The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.

Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Here we show that double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV-II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development. Received January 22, 2002; revised version accepted May 23, 2002. The mature neocortex is organized into six cell layers, each of which contains neurons with similar morphologies, molecular properties, and projection patterns. The development of this neocortical structure depends on a highly ordered pattern of neuronal production and migration. Cortical neurons that comprise each layer are sequentially produced in the ventricular zone of the dorsal telencephalon (Angevine and Sidman 1961;Takahashi et al. 1999). Although the regulatory factors that function in this sequential production of a variety of layer-specific neurons have not been identified in mammals, in Drosophila the successive production of different types of cells from neuroblasts has been found to require a temporally stereotyped pattern of expression of a set of transcription factors including the Drosophila POU transcription factors Pdm1 and Pdm2 (Isshiki et al. 2001). In mammals, newly produced neurons leave their birthplace, migrate toward the cortical surface, and form cortical layers in an inside-out pattern with respect to their time of birth (Angevine and Sidman 1961;Rakic 1972). Recent genetic studies have identified large numbers of functional molecules involved in the migration/ positioning of neocortical neurons (for review, see Rice and Curran 1999).Brn-1 and Brn-2, members of the mammalian class III POU transcription factor family, are prominently expressed in the embryonic brain, including the neocortex (He et al. 1989). Each single mutant, however, shows abnormalities only in limited brain regions. In Brn-2 mutant neonates, neuronal loss was observed only in the hypothalamic supraoptic and paraventricular nuclei, where Brn-1 is not expressed (Nakai et al. 1995;Schonemann et al. 1995). In Brn-1 mutants, remarkable changes in brain morphology were observed only in the hippocampus, where Brn-2 expression is barely detectable (data not shown). In the neocortex, where both Brn-1 and Brn-2 are expressed, no overt developmental defects were seen in either single mutant. These observations suggest functional complementation between Brn-1 and Brn-2 in neocortical development. Results and DiscussionTo explore their possible overlapping functions in neocortical development, we generated Brn-1/Brn-2 double homozygous mutants by intercrossing double heterozygotes that were healthy and fertile, wi...

The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ-and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1 ؉/؊ ) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1 ؉/؊ mice was apparently slower than that in Tsc2 ؉/؊ mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.T uberous sclerosis (TS) is an autosomal dominantly inherited disease characterized by the development of hamartomas and benign tumors in various organs such as brain, kidney, and heart (1). A germ-line mutation in either TSC1 or TSC2 genes (2, 3), both of which act as tumor suppressors (4, 5), is a genetic factor responsible for pathogenesis of TS. The similar symptoms of TS patients associated with TSC1 or TSC2 mutations suggest that the products of TSC1 and TSC2 are involved in a common physiological pathway (1, 6). TSC1 encodes a protein with a molecular mass of Ϸ130 kDa, hamartin, which contains a coiled-coil domain in the carboxyl-terminal half (3). TSC2 encodes tuberin, a rap1-GTPase activating protein homology domain-containing protein with a molecular mass of Ϸ180 kDa (2). Although several studies concerned with functions of these products have been reported, in vivo functions of them remain to be elucidated (1,7,8).The tumor suppressor function of TSC2 became evident by studies of rodents with a germ-line Tsc2 mutation such as the Eker rat (9-13) and Tsc2 knockout mice (14, 15). Both heterozygous Tsc2 mutant rats and mice develop hereditary renal tumors and extra-renal tumors carrying a second hit of Tsc2 gene (14-17). Homozygosity of Tsc2 mutation leads to the embryonic lethality both in rats (9, 18) and mice (15,16), indicating that the function of tuberin is essential for mammalian development.We also isolated a rat homologue of TSC1 (Tsc1) and analyzed its mutation in chemically induced renal tumors in wild-type rats, in which Tsc2 mutations were found with high frequency (Ϸ50%) (19). In those tumors, we found Tsc1 mutations in a case with no Tsc2 mutation (19). These results suggest that mutations of Tsc1 and Tsc2 are involved in the development of chemically induced renal tumors in rats, although the latter is more common. These systems o...

DFN3, an X chromosome-linked nonsyndromic mixed deafness, is caused by mutations in the BRN-4 gene, which encodes a POU transcription factor. Brn-4-deficient mice were created and found to exhibit profound deafness. No gross morphological changes were observed in the conductive ossicles or cochlea, although there was a dramatic reduction in endocochlear potential. Electron microscopy revealed severe ultrastructural alterations in cochlear spiral ligament fibrocytes. The findings suggest that these fibrocytes, which are mesenchymal in origin and for which a role in potassium ion homeostasis has been postulated, may play a critical role in auditory function.

Brain-type fatty acid-binding protein (B-FABP) belongs to a family of intracellular lipid-binding proteins. B-FABP exhibits a binding affinity to long-chain fatty acids (FAs) whose effects on brain functions including development, emotion, learning and memory have been proposed. B-FABP is localized in the ventricular germinal cells in embryonic brain and astrocytes in developing and mature brain of rodents. In the present study we generated the mouse harboring a null mutation in the B-FABP gene and studied its phenotype. B-FABP mutant mice exhibited the enhanced anxiety and increased fear memory as well as the decreased content of docosahexaenoic acid (DHA) in their brain during the neonatal period without detection of any histological changes in the brain. In the adult brain, B-FABP was localized more numerously to the astrocytes in the amygdala and septal area than to those in the hippocampal area. Analysis of FA content in the amygdala of adult brain revealed that arachidonic and palmitic acids increased significantly in the mutant mice compared with wild-type. Furthermore, the response of N-methyl-d-aspartate receptor-mediated current to DHA in isolated neurons from B-FABP mutant brain was significantly decreased compared with that of wild-type, while no significant differences were detected in behavioral responses related to the spatial learning/memory or in the hippocampal long-term potentiation. These data indicate that B-FABP is crucially involved in the fear memory and anxiety through its binding with FAs and/or its own direct effects on pertinent metabolism/signaling of FAs.

This study identifies a role for the gene for the POU transcription factor Brn1 in distal tubule formation and function in the mammalian kidney. Normal development of Henle's loop (HL), the distal convoluted tubule and the macula densa was severely retarded in Brn1-deficient mice. In particular, elongation and differentiation of the developing HL was affected. In the adult kidney, Brn1 was detected only in the thick ascending limb (TAL) of HL. In addition, the expression of a number of TAL-specific genes was reduced in the Brn1 +/-kidney, including Umod, Nkcc2/Slc12a1, Bsnd, Kcnj1 and Ptger3. These results suggest that Brn1 is essential for both the development and function of the nephron in the kidney.

The ryanodine receptor type 3 (RyR-3) functions as a Ca 2؉-induced Ca 2؉ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca 2؉ -induced Ca 2؉ release from intracellular Ca 2؉ stores of the mutant skeletal muscle differed in Ca 2؉ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca 2؉ signaling results in abnormalities of certain neurons in the central nervous system.