RESUMO
DLX1 and DLX2 transcription factors are necessary for forebrain GABAergic neuron differentiation, migration, and survival. We generated transgenic mice that express Cre-recombinase under the control of two ultra-conserved DNA elements near the Dlx1 and 2 locus termed I12b and URE2. We show that Cre-recombinase is active in a "Dlx-pattern" in the embryonic forebrain of transgenic mice. I12b-Cre is more active than URE2-Cre in the medial ganglionic eminences and its derivatives. Fate-mapping of EGFP+ cells in adult Cre;Z/EG animals demonstrated that GABAergic neurons, but not glia, are labeled. Most NPY+, nNOS+, parvalbumin+, and somatostatin+ cells are marked by I12b-Cre in the cortex and hippocampus, while 25-40% of these interneuron subtypes are labeled by URE2-Cre. Labeling of neurons generated between E12.5 to E15.5 indicated differences in birth-dates of EGFP+ cells that populate the olfactory bulb, hippocampus, and cortex. Finally, we provide the first in vivo evidence that both I12b and URE2 are direct targets of DLX2 and require Dlx1 and Dlx2 expression for proper activity.
Assuntos
Elementos Facilitadores Genéticos , Proteínas de Homeodomínio , Integrases/metabolismo , Interneurônios/fisiologia , Camundongos Transgênicos , Fatores de Transcrição , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Linhagem da Célula , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Integrases/genética , Interneurônios/citologia , Masculino , Camundongos , Neuroglia/citologia , Neuroglia/fisiologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransgenesRESUMO
The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase beta(1) subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase beta(1) subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase beta(1) subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase beta(1)-subunit.