Page 81 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
Stem Cells and Germ Cells
Differential regulation of Sox10 during the maintenance of the melanocyte lineage and establishment of the melanocyte
stem cell. Melissa L Harris
William J Pavan. Genetic Disease Research Branch, National Human Genome Research Institute,
NIH, Bethesda, MD.
During embryogenesis SOX10 functions within neural crest cells to upregulate master switch transcription factors needed for
specifying different neural crest sublineages. Melanocytes, being a neural crest derivative, use SOX10 to initiate the expression
of the transcription factor, Mitf. SOX10 and MITF together then drive the survival and differentiation of the melanocyte lineage
embryonically. Postnatally, melanocytes are incorporated into the hair follicle and those that reside in a region called the hair
bulge give rise to the melanocyte stem cells (McSCs). These McSCs replenish the melanocyte system of the hair follicle
throughout adult hair cycling, however it is unknown how this population is established. We hypothesize that through the
differential regulation of Sox10, postnatal melanocytes can maintain their lineage specification while also allowing a portion of
them to acquire the role of a McSC. In support of this idea, we find that McSCs express SOX10 and MITF but remain
undifferentiated. By knocking out Sox10 in the melanocyte lineage postnatally (Sox10fl; Tg(Tyr::CreER)), we also show that
hair follicle melanocytes need Sox10 for their survival. However, by gain-of-function analysis (Tg(DctSox10)) we demonstrate
that overexpression of Sox10 results in premature differentiation of the McSC, their eventual loss, and consequently leads to
early hair graying. This suggests that Sox10 must be downregulated in order for the McSC to be established. In an attempt to
dissect whether SOX10s role in McSCs is simply to regulate Mitf we asked whether haploinsufficiency for Mitf (Mitfvga9) can
rescue hair graying in Tg(DctSox10) animals. Surprisingly, the combination of Mitfvga9 and Tg(DctSox10) exacerbates hair
graying and suggests that MITF negatively regulates Sox10 in McSCs. Together these data suggest a mechanism where SOX10
can be present to support the maintenance of the melanocyte lineage while also be inhibited from driving differentiation in the
McSC population. These data illustrate how tissue-specific stem cells can arise from lineage-specified precursors, and how this
can occur through the regulation of the very transcription factors important in defining that lineage.
Systems-level analysis of embryonic pluripotency and lineage-specific differentiation. David Emlyn Parfitt
Mariano Alvarez
Celine Lefebvre
Andrea Califano
Michael Shen
. 1)
Herbert Irving Comprehensive
Cancer Center, Columbia University, New York, NY; 2) Department of Genetics and Development; 3) Joint Centers for Systems
Biology; 4) Department of Biomedical Informatics; 5) Herbert Irving Comprehensive Cancer Center, Columbia University
Medical Center, New York, NY.
The molecular mechanisms of pluripotency maintenance in cell culture and its loss during embryonic development in vivo are
of central importance in stem cell biology. To understand these mechanisms, it is essential to perform a complete analysis of their
master regulators, both known as well as novel. For this purpose, we have constructed the first genome-wide molecular
interaction networks (interactomes) for mouse epiblast stem cells (EpiSC) and embryonic stem cells (ESC). To generate these
interactomes, we applied the experimentally validated reverse-engineering algorithms ARACNe and MINDy to expression data
gathered from EpiSCs and ESCs undergoing a broad range of differentiation events. This methodology enables the unbiased de
novo inference of transcriptional and post-translational gene interactions from large datasets of gene expression profiles. To
determine which genes in these networks are most critical for regulating specific ES and EpiSC properties, thus corresponding to
master regulators, we are conducting analyses of gene expression signatures using the MARINa algorithm. Using these signatures
to interrogate the EpiSC and ESC interactomes, we can identify master regulators of in vivo and in vitro cellular processes of
interest. In particular, we have generated gene expression signatures that capture the cascade of molecular changes associated
with transitions from pluripotent states towards specific lineage-restricted cell types. For example, we have generated time course
signatures from EpiSCs treated with the Nodal inhibitor SB431542, in order to understand how Nodal/Activin signaling is
involved in ESC and EpiSC maintenance. Similarly, we have generated signatures corresponding to post-implantation embryos to
investigate the mechanisms regulating the different states of pluripotency that exist before and after the onset of gastrulation. We
anticipate that these analyses will highlight known and/or novel genes that are most capable of faithfully recapitulating the
transcriptional and epigenetic signatures of pluripotency, either individually or in combination, and thereby provide molecular
insights into somatic cell reprogramming and transdifferentiation.
Isolation of a Genetically Stable ES Cell Subline from Chromosomally Instable C57BL/6 ES Cells. Thomas L Saunders
Virginia Zawistowski
Keith Childs
Elizabeth Hughes
. 1)
Transgenic Animal Model Core, University of Michigan, Ann
Arbor, MI; 2) Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan Medical
School, Ann Arbor, MI.
Embryonic Stem (ES) cells derived from the mouse are a powerful tool to manipulate the mouse genome and generate new
mouse models of human disease. Gene targeting in C57BL/6 mouse ES cells produces genetically engineered mice on a C57BL/6