E2f4 and E2f5 are essential for the development of the male reproductive system

The E2F transcription factors are primarily implicated in the regulation of entry and exit from the cell cycle. However, in vivo studies have established additional roles for E2Fs during organ development and homeostasis. With the goal of addressing the intestinal requirements of E2f4 and E2f5, we crossed mice carrying Vil-cre, E2f4 conditional and E2f5 germline alleles. E2f4 deletion had no detectable effect on intestinal development. However, E2f4f/f;E2f5+/−;Vil-cre males, but not E2f4f/f;Vil-cre littermates, were unexpectedly sterile. This defect was not due to defective spermatogenesis. Instead, the seminiferous tubules and rete testes showed significant dilation, and spermatozoa accumulated aberrantly in the rete testis and efferent ducts. Our data show that these problems result from defective efferent ducts, a tissue whose primary function is to concentrate sperm through fluid absorption. First, Vil-cre expression, and consequent E2F4 loss, was specific to the efferent ducts and not other reproductive tract tissues. Second, the E2f4f/f;E2f5+/−;Vil-cre efferent ducts had completely lost multiciliated cells and greatly reduced levels of critical absorptive cell proteins: aquaporin1, a water channel protein, and clusterin, an endocytic marker. Collectively, the observed testis phenotypes suggest a fluid flux defect. Remarkably, we observed rete testis dilation prior to the normal time of seminiferous fluid production, arguing that the efferent duct defects promote excessive secretory activity within the reproductive tract. Finally, we also detect key aspects of these testis defects in E2f5−/− mice. Thus, we conclude that E2f4 and E2f5 display overlapping roles in controlling the normal development of the male reproductive system.National Institutes of Health (U.S.) (Grant NIH-P01 CA42063

Loss of pRB and p107 disrupts cartilage development and promotes enchondroma formation

The pocket proteins pRB, p107 and p130 have established roles in regulating the cell cycle through the control of E2F activity. The pocket proteins regulate differentiation of a number of tissues in both cell cycle-dependent and -independent manners. Prior studies showed that mutation of p107 and p130 in the mouse leads to defects in cartilage development during endochondral ossification, the process by which long bones form. Despite evidence of a role for pRB in osteoblast differentiation, it is unknown whether it functions during cartilage development. Here, we show that mutation of Rb in the early mesenchyme of p107-mutant mice results in severe cartilage defects in the growth plates of long bones. This is attributable to inappropriate chondrocyte proliferation that persists after birth and leads to the formation of enchondromas in the growth plates as early as 8 weeks of age. Genetic crosses show that development of these tumorigenic lesions is E2f3 dependent. These results reveal an overlapping role for pRB and p107 in cartilage development, endochondral ossification and enchondroma formation that reflects their coordination of cell-cycle exit at appropriate developmental stages.Virginia and D.K. Ludwig Fund for Cancer Research (Fellowship)National Cancer Institute (U.S.) (Grant CA121921

Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1[superscript −/−] mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1[superscript −/−] adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.Virginia and D.K. Ludwig Fund for Cancer ResearchNational Science Foundation (U.S.)National Cancer Institute (U.S.

E2F4 cooperates with pRB in the development of extra-embryonic tissues

August 1, 2010The retinoblastoma gene, RB-1, was the first identified tumor suppressor. Rb[superscript −/−] mice die in mid-gestation with defects in proliferation, differentiation and apoptosis. The activating E2F transcription factors, E2F1–3, contribute to these embryonic defects, indicating that they are key downstream targets of the retinoblastoma protein, pRB. E2F4 is the major pRB-associated E2F in vivo, yet its role in Rb[superscript −/−] embryos is unknown. Here we establish that E2f4 deficiency reduced the lifespan of Rb[superscript −/−] embryos by exacerbating the Rb mutant placental defect. We further show that this reflects the accumulation of trophectoderm-like cells in both Rb and Rb;E2f4 mutant placentas. Thus, Rb and E2f4 play cooperative roles in placental development. We used a conditional mouse model to allow Rb[superscript −/−];E2f4[superscript −/−] embryos to develop in the presence of Rb wild-type placentas. Under these conditions, Rb[superscript −/−];E2f4[superscript −/−] mutants survived to birth. These Rb[superscript −/−];E2f4[superscript −/−] embryos exhibited all of the defects characteristic of the Rb and E2f4 single mutants and had no novel defects. Taken together, our data show that pRB and E2F4 cooperate in placental development, but play largely non-overlapping roles in the development of many embryonic tissues.David H. Koch Institute for Integrative Cancer Research at MIT. Pearl Staller Graduate Student FundNational Institutes of Health (U.S.) (Grant GM53204)National Institutes of Health (U.S.) (Grant CA121921

