Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics1. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

The mutation brachypodism (bp) alters the length and number of bones in the limbs of mice but spares the axial skeleton. It illustrates the importance of specific genes in controlling the morphogenesis of individual skeletal elements in the tetrapod limb. We now report the isolation of three new members of the transforming growth factor-beta (TGF-beta) superfamily (growth/differentiation factors (GDF) 5,6 and 7) and show by mapping, expression patterns and sequencing that mutations in Gdf5 are responsible for skeletal alterations in bp mice. GDF5 and the closely related GDF6 and GDF7 define a new subgroup of factors related to known bone- and cartilage-inducing molecules, the bone morphogenetic proteins (BMPs). Studies of Bmp5 mutations in short ear mice have shown that at least one other BMP gene is also required for normal skeletal development. The highly specific skeletal alterations in bp and short ear mice suggest that different members of the BMP family control the formation of different morphological features in the mammalian skeleton.

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5 cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5 cells that continuously attempt to replenish the Lgr5 CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

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Mouse embryos bearing hypomorphic and conditional null Fgf8 mutations have small and abnormally patterned telencephalons. We provide evidence that the hypoplasia results from decreased Foxg1 expression, reduced cell proliferation and increased cell death. In addition, alterations in the expression of Bmp4, Wnt8b, Nkx2.1 and Shh are associated with abnormal development of dorsal and ventral structures. Furthermore, nonlinear effects of Fgf8 gene dose on the expression of a subset of genes, including Bmp4 and Msx1, correlate with a holoprosencephaly phenotype and with the nonlinear expression of transcription factors that regulate neocortical patterning. These data suggest that Fgf8 functions to coordinate multiple patterning centers, and that modifications in the relative strength of FGF signaling can have profound effects on the relative size and nature of telencephalic subdivisions.KEY WORDS: Fgf8, Forebrain, Patterning, Mouse Development 133, 1831Development 133, -1844Development 133, (2006 The previous studies concentrated on the phenotype of the Fgf8 TelKO and Fgf8 Null/Neo mutant telencephalon beginning at E10.5 and did not examine primary phenotypes in the neural plate or just following neural tube closure. Because prosencephalic expression of Fgf8 begins at neural plate stages (Crossley and Martin, 1995;Shimamura and Rubenstein, 1997; Crossley et al., 2001), it is essential to investigate the mutant phenotypes shortly after this stage to elucidate the mechanisms underlying Fgf8 TelKO and Fgf8 Null/Neo phenotypes. Therefore, here we report studies of Fgf8 dose-dependent effects on neural plate and early post-neurulation stage embryos. Furthermore, Storm et al. (Storm et al., 2003) focused on the effects of reducing Fgf8 dose on telencephalic midline development; here we concentrate on the effect of reducing Fgf8 dose on telencephalic patterning centers, regionalization and growth.We report our finding that specification of the prosencephalon is intact in Fgf8 mutants; however, a major reduction in Foxg1 expression, a reduced mitotic index, and increased apoptosis contribute to telencephalic hypoplasia. We also demonstrate that Fgf8 regulates the expression of Bmp4, Wnt8b and Shh, which in turn affect patterning of both dorsal and ventral structures. Nonlinear effects of Fgf8 dose on Bmp4 expression correlate with a holoprosencephaly phenotype and alterations in the expression of transcription factors that regulate neocortical patterning. The nexus of regulatory interactions between patterning centers that control gradients of transcription factor expression demonstrates that modifications in the relative strength of FGF/BMP/WNT/SHH signaling have profound effects on the relative size and nature of telencephalic subdivisions that are likely to contribute to their phylogenetic and intra-individual diversity. MATERIALS AND METHODS Mice and genotypingAll Fgf8 mutant alleles were maintained on a mixed 129/CD1 Swiss genetic background. Fgf8 Null/+ )embryos. PCR genotyping was performed as described pr...

