Page 59 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA.
The highly conserved T-box transcription factor Tbx18 is expressed in a complex pattern during development of the vertebrate
embryo. Tbx18-null phenotypes, including loss of somite polarity, decreased proliferation of the cardiac conduction system, and
patterning pathologies of the ureter suggest a complex regulatory mechanism in which Tbx18 acts in these different tissues. In
concordance with these known phenotypes, here we describe a new Tbx18 mutation, 12Gso, a chromosome translocation located
nearly 80kb downstream of the Tbx18 gene that perturbs the spatio-temporal expression pattern of the gene. We show that this
translocation physically separates enhancers necessary for proper expression in the somites and urinary system. Using a LacZ
reporter system in transgenic mice we identified a urinary enhancer that confers the proper expression of Tbx18 beginning at
E9.0 in the urogenital mesenchyme and persisting through birth in a diminishing proximal-distal wave of the ureter smooth
muscle layer. To further understand the genetic pathways through which Tbx18 executes its role during development, we
performed chromatin immunoprecipitiation using a custom Tbx18 antibody in C2C12 mouse cells, which have properties similar
to mesenchymal stem cells, followed by hybridization to extended promoter arrays (ChIP-chip). To complement this ChIP-chip
study, we performed siRNA knockdown of Tbx18 mRNA in C2C12 followed by hybridization to expression arrays. We
combined the results of these two experiments to identify putative direct targets of Tbx18. We identified significant enrichment
in genes involved in cell cycle regulation, tissue patterning, and cell proliferation/apoptosis, among other biological processes.
The targets identified in this study support a role for Tbx18 in regulation of cell proliferation/apoptosis as previously reported by
others, but also suggest additional roles in regulating components of the cell cycle. These newly discovered targets suggest a
potential role for Tbx18 in other types of biology and may hint towards previously undiscovered phenotypes.
Somatic mosaicism detected using the Mouse Diversity Genotyping Array reveals tissue-specific mutation patterns
associated with the
phenotype. Susan T. Eitutis
Andrea E. Wishart, Kathleen A. Hill. Department of Biology, The
University of Western Ontario, London, ON, Canada.
A genomic perspective to studying somatic mosaicism can be approached using the Mouse Diversity Genotyping Array
MDGA). First, to establish an optimal genome-wide target, an accurate and complete annotation of single nucleotide
polymorphism (SNP) probes was completed. This final probe list was established by selecting only probes 25 nucleotides in
length that mapped uniquely to the mouse genome. Probe sets included in the final list had to fit these criteria for both the
forward and reverse strand probes for each SNP. The selected probes have an average spacing of 4887 nucleotides. Genotypes for
the spleen and the cerebellum, tissues that differ in cell type and proliferation rate, were determined using the final list of 526,808
SNP probes for two premature aging
mice and two wild-type (WT) mice. Post genotyping, probes were again
filtered based on genotype cluster analysis and the possible effects of copy number variation were considered. This generated a
relaxed list of 497,333 SNP probes and a stringent list of 470,147 SNP probes. Differences in genotype calls between tissues
were interpreted as putative mutations. In WT compared to
mice, there is a greater difference in the number of mutations
between tissues (p0.001). Each sample had a unique distribution of mutations across the genome (p0.001). There is an
underrepresentation of putative mutations on the X chromosome consistent with the predicted negative selection. Chromosomes 9
and 17 had an overrepresentation of mutations. Chromosomes 1, 8, and 12 had an overrepresentation of mutations in
Spleen of
mice had an overrepresentation of mutations on chromosomes 12, 13, 14, and 19. The
cerebellum had an
overrepresentation of putative mutations on chromosome 13. Two mutations that were specific to the
cerebellum are predicted
to affect two genes on chromosomes 8 and 12. The
spleen-specific mutations potentially affect nine genes on seven
chromosomes. Identification of putative mutations unique to one tissue and one genotype, and those potentially affecting gene
function is the essential advance in understanding origins and mechanisms of mutations during development.
CRY1-PHR(313-426): A Key Domain for Negative Feedback Repression and Circadian Clock Function in Mice. Sanjoy
Kumar Khan
Haiyan Xu
Maki Ukai-Tadenuma
Hiroki Ueda
Andrew Liu
. 1)
Biological Sciences, University of
Memphis, Memphis, TN; 2) RIKEN Center for Developmental Biology, Kobe, Japan.
In mammals, daily physiological processes such as sleep/wake cycle, hormone production and metabolism are driven by an
endogenous time-keeping system, namely the circadian clock. The suprachiasmatic nuclei (SCN) of the anterior hypothalamus
are the master oscillator that regulates circadian behavioral and physiological activities. The circadian clock mechanism is based
on an autoregulatory negative feedback loop, in which two transcriptional activators BMAL1 and CLOCK form a heterodimer
and activate the rhythmic transcription of genes including period (Per1, Per2 and Per3) and cryptochromes (Cry1 and Cry2); the
resultant PER and CRY proteins translocate to the nucleus where CRYs act as repressors to inhibit transcription by directly
interacting with the BMAL1/CLOCK heterodimer. Despite similarities in sequence, domain structure and biochemical activity,
CRY1 and 2 play distinct roles in clock function. Loss-of-function studies show that single knockout mice have opposing
circadian phenotypes; Cry1-/- mice display shorter behavioral period length, whereas Cry2-/- mice display longer period. In cell-
autonomous clock models, Cry1 deletion resulted in arrhythmicity or transient rhythms, whereas Cry2 knockout resulted in
longer period and higher amplitude. Recently, using a novel genetic complementation assay in Cry1-/-:Cry2-/- mouse fibroblasts,
we demonstrated that Cry1 alone is able to maintain cell-autonomous circadian rhythms, while Cry2 cannot (Cell, 2011). We
further identified a domain within CRY1s photolyase homology region (PHR), designated as CRY1-PHR(313-426), that is
required for clock function and differentiates CRY1 from CRY2 (JBC, 2012). Our studies also revealed that the C-terminal tail
domain is critical for regulating period length, which provides a mechanistic basis for the opposing period length phenotypes of