Supplementary Figure 1 and Tables 1-2 from Medulloblastomas Derived from Cxcr6 Mutant Mice Respond to Treatment with a Smoothened Inhibitor

To what extent dorsal horn interneurons contribute to the modality specific processing of pain and itch messages is not known. Here, we report that loxp/cre-mediated CNS deletion of TR4, a testicular orphan nuclear receptor, results in loss of many excitatory interneurons in the superficial dorsal horn but preservation of primary afferents and spinal projection neurons. The interneuron loss is associated with a near complete absence of supraspinally integrated pain and itch behaviors, elevated mechanical withdrawal thresholds and loss of nerve injury-induced mechanical hypersensitivity, but reflex responsiveness to noxious heat, nerve injury-induced heat hypersensitivity, and tissue injury-induced heat and mechanical hypersensitivity are intact. We conclude that different subsets of dorsal horn excitatory interneurons contribute to tissue and nerve injury-induced heat and mechanical pain and that the full expression of supraspinally mediated pain and itch behaviors cannot be generated solely by nociceptor and pruritoceptor activation of projection neurons; concurrent activation of excitatory interneurons is essential.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

PURPOSE:Goals of this study were to determine if pharmacological or genetic inhibition of Rho-associated coiled coil containing protein kinases (known as ROCK1 and ROCK2) alters intraocular pressure (IOP) in mice.METHODS:Micro-cannulation of the anterior chamber was used to measure IOP in wild-type B6.129 hybrid mice following treatment with ROCK inhibitors Y-27632 or Y-39983. For comparative purposes, wild-type mice were also treated with timolol, acetazolamide, pilocarpine, or latanoprost. Mice deficient in either Rock1 or Rock2 were generated by homologous recombination or gene trapping, respectively, and their IOP was determined using identical methods employed in the pharmacology studies.RESULTS:Treatment of wild-type B6.129 hybrid mice with ROCK inhibitors (Y-27632 and Y-39983) resulted in significant reductions in IOP. The magnitude of IOP reduction observed with topical Y-39983 was comparable to timolol, and exceeded the IOP effects of latanoprost in this study. Pilocarpine had no discernible effect on IOP in mice. Moreover, mice deficient in either Rock1 or Rock2 exhibited a significant decrease in IOP compared to their B6.129 wild-type littermates.CONCLUSIONS:Pharmacological or genetic inhibition of ROCKs results in decreased IOP in mice. The magnitude of IOP reduction is significant as demonstrated with comparative pharmacology using agents that lower IOP in humans. These studies support the ROCK pathway as a therapeutic target for treating ocular hypertension.

We developed a high‐throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual‐energy X‐ray absorptiometry (DXA) technology estimates body‐fat stores in eight KO and four wild‐type (WT) littermate chow‐fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body‐fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were ≥1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body‐fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body‐fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost‐effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

The development of molecular biological tools for mutant-mouse production signaled the new era of contemporary mammalian genetics. Recent technology advances coupled with completed sequence of the mouse and human genomes are converging synergistically such that today's scientists are exploring their fields of interest in arguably the most productive period for research and discovery of modern biological science. Scientists can now take advantage of an unprecedented ability to manipulate the mouse genome using versatile techniques that afford both custom genetic alteration and random mutagenesis approaches. Together, these strategies can be employed to quickly and comprehensively saturate the genome with a high percentage of gene coverage. Productivity estimates of the leading commercial biotechnology groups that have knockout mouse production and analysis capabilities, together with published knockout reports, suggest that roughly one-fourth of the approximate 25,000 genes in the mouse genome have been knocked out and studied to varying degrees. The international mouse-genomics community has recently begun an initiative to produce knockout alleles for all mouse genes and to develop a system for open access to data and reagents for the broader scientific community. Clearly we can look forward to an escalation of discovery productivity in the upcoming years. It will be even more exciting to track how mouse gene-function discoveries will drive the drug discovery industry's efforts to develop the next generation of therapies for human disease.

Abstract The sonic hedgehog (Shh) pathway is activated in ∼30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes. [Cancer Res 2007;67(8):3871–7]

Ref-1 participates in DNA repair as well as in redox regulation of transcription factor function. The redox function of Ref-1 involves reduction of oxidized cysteine residues within the DNA binding domains of several transcription factors, including Fos and Jun. Reduction of these residues is required for DNA binding, providing a redox-dependent mechanism for regulation of target gene expression. Previous in vitro studies implicated cysteine 65 of human Ref-1 (cysteine 64 of mouse Ref-1) as the redox catalytic site. We analyzed the in vivo role of cysteine 64 in redox regulation of AP-1 activity by introducing a cysteine-to-alanine point mutation into the endogenous mouse Ref-1 gene (ref-1(C64A)). Unlike Ref-1 null mice, which die very early in embryonic development, homozygous ref-1(C64A) mice are viable, they survive to normal life expectancy, and they display no overt abnormal phenotype. Although Ref-1 provides the major AP-1-reducing activity in murine cells, ref-1(C64A) cells retain normal levels of endogenous AP-1 DNA binding activity in vivo as well as normal Fos- and Jun-reducing activity in vitro. These results demonstrate that Ref-1 cysteine 64/65 is not required for redox regulation of AP-1 DNA binding in vivo, and they challenge previous hypotheses regarding the mechanism by which Ref-1 regulates the redox-dependent activity of specific transcription factors.

Cloning of the fukutin gene reveals that an ancient retrotransposon insertion causes Fukuyama-type congenital muscular dystrophy, a disease with high prevalence among the Japanese.