Page 51 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
51
uncommitted lung mesenchymal cells. Mouse embryonic lung that was induced to overexpress FGF9 develops mesenchymal
hyperplasia with histology that mimics that of type I PPB, suggesting that FGF9 might be involved in the pathogenesis of PPB.
The genetic origins of PPB were mapped to loss-of-function mutations in the microRNA (miRNA) processing gene, DICER1.
We find that Dicer1 ablation in developing lung epithelium mimics the early cystic stage of PPB. Immunohistochemical studies
of human PPB tumors often show decreased DICER1 in lung epithelium. These observations suggest a model to explain the
pathogenesis of PPB in which decreased epithelial miRNAs result in overexpression of an epithelial gene(s) and production of a
factor(s) that stimulates the proliferation of adjacent mesenchyme, predisposing the mesenchyme to neoplastic transformation.
Preliminary studies show that Fgf9 is directly regulated by DICER1-mediated miRNA pathways and could initiate early
pathogenic events that lead to PPB.
48
Genome-wide microRNA and mRNA profiling in mouse liver development implicates mir302b and mir20a in repressing
TGFb signaling.
Wei Wei
1
,
Juan Hou
1
,
Olivia Alder
1
,
Xin Ye
3
,
Sam Lee
1,4
,
Rebecca Cullum
1
,
Andy Chu
2
,
Yongjun Zhao
2
,
Stephanie Warner
3,4
,
Darryl Knight
3,4
,
Decheng Yang
3,4
,
Steven Jones
2,4
,
Marco Marra
2,4
,
Pamela Hoodless
1,4
. 1)
Terry Fox
Laboratory, BC Cancer Agency, Vancouver, BC, Canada; 2) Genome Sciences Centre, BC Cancer Agency, Vancouver, BC,
Canada; 3) The Institute for Heart and Lung Health, St. Paul’s Hospital, Vancouver, BC, Canada; 4) University of British
Columbia, Vancouver, BC, Canada.
During mouse embryonic development, liver progenitor cells are specified from definitive endoderm at the 7-8 somite stage
(
embryonic day (E) 8.5). At E9.5, cells in the liver domain, known as hepatoblasts, undergo epithelial-to-mesenchymal transition
(
EMT), invade the surrounding septum transversum, form the liver bud and rapidly proliferate. Around E14.5-15.5, hepatoblasts
start to differentiate into hepatocytes or cholangiocytes, which mature into the major functional cells of the liver. MicroRNAs
(
miRNAs) are small, non-coding RNA molecules that can regulate gene expression by binding to complementary sequences
within mRNAs. MicroRNAs have been shown to regulate developmental processes, such as proliferation, differentiation and
apoptosis; however, the identity of miRNAs and their functions during liver development are largely unknown. We investigated
the miRNA and gene expression profiles for E8.5 endoderm, E14.5 DLK1+ liver cells (hepatoblasts), and adult liver by
employing Illumina sequencing. miRNAs were found to be abundantly expressed at all three stages. Using K-means clustering
analysis, thirteen miRNA clusters with distinct temporal expression patterns were identified. mir302b, an endoderm-enriched
miRNA, was identified as a miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm.
We validated the expression of mir302b in the endoderm by whole-mount in situ hybridization. Interestingly, mir20a, the most
highly expressed miRNA in the endoderm library, was also predicted to regulate some of the same targets as mir302b. By using
luciferase and cell-based assays, we show that mir302b and mir20a are able to inhibit TGF-mediated epithelial-to-mesenchymal
transition (EMT)through targeting Tgfbr2, suggesting their potential roles in maintaining the epithelial status of endoderm.
Moreover, TGF1 can repress liver specification in an embryonic stem cell differentiation model. Collectively, we have identified
dynamic patterns of individual miRNAs during liver development, as well as miRNA networks that could be essential for the
specification and differentiation of liver progenitors.
49
Taking a different pathway: Notchless plays with p53 and Wnt during embryogenesis. Amy C. Lossie
1,2
,
Chiao-Ling Lo
1,3
,
Jeremy Sherrill
4
. 1)
Dept Animal Sci, Purdue Univ, West Lafayette, IN; 2) Dept of Medicine, Indiana University School of
Medicine, Indianapolis, IN; 3) PULSe Interdisciplinary Graduate Program, Purdue University, West Lafayette, IN; 4)
Department of Biological Sciences, Purdue University, West Lafayette, IN.
Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-
fetal interactions during pregnancy. Through a positional cloning strategy, we discovered that mutations in
Notchless
(
Nle1
)
lead
to embryonic lethality during peri-implantation in mice. Nle1 is a member of the WD40-repeat protein family, and is thought to
signal via the canonical Notch pathway. In invertebrates and lower vertebrates, the Notch pathway is critical for directing cell
fate prior to gastrulation. However, the role of Notch signaling during the earliest stages of mammalian development is unclear,
as gene targeting studies of Notch family members and factors that are necessary for Notch signal transduction, demonstrate that
Notch signaling is dispensable for gastrulation in mice. The phenotype of
Nle1
mutant embryos is much more severe than
single
Notch
receptor mutations or even in animals where Notch signaling is blocked. To test the hypothesis that
Nle1
functions
in multiple signaling pathways during pre-implantation development, we examined expression of multiple
Notch
downstream
target genes, as well as select members of the
Wnt
and
Trp53
pathways in wild-type and mutant embryos. Surprisingly, we saw
no indication that the
Notch
pathway is disrupted in mutant embryos;
Notch
receptors, ligands and downstream targets showed
normal expression levels. Instead, we found that members of the
Wnt
pathway are downregulated in
Nle1
mutants,
while
Trp53
and
Cdkn1a
were upregulated in a stage-specific manner. Induction of
Cdkn1a
was highest in morulae and full
blastocysts, while
Trp53
overexpression was confined to hatched blastocysts. Our results refute the possibility that Nle1 is a
negative regulator of Notch signaling during mammalian pre-implantation development, as mutation of
Nle1
does not lead to
increased expression of downstream target genes. Instead, our data implicate Nle1 in Wnt signaling, cell cycle arrest via Cdkn1a,
and p53-mediated apoptosis. Although Notch signaling is dispensable in mice prior to gastrulation, Wnt signaling is not. Deletion
of
Wnt3
leads to failure prior to primitive streak formation, and multiple ligands and receptors are detected in blastocysts and the
uterus during peri-implantation. As p53 inhibits self-renewal and promotes differentiation in ES cells, Nle1 and its co-opted Wnt
and p53 pathways could provide novel targets for the design of therapeutic interventions for infertility.