The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice homozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.[Key Words: T/EBP; gene targeting; thyroid; lung; ventral forebrain; pituitary] Received August 22, 1995; revised version accepted November 10, 1995.Thyroid-specific enhancer-binding protein (T/EBP) binds to an enhancer element located -5.5 kb upstream of the human thyroid peroxidase gene transcription start site and regulates thyroid-specific gene expression (Kikkawa et al. 1990;Mizuno et al. 1991). T/EBP, also named thyroid-specific transcription factor 1 (TTF-1)or Nkx-2.1, was originally described to govern thyroid-specific expression of the rat thyroglobulin gene (Civitareale et al. 1989). Several studies have established the role of T/EBP in expression of genes encoding thyroid peroxidase (Kikkawa et al. 1990;Mizuno et al. 1991;Abramowicz et al. 1992;Francis-Lang et al. 1992), thyroglobulin (Civitareale et al. 1989), and the thyrotropin (TSH) receptor tCivitareale et al. 1993;Shimura et al. 1994). All three proteins are essential for thyroid hormone biosynthesis (DeGroot and Niepomniszcze 1977). T/EBP is also expressed in the lung (Guazzi et al. 1990;Mizuno et al. 1991), and it has been recently demonstrated that the expression of genes encoding the lung surfactant proteins A and B is regulated by this DNA-binding protein (Bohinski et al. 1994;Bruno et al. 1995). T/ebp ) is the first member of the mouse Nkx-2 gene family that is closely related to Drosophila NK-2 in their homeo domain sequences (68%-95% similarity) (Kim and Nirenberg 1989;Guazzi et al. 1990;Price et al. 1992;Lints et al. 1993). Members of the Nkx-2 family also share a highly conserved 17-aminoacid motif that is located on the carboxyl-terminal side of the homeo domain. From the six members of this family characterized to date, the expression patterns of three genes, T/ebp (Nkx-2.1), Nkx-2.2, and Nkx-2.5, have been studied. Lazzaro et al. (1991) have established T/ebp(Nkx-2.1) gene expression at -10.5 days postcoitum (El0.5) in the ...

The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex.

Ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice results in severe hydrocephalus with enlargement of the lateral and third ventricles. All B(-)/B(-) mice died either during embryonic development or on the day of birth (PO). Neurons cultured from superior cervical ganglia of B(-)/B(-) mice between embryonic day (E) 18 and P0 showed decreased rates of neurite outgrowth, and their growth cones had a distinctive narrow morphology compared with those from normal mice. Serial sections of E12.5, E13.5, and E15 mouse brains identified developmental defects in the ventricular neuroepithelium. On E12.5, disruption of the coherent ventricular surface and disordered cell migration of neuroepithelial and differentiated cells were seen at various points in the ventricular walls. These abnormalities resulted in the formation of rosettes in various regions of the brain and spinal cord. On E13.5 and E15, disruption of the ventricular surface and aberrant protrusions of neural cells into the ventricles became more prominent. By E18.5 and P0, the defects in cells lining the ventricular wall resulted in an obstructive hydrocephalus due to stenosis or occlusion of the third ventricle and cerebral aqueduct. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC-B is essential for both early and late developmental processes in the mammalian brain.

We generated mice harboring a single amino acid mutation in the motor domain of nonmuscle myosin heavy chain II-B (NMHC II-B). Homozygous mutant mice had an abnormal gait and difficulties in maintaining balance. Consistent with their motor defects, the mutant mice displayed an abnormal pattern of cerebellar foliation. Analysis of the brains of homozygous mutant mice showed significant defects in neuronal migration involving granule cells in the cerebellum, the facial neurons, and the anterior extramural precerebellar migratory stream, including the pontine neurons. A high level of NMHC II-B expression in these neurons suggests an important role for this particular isoform during neuronal migration in the developing brain. Increased phosphorylation of the myosin II regulatory light chain in migrating, compared with stationary pontine neurons, supports an active role for myosin II in regulating their migration. These studies demonstrate that NMHC II-B is particularly important for normal migration of distinct groups of neurons during mouse brain development.

Prepulse inhibition (PPI) is a compelling endophenotype (biological markers) for mental disorders including schizophrenia. In a previous study, we identified Fabp7, a fatty acid binding protein 7 as one of the genes controlling PPI in mice and showed that this gene was associated with schizophrenia. We also demonstrated that disrupting Fabp7 dampened hippocampal neurogenesis. In this study, we examined a link between neurogenesis and PPI using different animal models and exploring the possibility of postnatal manipulation of neurogenesis affecting PPI, since gene-deficient mice show biological disturbances from prenatal stages. In parallel, we tested the potential for dietary polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA) and/or docosahexaenoic acid (DHA), to promote neurogenesis and improve PPI. PUFAs are ligands for Fabp members and are abundantly expressed in neural stem/progenitor cells in the hippocampus. Our results are: (1) an independent model animal, Pax6 (+/−) rats, exhibited PPI deficits along with impaired postnatal neurogenesis; (2) methylazoxymethanol acetate (an anti-proliferative drug) elicited decreased neurogenesis even in postnatal period, and PPI defects in young adult rats (10 weeks) when the drug was given at the juvenile stage (4–5 weeks); (3) administering ARA for 4 weeks after birth promoted neurogenesis in wild type rats; (4) raising Pax6 (+/−) pups on an ARA-containing diet enhanced neurogenesis and partially improved PPI in adult animals. These results suggest the potential benefit of ARA in ameliorating PPI deficits relevant to psychiatric disorders and suggest that the effect may be correlated with augmented postnatal neurogenesis.

