Page 69 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
prospectively. We have utilized a novel mouse model that involves an out-of-frame Cre allele that sporadically becomes activated
by frameshift reversion in single, isolated cells. Upon cre activation target genes are altered and marked by beta-galactosidase
expression. Thus, by combining our mouse with floxed target genes and a Cre-reporter gene, we can follow the fate of mutant
intestinal stem cells even in the absence of any visible phenotypic changes. Our previous studies demonstrated phenotypic
plasticity following Apc loss in that, while adenoma formation could ensue, the majority of Apc-deficient intestinal crypts
exhibited a normal phenotype. Interestingly, a significant fraction of the normal, Apc-deficient crypts exhibited a growth
advantage raising the possibility of crypt fission as an important, albeit occult, intermediate in tumorigenesis. Here we show that
mutations occurring during intestinal growth/development are more likely to spread via crypt fission resulting in a field of mutant
crypts and hence an altered microenvironment, which is more conducive to transformation. In contrast, later occurring mutant
crypts are more likely to remain isolated, resulting in a microenvironment that is not conducive to transformation. Specifically,
we show that either early Apc loss or Kras activation in intestinal stem cells results in increased crypt fission, but inefficient
transformation. Not surprisingly, the combination of early Apc loss and Kras activation is highly conducive to transformation.
Unexpectedly, late Apc loss and Kras activation was inefficient at altering either crypt fission or transformation. Finally, our
results show that the NSAID, sulindac, can act as a chemopreventive, not only through increasing apoptosis, but also by
inhibiting the increased crypt fission associated with Apc loss. Based on these findings, we suggest that 1) isolated mutant crypts
are not sufficient for tumorigenesis, whereas a field of Apc-deficient crypts creates a rich microenvironment that is highly
conducive to transformation and 2) limiting Apc-deficient field size is a previously unappreciated mechanism of
Neural Tube Defects and Epigenetics of Gene Expression in Non-obese Diabetic (NOD) Mice. Claudia Kappen
Claudia Kruger
J. Michael Salbaum
. 1)
Developmental Biology, Pennington Biomedical Research Center, Baton
Rouge, LA; 2) Laboratory of Gene Regulation, Pennington Biomedical Research Center, Baton Rouge, LA.
Neural tube defects (NTDs) and heart defects are the most prominent congenital defects in pregnancies affected by maternal
diabetes. Using a chemical induction model of diabetes by administration of Streptozotocin (STZ) to FVB mice, we found NTDs
with a frequency of 5.6% in diabetic dams fed regular chow, while a diet specifically formulated for pregnancy elevated the rate
of NTDs to 21.6%. These results indicated not only that NTDs occur with incomplete penetrance, but also that NTD frequency is
epigenetically regulated. Genome-wide expression profiling demonstrated altered gene expression in diabetes-exposed embryos,
which was also accompanied by an increase in variability of gene expression levels, and by altered chromatin marks. To
investigate whether elevated variability of gene expression is a general epigenetic feature associated with maternal diabetes, we
employed a model in which diabetes occurs spontaneously, the non-obese diabetic (NOD) strain of mice. We observed normal
embryonic development in normoglycemic NOD dams, while in diabetic dams, 39% of the embryos had NTDs, confirming
incomplete penetrance. Microarrays and next-generation sequencing revealed deregulation of gene expression in diabetes-
exposed embryos at embryonic day 10.5, and at day 8.5, during neural tube closure. Similar to the STZ model, transcription and
chromatin-binding factors were enriched among differentially expressed genes, again implicating epigenetic mechanisms. A
global increase in variation of gene expression was again evident within the group of diabetes-exposed embryos. Intriguingly,
while embryos from normal pregnancies were distinguishable from those of diabetic dams by virtue of their differential
expression repertoires, the diabetes-exposed embryos with NTDs displayed distinct patterns only when clustering approaches
were based on the characteristic of variability. We therefore developed a variation-based model that can explain the incomplete
penetrance of the NTD phenotype in embryos of diabetic FVB and NOD mice, and by inference, the phenomenon of incomplete
penetrance in inbred strains in general. This model provides a new paradigm for discovery of genes that are involved, causally or
as modifiers, in the pathogenesis of neural tube defects and other pathologies.
Impaired Maternal Cardiovascular Adaptation to Pregnancy in HcB-8 Mice. Jasmin Kristianto
Jacqueline Fisher,
Shannon Phillips, Michael Johnson, Suzanne Litscher, Robert Blank. University of Wisconsin-Madison, Madison, WI.
Preeclampsia (~8% of all pregnancies), fetal loss (~15% of all pregnancies) and intrauterine growth restriction (IUGR) (~10%
of newborns) are common pregnancy complications. Our laboratory has isolated a pleiotropic quantitative trait locus (QTL) in
mouse chromosome 4 in recombinant congenic mice (HcB-8 and HcB-23) mice that harbors differentially expressed Ece1 (the
gene encoding Endothelin Converting Enzyme 1) alleles resulting in differences in BP, litter size, birth weight, and frequency of
fetal loss. Reduced Ece1 expression in HcB-8 male mice is associated with increase in blood pressure (BP), larger heart size, less
compliant and smaller carotid arteries diameter than HcB-23. The non pregnant HcB-8 females also have higher BP than HcB-23
(116 7/90 4
v 103 3/75 3, p =0.016 (systolic)/p= 0.007 (diastolic)). Furthermore, HcB-8 litters are smaller than HcB-23 litters
+ 1.8 v 5.8 + 2.0, p 10-3) and HcB-8 pups are lighter at birth (1.2 + 0.2 g v 1.5 + 0.2 g, p 10-20). We hypothesized that
chromosome 4 QTL mediates vascular adaptation in response to pregnancy and that differential Ece1 expression manifests in
differences in reproductive phenotypes. HcB-8 females experience higher rate of pregnancy loss (PL) (5 PL/7 dams v 1 PL/8
dams, p= 0.04) based on the finding of vaginal plugs and the failure to produce litter after successful copulation. Placental
insufficiency may be the contributing factor. Pregnant HcB-8 females harvested at 17.5 dpc have smaller placental weight than
HcB-23 17.5 dpc females (0.085 0.006 v 0.10 0.010, p =0.010). However, pregnant HcB-8 females have larger fetal to placental
ratio than HcB-23 (9 1 v 6 0.5, p = 0.009). Pregnant HcB-8 females also have larger fractional heart sizes than HcB-23
(0.006 0.0007
v 0.004 0.0003, p=0.001). Non-pregnant HcB-8 females expressed nearly ~ 5 fold higher brain natriuretic peptide,