Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary

AbstractThe number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-1) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-1-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination

K-Ras and β-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder

The human fibroblast growth factor receptor 3 (FGFR3) gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 activating mutations as a ‘driver’ of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age. Furthermore, even when the Fgfr3 mutations were introduced together with K-Ras or β-catenin (Ctnnb1) activating mutations, no urothelial dysplasia or UCC was observed. Interestingly, however, owing to a sporadic ectopic Cre recombinase expression in the skin and lung of these mice, Fgfr3 mutation caused papilloma and promoted lung tumorigenesis in cooperation with K-Ras and β-catenin activation, respectively. These results indicate that activation of FGFR3 can cooperate with other mutations to drive tumorigenesis in a context-dependent manner, and support the hypothesis that activation of FGFR3 signaling contributes to human cancer

Canonical Wnt Signaling Induces BMP-4 to Specify Slow Myofibrogenesis of Fetal Myoblasts

Background The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes. Methods We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses. Results We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts. Conclusions These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype

Loss of pancreas upon activated Wnt signaling is concomitant with emergence of gastrointestinal identity

Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that β -catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries

Optic cup and facial patterning defects in ocular ectoderm β-catenin gain-of-function mice

BACKGROUND: The canonical Wnt signaling pathway has a number of critical functions during embryonic development and, when activated aberrantly, in the genesis of cancer. Current evidence suggests that during eye development, regulation of Wnt signaling is critical for patterning the surface ectoderm that will contribute to multiple components of the eye. Wnt signaling loss-of-function experiments show that a region of periocular ectoderm will form ectopic lentoid bodies unless the Wnt pathway modifies its fate towards other structures. Consistent with this, Wnt signaling gain of function in the ocular region ectoderm results in a suppression of lens fate. RESULTS: Here we demonstrate that ectoderm-specific Wnt signaling gain-of-function embryos exhibit additional defects besides those noted in the lens. There are profound facial defects including a foreshortened snout, malformation of the nasal region, and clefting of the epidermis along the ocular-nasal axis. Furthermore, despite the restriction of Wnt pathway gain-of-function to the surface ectoderm, the optic cup is inappropriately patterned and ultimately forms a highly convoluted, disorganized array of epithelium with the characteristics of retina and retinal pigmented epithelium. CONCLUSION: We suggest that activation of the Wnt pathway in surface ectoderm may disrupt the normal exchange of signals between the presumptive lens and retina that coordinate development of a functional eye

Promoter methylation regulates cyclooxygenase expression in breast cancer

INTRODUCTION: Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract. METHODS: Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential. RESULTS: We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis). CONCLUSIONS: These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype

β-catenin Initiates Tooth Neogenesis in Adult Rodent Incisors

β-catenin signaling is required for embryonic tooth morphogenesis and promotes continuous tooth development when activated in embryos. To determine whether activation of this pathway in the adult oral cavity could promote tooth development, we induced mutation of epithelial β-catenin to a stabilized form in adult mice. This caused increased proliferation of the incisor tooth cervical loop, outpouching of incisor epithelium, abnormal morphology of the epithelial-mesenchymal junction, and enhanced expression of genes associated with embryonic tooth development. Ectopic dental-like structures were formed from the incisor region following implantation into immunodeficient mice. Thus, forced activation of β-catenin signaling can initiate an embryonic-like program of tooth development in adult rodent incisor teeth

The development of a knowledge base for basic active structures: an example case of dopamine agonists

<p>Abstract</p> <p>Background</p> <p>Chemical compounds affecting a bioactivity can usually be classified into several groups, each of which shares a characteristic substructure. We call these substructures "basic active structures" or BASs. The extraction of BASs is challenging when the database of compounds contains a variety of skeletons. Data mining technology, associated with the work of chemists, has enabled the systematic elaboration of BASs.</p> <p>Results</p> <p>This paper presents a BAS knowledge base, BASiC, which currently covers 46 activities and is available on the Internet. We use the dopamine agonists D1, D2, and Dauto as examples and illustrate the process of BAS extraction. The resulting BASs were reasonably interpreted after proposing a few template structures.</p> <p>Conclusions</p> <p>The knowledge base is useful for drug design. Proposed BASs and their supporting structures in the knowledge base will facilitate the development of new template structures for other activities, and will be useful in the design of new lead compounds via reasonable interpretations of active structures.</p