We describe a novel spontaneous mouse mutant, laggard (lag), characterized by a flat head, motor impairment and growth retardation. The mutation is inherited as an autosomal recessive trait, and lag/lag mice suffer from cerebellar ataxia and die before weaning. lag/lag mice exhibit a dramatic reduction in brain size and slender optic nerves. By positional cloning, we identify a splice site mutation in Kif14. Transgenic complementation with wild-type Kif14-cDNA alleviates ataxic phenotype in lag/lag mice. To further confirm that the causative gene is Kif14, we generate Kif14 knockout mice and find that all of the phenotypes of Kif14 knockout mice are similar to those of lag/lag mice. The main morphological abnormality of lag/lag mouse is severe hypomyelination in central nervous system. The lag/lag mice express an array of myelin-related genes at significantly reduced levels. The disrupted cytoarchitecture of the cerebellar and cerebral cortices appears to result from apoptotic cell death. Thus, we conclude that Kif14 is essential for the generation and maturation of late-developing structures such as the myelin sheath, cerebellar and cerebral cortices. So far, no Kif14-deficient mice or mutation in Kif14 has ever been reported and we firstly define the biological function of Kif14 in vivo. The discovery of mammalian models, laggard, has opened up horizons for researchers to add more knowledge regarding the etiology and pathology of brain malformation.

Phospholipase C is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase C is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase C. These results suggest a crucial role of phospholipase C downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phosphoinositide-specific phospholipase C (PLC) is a key event triggering intracellular signal transduction from various receptor molecules at the plasma membrane by yielding two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which induce activation of protein kinase C and mobilization of Ca 2ϩ from intracellular stores, respectively (9). Concurrently, reduction in PIP 2 concentration appears to be an important signal because activities of various actin-binding proteins and pleckstrin homology domain-containing proteins are modulated through interaction with PIP 2 (25). More than 12 mammalian PLC isoforms have been identified and organized into five classes (␤, ␥, ␦, ε, and ), which are regulated through distinct mechanisms (9). PLCε is characterized by possession of two Ras-associating domains and a CDC25 homology domain. The Ras-associating domains are responsible for activation of PLCε through direct association with the GTP-bound active forms of the small GTPases Ras (15, 23), Rap1 (23,24), and Rap2 (20). Stimulation of cells by growth factors, such as platelet-derived growth factor, induces persistent activation of PLCε through activation of Ras and Rap1 (24). The rapid and initial phase of this activation is mediated by Ras at the plasma membrane, whereas Rap1 is responsible for the prolonged activation mainly at the Golgi complex (24). The CDC25 homology domain acts as a guanine nucleotide exchange factor for Rap1 and is crucial for the prolonged activation of PLCε by Rap1 (14,24). The involvement of other factors, such as the ␣ subunits of the G 12 and G 13 families or the ␤ 1 ␥ 2 subunits ...

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A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1(-/-) mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1(-/-) yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1(-/-) embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.

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