Variations in dysfunction of sister chromatid cohesion in esco2 mutant zebrafish reflect the phenotypic diversity of Roberts syndrome

Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC), cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes

Five-year costs from a randomised comparison of bilateral and single internal thoracic artery grafts

Background: The use of bilateral internal thoracic arteries (BITA) for coronary artery bypass grafting (CABG) may improve survival compared with CABG using single internal thoracic arteries (SITA). We assessed the long-term costs of BITA compared with SITA. Methods: Between June 2004 and December 2007, 3102 patients from 28 hospitals in seven countries were randomised to CABG surgery using BITA (n=1548) or SITA (n=1554). Detailed resource use data were collected from the initial hospital episode and annually up to 5 years. The associated costs of this resource use were assessed from a UK perspective with 5 year totals calculated for each trial arm and pre-selected patient subgroups. Results: Total costs increased by approximately £1000 annually in each arm, with no significant annual difference between trial arms. Cumulative costs per patient at 5-year follow-up remained significantly higher in the BITA group (£18 629) compared with the SITA group (£17 480; mean cost difference £1149, 95% CI £330 to £1968, p=0.006) due to the higher costs of the initial procedure. There were no significant differences between the trial arms in the cost associated with healthcare contacts, medication use or serious adverse events. Conclusions: Higher index costs for BITA were still present at 5-year follow-up mainly driven by the higher initial cost with no subsequent difference emerging between 1 year and 5 years of follow-up. The overall cost-effectiveness of the two procedures, to be assessed at the primary endpoint of the 10-year follow-up, will depend on composite differences in costs and quality-adjusted survival

Single-molecule transcript counting of stem-cell markers in the mouse intestine

available in PMC 2012 July 1.Determining the molecular identities of adult stem cells requires technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies indicated that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but a detailed analysis of their spatial co-expression has not been feasible. Here we apply three-colour single-molecule fluorescent in situ hybridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, ageing and regeneration. We find that the expression of all markers overlaps at crypt-base cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl1 tuft cells, distributed throughout the crypt axis, co-express Lgr5 and other stem-cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the markers following irradiation, indicating their potential role in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and in tumours, guiding complementary functional studies to evaluate their stem-cell properties.National Institutes of Health (U.S.) (U54CA143874)National Cancer Institute (U.S.) (Physical Sciences Oncology Center at MIT (U54CA143874))National Institutes of Health (U.S.) (NIH Pioneer award (1DP1OD003936))National Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051)European Molecular Biology Organization (postdoctoral fellowship)Human Frontier Science Program (Strasbourg, France)Machiah FoundationHoward Hughes Medical Institute (Gilliam fellowship

Zebrafish Hagoromo mutants upregulate fgf8 post-embryonically and develop neuroblastoma

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain–containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841–50)National Cancer Institute (U.S.) (Grant CA106416

Land‐use change differentially affects endemic, forest and open‐land butterflies in Madagascar

The conversion of tropical forests into agriculture reduces biodiversity dramatically. However, species might differ in their responses, depending on their habitat specialisation and geographic origin. In this study, we assess how butterfly assemblages differ between old-growth forests, forest fragments, forest-derived vanilla agroforests, fallow-derived vanilla agroforests, woody fallows, herbaceous fallows, and rice paddies in Madagascar. We recorded 88 butterfly species, of which 65 species are endemic to Madagascar. Land-use types with woody vegetation sustained many endemic (mean: 6.8 species) and forest butterfly species (mean: 4.8 species). Rice paddies and herbaceous fallows were richer in open-land species (mean: 7.6 species) and poorer in forest species (mean: 1.7 species) compared to other land-use types. Compared to herbaceous fallows, fallow-derived vanilla agroforests hosted more endemic (+164%) and forest (+239%) species. Richness of open-land species in forest-derived vanilla agroforests was six times higher than in forest fragments. Overall, 27% of species occurred exclusively in one land-use type and 19% of all species when old-growth forests were excluded. We found the highest number of exclusive species in forest-derived vanilla agroforests. We conclude that all studied land-use types contribute to butterfly conservation in our study region. Especially the woody vegetation in forest fragments, vanilla agroforests, and woody fallows supports a high butterfly diversity and is pivotal for maintaining a broad diversity of forest butterflies in the agricultural matrix. Our study highlights the importance of preserving the diversity of small-scale land-use types, including agroforestry, forests and fallow land in this tropical biodiversity hotspot