A functional skeletal system requires the coordinated development of many different tissue types, including cartilage, bones, joints, and tendons. Members of the Bone morphogenetic protein (BMP) family of secreted signaling molecules have been implicated as endogenous regulators of skeletal development. This is based on their expression during bone and joint formation, their ability to induce ectopic bone and cartilage, and the skeletal abnormalities present in animals with mutations in BMP family members. One member of this family, Growth/differentiation factor 5 (GDF5), is encoded by the mouse brachypodism locus. Mice with mutations in this gene show reductions in the length of bones in the limbs, altered formation of bones and joints in the sternum, and a reduction in the number of bones in the digits. The expression pattern of Gdf5 during normal development and the phenotypes seen in mice with single or double mutations in Gdf5 and Bmp5 suggested that Gdf5 has multiple functions in skeletogenesis, including roles in joint and cartilage development. To further understand the function of GDF5 in skeletal development, we assayed the response of developing chick and mouse limbs to recombinant GDF5 protein. The results from these assays, coupled with an analysis of the development of brachypodism digits, indicate that GDF5 is necessary and sufficient for both cartilage development and the restriction of joint formation to the appropriate location. Thus, GDF5 function in the digits demonstrates a link between cartilage development and joint development and is an important determinant of the pattern of bones and articulations in the digits.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

FGF8 is known to be an important regulator of forebrain development. Here, we investigated the effects of varying the level of Fgf8 expression in the mouse forebrain. We detected two distinct responses, one that was proportionate with Fgf8 expression and another that was not. The latter response, which led to effects on cell survival, displayed a paradoxical relationship to Fgf8 dosage. Either eliminating or increasing Fgf8 expression increased apoptosis, whereas reducing Fgf8 expression had the opposite effect. To explain these counterintuitive observations, we suggest that an FGF8-dependent cell-survival pathway is negatively regulated by intracellular inhibitors produced in proportion to FGF8 concentration. Our data provide insight into the function of FGF8 in forebrain development and underscore the value of using multiple alleles and different experimental approaches to unravel the complexities of gene function in vertebrate development. It is now well established that the fibroblast growth factor (FGF) family of intercellular signaling molecules plays a central role in vertebrate embryogenesis. At present, 22 different mouse͞human genes are classified as FGF family members because the proteins they encode contain a conserved core sequence of Ϸ120 aa that includes FGF receptor-binding and heparin-binding domains (1). Most FGFs are secreted proteins that bind to high-affinity receptor tyrosine kinases, leading to the activation of multiple signal transduction pathways, including the RAS͞MAPK, PLC-␥, PI3 kinase, and STAT1 pathways (2, 3). One response to FGF receptor activation is the production of antagonists of FGF signaling (4-7). Thus, cellular responses to FGF depend on which signaling pathways are activated and the degree to which they are affected by FGF-induced inhibitors.Fgf8 is an FGF family member that is essential for normal development of the forebrain. At early stages, Fgf8 is expressed along the apex of the anterior neural ridge (ANR), the rostralmost portion of the neural plate, which contains the progenitors of much of the anterior forebrain (telencephalon; ref. 8). After neural tube closure, Fgf8 expression is localized in a domain that encompasses the rostral midline of the telencephalon (9, 10). Surgical removal of the ANR or treatment of forebrain explants with inhibitors of FGF signaling causes a loss of expression of molecular markers of the telencephalon, and beads soaked in FGF8 protein can prevent this effect (11, 12). Moreover, experimentally changing the level of Fgf8 expression at later stages of forebrain development alters telencephalic patterning, suggesting that FGF8 plays a role in specifying positional information in the developing forebrain (13). Analysis of Fgf8 mutants also demonstrates an important role for FGF8 in forebrain development. Although mouse embryos homozygous for an Fgf8-null allele fail to gastrulate and die without forming organs (14, 15), embryos carrying an Fgf8 hypomorphic allele survive to birth and have telencephalic defects (14, 35). Likewise, zebrafi...