Page 43 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
these possibilities, we examined gonadal development in XY Fgfr2IIIc knockout mice at embryonic days (E) 13.5 and 15.5.
Instead of normal testes, XY Fgfr2IIIcKO mice developed ovotestes or ovaries, phenocopying the gonadal defects of Fgf9KO
and Fgfr2KO mice (on a mixed genetic background). The ovarian regions of XY Fgfr2IIIcKO gonads lacked expression of the
Sertoli cell marker AMH and expressed the female somatic cell marker FOXL2. XY sex reversal extended to germ cells which
expressed SCP3, a marker of meiotic prophase. Subsequent analysis of adult XY Fgfr2IIIc knockout mice revealed that around
were phenotypic females. These data suggest that the XY Fgfr2IIIc knockout embryos with ovotestes develop into the
reported fertile males, while the XY Fgfr2IIIc knockout embryos with ovaries develop into infertile females. Genes such as Wt1
and Sox9 that are critical for gonadal development or sex determination often have subsequent roles in testicular development
and function such as in the maintenance of Sertoli cell identity or in nursing germ cells through spermatogenesis. To examine the
role of FGFR2 in Sertoli cells after the sex determination phase, we conditionally inactivated Fgfr2 (both isoforms) in Sertoli
cells at E13.5 using the AMH-Cre mouse line. These mice did not show obvious defects in testis cord differentiation and
spermatogenesis. Moreover, expression of selected male and female markers was unchanged when compared to control mice. In
summary, FGFR2IIIc is required for testis determination, but is dispensable for subsequent testis cord differentiation.
System-level analysis of sperm maturation in the epididymis. Timothy Karr
Sheri Skerget
Konstantino Petritis
. 1)
Biodesign Inst, PO Box 875001, Arizona State Univ, Tempe, AZ; 2) Center for Proteomics, Translational
Genomics Research Institute, Phoenix, AZ 85004.
The molecular components of complex cellular networks are being identified at a rapidly increasing rate. A key aspect of this
effort is to obtain an understanding of interrelated functional elements and pathways using systems biology approaches. Systems
levels analyses of transcriptomic and proteomic data is central to this effort, but the complexity of these datasets presents a
variety of barriers to achieving a functionally meaningful understanding of system level properties. Our approach has been to
leverage existing knowledge of a tractable system, the sperm proteome, to allow the integration of transcriptomic and proteomic
landscapes within the epididymus, a tissue through which sperm must traverse to properly mature. This approach is based on our
recent studies characterizing the Drosophila, mouse, macaque and other sperm proteomes, coupled with our understanding of
transcriptome dynamics during spermatogenesis. We describe our recent results that have identified a surprising number of
protein changes occurring in the epididymis during transit and correlate these changes with primary gene/protein networks and
mechanisms operating during sperm transit which are functionally necessary for the acquisition of fertilization
Surprisingly, analysis of the three major morphological domains of the epididymis (caput, corpus and cauda)
identified over
protein additions to- and over
protein removals from- the sperm proteome during transit
The overarching
goal of our studies is to unify epididymal transcriptomic and sperm proteomic analyses to reveal how sperm sequentially interact
within different epididymal regions as part of their acquisition of motility and capacitation. The direct relevance of this approach
to humans is highlighted by high levels of functional homology (35%) between the Drosophila and mammalian sperm proteomes,
an approximately 50% overlap between mouse and human sperm proteomes, and a 10% orthology of mouse sperm proteins with
human associated disease genes. This research will therefore assist in obtaining a more complete molecular understanding of
disease gene biology as well as pioneer systems level integration of transcriptomic and proteomic dynamics within a tractable
cellular development process essential to sexual reproduction.
Sonic hedgehog-expressing basal stem cells are the cell-of-origin for bladder cancer. Philip A. Beachy
Kunyoo Shin, Agnes
Lim, Sally Kawano. Stanford Univ Sch Medicine, Stanford, CA.
The idea that human malignancies may originate from adult tissue stem cells derives from the intrinsic ability of stem cells to
self-renew, from their longevity and consequent ability to accrue multiple mutations, and from the phenotypic resemblance of
tumor-propagating cells to stem cells. Experimental tests of this hypothesis have relied on cell-specific genetic manipulation to
induce cancer in murine models, and such studies have produced varying conclusions regarding the cancer cell-of-origin. We
have recently identified the urothelial stem cell through lineage tracing and organoid culture, and have identified some of the
signals that control stem cell proliferative activity. Based on these findings, we examine here the origin of bladder cancer by
prospectively marking and tracking these stem cells in a murine model, selected for its similarity to human carcinogenesis, in
which invasive bladder carcinomas are induced by prolonged exposure to nitrosamine. In this model, we find that Shh-expressing
stem cells in the basal urothelium give rise to the carcinoma, although Shh expression within tumor cells is lost by the time
carcinomas develop. This malignant transformation progresses through epithelial hyperplasia, a precursor lesion consisting of a
thickened urothelium comprising many layers of cytokeratin 5 (CK5)-expressing basal-like cells. Shh expression occurs in basal
but not lumenal layers of this hyperplastic lesion, and these basal cells give rise to the Shh-negative tumor-propagating cells of
the full-fledged carcinoma. Confirming that tumor-propagating cells derive from basal stem cells but not more differentiated
lumenal cells, we showed that prior ablation of Shh-expressing cells rendered the bladder resistant to nitrosamine-induced
formation of epithelial hyperplasia or carcinoma. Our findings thus demonstrate that invasive carcinoma is initiated from the
basal urothelial stem cell and typically progresses through a hyperplastic lesion, thus confirming experimentally the view that
such lesions are not only associated with but actually give rise to epithelial cancers. Our findings further suggest that the
phenotypic properties of mature tumor cells, such as absence of Shh expression, may not provide a sound basis for inferring the
identity of the cancer cell-of-origin.