Congress Abstracts - Society for Developmental Biology

neural crest cells (hNCC)) human embryonic cell populations. In hESC, we uncovered a unique chromatin signature that identifies a
novel class of enhancers, which are inactive but poised in hESC and that become active upon differentiation in a lineage-specific
manner. Similarly, our epigenomic approach allowed us to characterize enhancers in hNCC, a hitherto largely inaccessible and
biochemically intractable vertebrate-specific embryonic cell population that contributes to the formation of multiple tissues and
organs, such as the peripheral nervous system and most of the facial bones and cartilages. Using the sequence information contained
within hNCC enhancers, we uncovered NR2F1 and NR2F2, two orphan nuclear receptors, as novel neural crest and craniofacial
regulators. Finally, I will briefly describe how the genomic characterization of human enhancers in relevant cell types might
streamline the identification of functional non-coding genetic variants, which can have far-reaching implications in our understanding
of the genetic basis of human complex diseases and human morphological evolution.
Program/Abstract # 6
Super-resolution imaging of regulatory chromatin dynamics in developing embryos
Alistair Boettiger, Xiaowei Zhuang (Harvard, USA)
The differentiation of embryonic cells into their appropriate developmental fates is mediated in part by fine scale structural changes to
chromatin. Developmental specific transcription factors may shape this chromatin structure through a variety of mechanisms, such as
re-positioning of histones (e.g. to restrict or increase access to DNA) or the generation of higher-order looped chromatin structures
(e.g. to facilitate looping of distal regulatory sequences to target sites). These fine scale structural changes are mostly too small (10s of
nanometers) to be observed with conventional microscopy techniques (limited to several hundred nanometer resolution), and have so
far evaded in vivo observation in intact embryonic tissue. We present super-resolution imaging techniques which allow for the
detection of changes in chromatin on the scale of tens of nanometers in developing embryos at gene locations of interest. We can
detect locus-specific clusters of modified histones and regulatory chromatin proteins, and resolve sub-diffraction structural details of
the chromatin region of interest. Because chromatin states are studied within the intact embryo, cell identity and the spatial relation of
the cell to its neighbors and embryonic signals are still maintained. This allows us to follow how these structures and their protein
composition change as a cell progresses through different stages of development, or as its daughter cells diverge into different fates.
This approach allows a detailed view of regulatory modifications at the single cell level. Single cell analysis facilitates inference of
causal relations between expression states and modification. It also allows for variation between identical populations to be measured
and the frequency of each state within the population to be determined.
Program/Abstract # 7
HoxA genes regulation in developing limbs: from long-range control to cross-regulation
Marie Kmita (IRCM, Canada)
In most developing embryos, genes of each Hox cluster are activated sequentially in time and their expression patterns are
differentially distributed along the main body plan. A similar “collinearity rule” applies in evolutionary novel structures such as limbs.
Thorough analysis of HoxD genes regulation has revealed numerous remote enhancers triggering HoxD expression in limbs. In
contrast to HoxD genes, HoxA expression does not follow the “collinearity rule” in limbs as Hoxa13 and Hoxa10 are robustly
expressed distally while Hoxa11 is completely excluded from this domain. Such discrepancy between HoxA and HoxD expression
raises the possibility that the regulation of HoxA genes in distal limb is distinct from that of HoxD genes. Mapping of genomic loci
bound by proteins enriched at active enhancers allowed us to locate several putative enhancers, the transcriptional activity of which
was confirmed using transgenic assays. These enhancers were tested for potential interaction with Hoxa13 using Chromosome
Conformation Capture Carbon Copy (5C), which identified 10 remote enhancers “contacting” Hoxa13 . In addition, we identified one
enhancer located in Hoxa11 intron. Interestingly, previous work uncovered the existence of Hoxa11 antisense RNA in distal limbs
raising the possibility that this antisense transcription prevents Hoxa11 expression distally. By deleting Hoxa11 intron, we found that
disruption of the antisense transcription results in ectopic Hoxa11 sense transcription in distal buds. We also found that Hoxa13 and
Hoxd13 proteins trigger this antisense transcription revealing a cross-regulation mechanism in the control of Hoxa11 expression. Our
data will be discussed in the context of limb development and evolution.
Program/Abstract # 8
Cell shape and morphogenesis: sub cellular and supra-cellular mechanisms
Matteo Rauzi, Uros Krzic, Timothy Saunders, Lars Hufnagel, Maria Leptin (EMBO, Germany)
The invagination of the ventral furrow during gastrulation in Drosophila is probably the best-studied example of epithelial folding.
The genes that control the process are known, as are the mechanisms by which their products mediate the cell shape changes that bring
about the formation of the furrow. While the ventral cells act autonomously to create the initial furrow, the other parts of the
embryonic epithelium must participate to accommodate these changes, and perhaps to contribute to the evens that lead to the complete
internalisation of the mesoderm. To be able to correlate the behaviours of all cells in the embryo with those of the invaginating
mesoderm, and compare their shape changes and movements, it is necessary to follow them in the living embryo. In view of the speed
of the process, the analysis requires very high temporal and spatial resolution. We have recorded embryos expressing plasma
membrane-associated fluorescent markers and have made full 3D reconstructions at 20 second resolution. We find that the initial
formation of the furrow occurs without participation of non-mesodermal cells. Rather, the reduction in cell surface in the invaginating
cells is accommodated by an increase in the cell surface in the more lateral mesodermal cells. Once the mesodermal cells have
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