The potent neurotrophic factor glial cell-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-β (TGF-β) superfamily of proteins. We report a transcription factor that is the first nuclear protein known to be induced by GDNF, thus designated murine GDNF inducible factor (mGIF). The cDNA was cloned in the course of investigating transcription factors that bind to Sp1 consensus sequences, using thein situfilter detection method, and it was found to encode a protein having the same C2–H2zinc finger motif as Sp1. Sequence analysis indicated that mGIF is homologous to the human TGF-β inducible early gene (TIEG) and human early growth response gene-α (EGR-α). mGIF is widely distributed in the adult mouse with high mRNA levels in kidney, lung, brain, liver, heart, and testis. In the adult brain, mGIF is abundantly expressed in hippocampus, cerebral cortex, cerebellum, and amygdala with lower amounts in striatum, nucleus accumbens, olfactory tubercle, thalamus, and substantia nigra. During development, mGIF mRNA also has a wide distribution, including in cerebral cortex, cerebellar primordium, kidney, intestine, liver, and lung. GDNF induces the expression of mGIF rapidly and transiently both in a neuroblastoma cell line and in primary cultures of rat embryonic cortical neurons. Co-transfection of theDrosophilaSL2 cells using mGIF expression plasmid and reporter constructs having Sp1 binding sites indicated that mGIF represses transcription from a TATA-containing as well as from a TATA-less promoter. These observations suggest that the zinc finger transcription factor mGIF could be important in mediating some of the biological effects of GDNF.

Four mouse POU domain genomic DNA clones-Brain-i, Brain-2, Brain-4, and Scip-and Bran-2 cDNA, which are expressed in adult brain, were cloned and the coding and noncoding regions ofthe genes were sequenced. The amino acid sequences of the four PoU domains are highly conserved; sequences in other regions of the proteins also are conserved but to a lesser extent. The absence of introns from the coding regions of the four POU domain genes and the similarity of amino acid sequences of the corresponding proteins suggest that the coding region of the ancestral class HI POU domain gene lacked introns and therefore may have originated by reverse transcription of a molecule of POU domain mRNA followed by insertion ofthe cDNA into germ cell genomic DNA. Additional duplications ofthe ancestral class HI POU domain gene (or mRNA) would create the Brain-i, Brain-2, Brain-4, and Scip genes.POU domain proteins bind to specific nucleotide sequences in DNA and regulate gene expression (for reviews, see refs. 1 and 2). The POU domain is a conserved amino acid sequence =150 amino acid residues long. The initial region of 69-72 amino acid residues is termed the POU-specific domain, which is followed by a 15-to 25-amino acid residue linker region and a 60-amino acid residue POU homeodomain. Both the POU-specific domain and the homeodomain are required for specific high-affinity binding to DNA (3, 4).Rosenfeld and his colleagues have sorted POU domains into different groups on the basis of POU domain amino acid sequence similarity (2). Three mammalian class III POU domain cDNAs have been described-human (5) and rat (2) Brain-i and Brain-2 and rat (5-8) and mouse (9-11) Scip (also termed Oct-6 and Tst-J)-that have closely related POU domains and are expressed in embryonic and adult brain. Only the POU domain regions of human and rat Brain-i and Brain-2 cDNA have been sequenced thus far (2, 5), whereas the complete coding sequences of rat (6-8) and mouse (9-11) Scip cDNA have been reported. Scip RNA is expressed in a subset of neurons, oligodendroglia, Schwann cells, and in the testis (5-11). The expression of the Scip gene is promoted by cAMP (6, 7).In this report, a fourth class III mouse POU domain gene, Brain4, is described, which is also expressed in adult mouse brain. Brain4 is similar to the recently reported XLPOU 2 POU domain partial cDNA of Xenopus laevis (12). Three additional mouse class III POU domain genomic clonesBrain-i, Brain-2, and Scip-and Brain-2 cDNA were obtained and the nucleotide sequences of the coding and noncoding regions were determined. Comparison of the deduced amino acid sequences of the four POU domain proteins shows that the structure of the genes and the amino Fig. 4). DNA inserts were subcloned in pBluescript II SK+ and KS+. The nucleotide sequences of both strands of DNA were determined with universal or specific oligodeoxynucleotide primers and single-stranded DNA templates by the dideoxynucleotide chain-termination method (13).OlUgdeoxynucleotide Probes. Four oligodeoxynucleotides (48 bases...

Chemical synapses are specialized sites of the communication between neurons where information is processed and integrated. Electron microscopy has allowed morphological Received November 24, 2010; revised manuscript received December 8, 2010; accepted December 9, 2010. Address correspondence and reprint requests to Hiroyuki Sakagami, MD, PhD, Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan. E-mail: sakagami@med.kitasato-u.ac.jp Abbreviations used: Arf, ADP ribosylation factor; BRAG, brefeldin A-resistant Arf-GEF; DGC, dystrophin-associated glycoprotein complex; GABA A R, GABA A receptor; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GGA1, Golgi-localizing, c-adaptin ear homology domain, Arf-binding protein 1; GST, glutathione S-transferase; HA, hemagglutinin; IRSP, insulin receptor tyrosine kinase substrate of 53 kDa; MAGI, membrane-associated guanylate kinase with inverted orientation; PDZ, PSD-95/Discs large/Zona occludens 1; PSD, post-synaptic density; SDS, sodium dodecyl sulfate; SDS-PAGE, SDSpolyacrylamide gel electrophoresis; S-SCAM, synaptic scaffolding molecule; synArfGEF(Po), potential synaptic Arf-GEF; VGAT, vesicular c-aminobutyric acid transporter. AbstractSynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/ MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both b-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.