight into novel gene functions and new mouse models of human developmental disorders.
Program/Abstract # 261
MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq
Hill, Jonathon T.; Demarest, Bradley; Bisgrove, Brent; Gorsi, Bushra; Su, Yi-Chu; Yost, H. Joseph (University of Utah, USA)
Forward genetic screens in model organisms are vital for identifying novel genes essential for developmental or disease processes.
One drawback of these screens is the labor-intensive and sometimes inconclusive process of mapping the causative mutation. In order
to leverage high-throughput techniques to improve this mapping process, we have developed a Mutation Mapping Analysis Pipeline
for Pooled RNA-seq (MMAPPR) that works without parental strain information or requiring a pre-existing SNP map of the organism,
and adapts to differential recombination frequencies across the genome. MMAPPR accommodates the considerable amount of noise in
RNA-seq datasets, calculates allelic frequency by Euclidean distance followed by Loess regression analysis, identifies the region
where the mutation lies and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can exploit
RNA-seq datasets from isolated tissues or whole organisms that are utilized for gene expression and transcriptome analysis in novel
mutants. We tested MMAPPR on two known mutant lines in zebrafish, nkx2.5 and tbx1, and used it to map two novel ENU-induced
cardiovascular mutants, with mutations found in the ctr9 and cds2 genes. MMAPPR can be directly applied to other model organisms,
such as Drosophila and C. elegans, that are amenable to both forward genetic screens and pooled RNA-seq experiments. Thus,
MMAPPR is a rapid, cost-efficient, and highly automated pipeline, available to perform mutant mapping in any organism with a wellassembled
genome.
Program/Abstract # 262
The Role of Long Noncoding RNAs in Regulating Chicken Limb Patterning
Schwartz, Matthew G., (Harvard Med School, USA), Ulitsky, Igor; Bartel, David P. (Whitehead Institute for Biomedical Research,
MIT, Howard Hughes Medical Institute, USA); Tabin, Clifford J. (Harvard Med School, USA)
Recently, the known repertoire of functional RNAs has been expanded by the discovery that long noncoding RNAs (lncRNAs)
influence development and differentiation by regulating gene expression by a diverse array of mechanisms. In addition, it has become
clear that there is nearly pervasive transcription throughout the genome. While the number of protein-coding genes remains relatively
static across evolution, higher organisms tend to have a larger noncoding portion of the genome—suggesting a potential role for
lncRNAs in increasing organismal complexity. LncRNAs have been implicated in many developmental processes, but there remains
limited in vivo evidence for the functionality of lncRNAs in development. The developing chicken limb bud is readily accessible and
permissive to developmental manipulations in ovo, making it an excellent model for examining such roles. Using RNA-Seq and 3P-
Seq, we identified lncRNAs differentially expressed across seven stages of chicken embryonic development as well as in forelimbs
and hindlimbs at HH21/22 and HH25/26. Our analysis uncovered a subset of 7,197 candidate intergenic lncRNAs (lincRNAs), which
are enriched at genomic loci nearby to developmental transcription factors and many of which are spliced and/or have multiple
isoforms. Analysis of additional lncRNA candidates, including antisense and intronic lncRNAs, is currently ongoing. A whole-mount
in situ hybridization screen of candidate lncRNAs enriched in the limbs verified many novel lncRNAs with developmentally
interesting spatial and temporal expression patterns. Candidates are currently being functionally tested by overexpression and
knockdown via RCAS virus infection and in vivo electroporation in the developing chicken limb.
Program/Abstract # 263
Genome-wide approaches reveal dynamic Foxh1-mediated gene regulation during mesendoderm specification in Xenopus
tropicalis
Le, Rebekah Le; Chiu, William; Blitz, Ira; Cho, Ken (University of California-Irvine, USA)
The Nodal signaling pathway is necessary for vertebrate mesoderm and endoderm specification. The receptor-activated Smad2/4
complex functions together with the maternal transcription factor Foxh1 as key transcriptional activators of Nodal target genes. To
gain a comprehensive understanding of the Nodal signaling gene regulatory network, we employed a high-throughput sequencing
approach to investigate the role of Foxh1 during Xenopus tropicalis mesendoderm development. We performed mRNA-seq analysis to
identify Nodal signaling and Foxh1 targets in the gastrula embryo, where Nodal signaling was abrogated using the pharmacological
inhibitor SB-431542 and Foxh1 was knocked down using an antisense morpholino oligo. A comparison of regulated genes revealed a
critical, but not exclusive, role for Foxh1 in the transcriptional mediation of Nodal signaling. We also utilized ChIP-seq to investigate
in vivo Foxh1 binding patterns over the time course of mesendoderm development from blastula to early gastrula. Our current analysis
uncovered Foxh1 binding to cis-regulatory regions of over 5,000 genes at the blastula stage, and nearly 2,000 at the early gastrula
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