In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.

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.

P2Y receptors have been reported to modulate gastrointestinal functions. The newest family member is the nucleotide-sugar receptor P2Y14. P2ry14 mRNA was detected throughout the rat gut, with the highest level being in the forestomach. We investigated the role of the receptor in stomach motility using cognate agonists and knockout (KO) mice. In rat isolated forestomach, 100 microM UDP-glucose and 100 muM UDP-galactose both increased the baseline muscle tension (BMT) by 6.2+/-0.6 and 1.6+/-0.6 mN (P<0.05, n=3-4), respectively, and the amplitude of contractions during electrical field stimulation (EFS) by 3.7+/-1.7 and 4.3+/-2.5 mN (P<0.05, n=3-4), respectively. In forestomach from wild-type (WT) mice, 100 microM UDP-glucose increased the BMT by 1.0+/-0.1 mN (P<0.05, n=6) but this effect was lost in the KO mice (change of -0.1+/-0.1 mN, n=6). The 100 microM UDP-glucose also increased the contraction amplitude during EFS in this tissue from the WT animals (0.9+/-0.4 mN, P < 0.05, n=6) but not from the KO mice (0.0+/-0.2 mN, n=6). In vivo, UDP-glucose at 2,000 mg/kg ip reduced gastric emptying in rats by 49.7% (P<0.05, n=4-6) and in WT and KO mice by 56.1 and 66.2%, respectively (P<0.05, n=7-10) vs. saline-treated control animals. There was no significant difference in gastric emptying between WT and KO animals receiving either saline or d-glucose. These results demonstrate a novel function of the P2Y14 receptor associated with contractility in the rodent stomach that does not lead to altered gastric emptying after receptor deletion and an ability of UDP-glucose to delay gastric emptying without involving the P2Y14 receptor.

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 report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.

Our ability to genetically manipulate the mouse has had a great impact on medical research over the last few decades. Mouse genetics has developed into a powerful tool for dissecting the genetic causes of human disease and identifying potential targets for pharmaceutical intervention. With the recent sequencing of the human and mouse genomes, a large number of novel genes have been identified whose function in normal and disease physiology remains largely unknown. Government-sponsored multinational efforts are underway to analyze the function of all mouse genes through mutagenesis and phenotyping, making the mouse the interpreter of the human genome. A number of technologies are available for the generation of mutant mice, including gene targeting, gene trapping and transposon, chemical or radiation-induced mutagenesis. In this chapter, we review the current status of gene trapping technology, including its applicability to conditional mutagenesis.

ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (-/-) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (-/-) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.

P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.