Abstracts - Society for Developmental Biology

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combinations of transcription factors (TFs) and activate or repress the expression of a target gene. Despite detailed
analyses of a small number of enhancers, we lack a systematic picture of enhancers and their functions in animal genomes
and an understanding of the sequence basis for their activity. To map the regulatory landscape of Drosophila
melanogaster, we are performing an in vivo screen for enhancer activity during embryogenesis. We screen a large library
of several thousand transgenic flies that carry transcriptional reporter constructs with defined ~2kb candidate fragments
inserted at a defined genomic position (“VT-library”, currently being established in collaboration with the Dickson group
[IMP]). We assayed more than 4000 enhancer candidates, which represent around 10% of the entire non-coding
Drosophila genome. Interestingly, about half of all candidates functionas enhancers during embryogenesis, with diverse
temporal spatial patterns and a bias towards late embryonic stages. We apply bioinformatics and machine learning to
extract predictive signatures from regulatory DNA sequences. We identified the TAG team motif of Zelda as an important
feature of TF binding and enhancer function in the early embryo and the GAGA motif as a predictive feature of
Drosophila HOT regions. Taken together, we are systematically characterizing thousands of defined DNA sequence
fragments to create an unprecedented view on transcriptional regulation and the ‘regulatory code’ that governs it.
Program/Abstract # 61
Six1 expression is regulated by evolutionarily conserved enhancers
Sato, Shigeru; Ikeda, Keiko, Jichi Med Univ Ctr for Molecular Medicine, Japan; Shioi, Go; Nakao, Kazuki
(Kobe, Japan); Yajima, Hiroshi; Kawakami, Kiyoshi (Jichi Med Univ Ctr for Molecular Medicine, Japan)
Six1 homeobox gene plays critical roles in vertebrate organogenesis. Six1 knock-out mice show severe defects in organs
such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in
human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing
loss, branchial and kidney defects. In this study, we showed that seven conserved non-coding sequences retained in
tetrapod Six1 loci possessed distinct enhancer activities. Their activities were detected in all cranial placodes (excluding
the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-
expression domains were covered by the sum of the activities of these enhancers together with the previously identified
enhancer for the pre-placodal region and for egutendoderm. Thus, eight CNSs identified in a series of our study represent
major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. Mutational analysis of the
most conserved placode-specific enhancer, Six1-21, revealed that the enhancer integrates a variety of inputs from Sox,
Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. The involvement of Six protein-binding sites in Six1
regulation suggests the molecular basis of positive autoregulation. Analysis of Six1-21 enhancer and detailed expression
analysis of chick Six1 suggest that the function of Six1 may not be conserved during olfactory development between chick
and mouse, and raises the possibility of evolutionary changes in the olfactory developmental program.
Program/Abstract # 62
A conserved requirement of MED14 for the maintenance of stem cell populations.
Burrows, Jeffrey T., University of Toronto, Canada; Pearson, Bret (Hospital for Sick Children, Toronto, Canada); Scott,
Ian (University of Toronto, Canada)
The mediator complex links the RNA polymerase II transcriptional machinery to the enhancer bound regulatory factors
that in turn establish and maintain cell fate. However, the in vivo consequences of loss of function of many mediator
subunits are largely unknown. Through positional cloning analysis we have identified med14, a tail component of the
mediator complex, as the gene affected in a mutant isolated from an earlier ENU screen. A slight developmental delay is
noticeable by 1.5 days post fertilization (dpf) in med14 mutants and the gap in development compared to their wild-type
siblings continues to widen thereafter. Ultimately, med14-/- embryos progress only as far as the long-pec stage (normally
2.0 dpf) by 3.0 dpf and expire before the hallmark events of the hatching period commence (i.e. pectoral fine longation and
formation of the semi-circular canals). Interestingly, transcription is not broadly affected in mutants with only ~2%
(764/34858 with ≥ 2 fold difference) of genes assayed by mircoarray being differentially expressed relative to their wildtype
siblings at 2.25 dpf. More striking still, there is little difference in cell death or proliferation despite the observed
“arrest” in development. To gain further insight, RNAi knockdown of med14 in planarians was pursued. Homeostasis and
regeneration assays implicate med14 specifically in the maintenance of the planarian stem cell population. RNA insitu
analysis of zebrafish “stem cell” populations suggests that this could be an evolutionarily conserved role.
Program/Abstract # 63
Essential role of the chromatin remodeler Chd1 in mouse embryonic and placental development
Ramalho-Santos, Miguel; Guzman, Marcela; Koh, Fong Ming; Sachs, Michael; Lin, Chih-Jen, UC San Francisco, United
States