Congress Abstracts - Society for Developmental Biology

Disruption of presumptive enhancers downstream of the human short stature homeobox gene (hSHOX) is a frequent cause of the
zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse gene mShox2 is also
required for limb development, but in the more proximal stylopodium. In this study we used transgenic mice in a comparative
approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. We first analyzed the regulatory potential of
a genomic region containing a duplicated conserved noncoding element (dCNE) downstream of mShox2 and hSHOX. We identified a
strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene.
Interestingly, in order to drive strong limb expression this enhancer requires sequences only conserved in the mammalian lineage,
whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. In addition we found that DNase I
hypersensitive sites in limb bud samples (available through the ENCODE project) showed better correlation with limb enhancer
activity near mShox2 than did evolutionary conservation. Similarly, we found that a conserved element downstream of hSHOX
(CNE9) also functions as a neural enhancer in transgenic mice. However when the CNE9 transgenic construct was enlarged to include
adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the
limbs, as would be expected for a hSHOX enhancer. These data demonstrate that transgenic mice can be used as tools for
characterizing the enhancer deletions that cause LWD and other SHOX-deficient phenotypes.
Program/Abstract # 153
Importance of inhibitory mechanisms for deriving specific somatic lineages from the epiblast
Hisato Kondoh, Kazunari Matsuda, Tatsuya Takemoto (Osaka U, Japan)
Somatic development initiates from the epiblast in post-implantation embryos. Whereas ES cells are frequently used as a model for
studying somatic cell development, ES cells must actually go through the epiblast state before entering in somatic lineages, as
evidenced by the transient expression of the epiblast hallmark Fgf5. We utilized an epiblast stem cell (EpiSC) line, for the purpose of
characterizing gene regulatory networks that promote specific pathways of somatic development. It was found that when epiblast cells
select a single pathway of development, mechanisms to inhibit other developmental pathways are activated. Anterior neural plate
develops under the condition where endodermal, mesodermal and posterior neural development is inhibited. Endoderm development
characterized by Sox17 and Eomesodermin expression is permitted when the endoderm-inhibiting, and neuro-mesoderm-promoting
activity of Sox2 and Zic2/3 is suppressed. The paraxial mesoderm develops not directly from the epiblast but from an intermediate, the
axial stem cells, which have neuro-mesodermal bipotentiality, when the function of pro-mesodermal Tbx6 inhibits the expression of
pro-neural Sox2.
Program/Abstract # 154
Single-cell RNA-Seq reveals dynamic, random monoallelic gene expression in mammalian cells
Deng, Qiaolin, (, Sweden), Ramsköld, Daniel; Reinius, Björn; Sandberg, Rickard (Stockholm, Sweden)
In diploid eukaryotic organisms, the maternally and paternally derived copies of each gene are often assumed to be expressed
simultaneously at similar levels in the cell. Known exceptions to this include genes undergoing parent-of-origin imprinting, X-
chromosome inactivation, and the recently reported clonally inherited random monoallelic expression of around 10% of autosomal
genes. Here, we present genome-wide transcriptome analyses on 267 single cells derived from crossed mouse preimplantation
embryos (CAST/EiJ x C57BL/6J), containing oocytes to late blastocyst stages. This allowed us perform hitherto most comprehensive
analyses of allele-specific transcriptions in single mammalian cells by SNP (single nucleotide polymorphism) calling. Surprisingly, we
discovered that most autosomal genes are expressed monoallelicly in each cell. This monallelic expression is highly dynamic, random,
and not inherited after cell division. Our study has thus uncovered a novel mode of allelic gene expression characterized by
independent and random allelic transcription events, a phenomenon that is likely consequent to the stochastic nature of transcriptional
bursting. Moreover, our discovery has a fundamental implication for phenotypic variability and disease penetrance as well as severity.
Program/Abstract # 155
Molecular mechanisms underlying sex determination and reprogramming in the mouse
Sekido, Ryohei; Lovell-Badge, Robin (MRC National Institute for Medical Research, UK)
In mammals, sex relies strictly on the presence of the Y chromosome-linked testis-determining gene Sry. In mice, its transient
expression between E10.5 and E12.5 triggers the differentiation of Sertoli cells from the supporting cell precursor lineage, which
would otherwise give granulosa cells in ovaries. Our work has shed light for the first time on the role of SRY in the direct activation of
Sox9 expression. SRY synergistically acts with NR5A1/SF1 through a testis-specific enhancer of Sox9 (TES) to promote Sertoli cell
differentiation. SOX9 expression, once activated, is maintained in Sertoli cells throughout life.
On the other hand, ovarian development is established by an active repression of the testicular pathway rather than it depending
entirely on a passive default pathway. For example, FOXL2 directly binds to TES and represses its enhancer activity in granulosa
cells. Therefore, de-repression of TES activity by Foxl2 ablation in XX mice causes somatic sex reprogramming and eventually
transdifferentiation of ovaries into testes. To elucidate the intrinsic differences between Sertoli cells and granulosa cells by
characterising gene expression profiles, we have taken advantage of the transgenic mouse lines expressing the ecfp reporter gene,
which allow pure populations of Sertoli cells and granulosa cells to be isolated by FACS. We have identified a number of genes
showing sexual dimorphic expression by RNA-seq analyses, and are currently investigating the role(s) of those genes in supporting
cell differentiation.
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