ABSTRACT
During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a.
Subject(s)
Cell Differentiation/physiology , Gene Expression Regulation, Developmental/physiology , Interneurons/physiology , Neural Stem Cells/physiology , Wnt Proteins/metabolism , Animals , Animals, Newborn , Brain/cytology , Cell Differentiation/drug effects , Cells, Cultured , Chlorocebus aethiops , Chromatin Immunoprecipitation/methods , Coculture Techniques , Embryo, Mammalian , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Developmental/genetics , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/genetics , Homeodomain Proteins/genetics , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Mice , Mice, Transgenic , Models, Biological , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/drug effects , Olfactory Bulb/cytology , Protein Array Analysis/methods , Time Factors , Transfection/methods , Wnt Proteins/genetics , Wnt-5a Protein , beta Catenin/genetics , beta Catenin/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
A variety of signals governing early extension, guidance, and connectivity of olfactory receptor neuron (ORN) axons has been identified; however, little is known about axon-mesoderm and forebrain (FB)-mesoderm signals. Using Wnt-beta catenin reporter mice, we identify a novel Wnt-responsive resident cell population, located in a Frizzled7 expression domain at the surface of the embryonic FB, along the trajectory of incoming ORN axons. Organotypic slice cultures that recapitulate olfactory-associated Wnt-beta catenin activation show that the beta catenin response depends on a placode-derived signal(s). Likewise, in Dlx5-/- embryos, in which the primary connections fail to form, Wnt-beta catenin response on the surface of the FB is strongly reduced. The olfactory placode expresses a number of beta catenin-activating Wnt genes, and the Frizzled7 receptor transduces the "canonical" Wnt signal; using Wnt expression plasmids we show that Wnt5a and Wnt7b are sufficient to rescue beta catenin activation in the absence of incoming axons. Finally, blocking the canonical Wnt pathway with the exogenous application of the antagonists Dikkopf-1 or secreted-Frizzled-receptor protein-2 prevents ORN axon contact to the FB. These data reveal a novel function for Wnt signaling in the establishment of periphery-CNS olfactory connections and highlight a complex interplay between cells of different embryonic origin for ORN axon connectivity.
Subject(s)
Axons/physiology , Olfactory Receptor Neurons/physiology , Prosencephalon/cytology , Prosencephalon/physiology , Wnt Proteins/physiology , beta Catenin/physiology , Animals , Embryo, Mammalian , Frizzled Receptors/genetics , Frizzled Receptors/physiology , Gene Expression Regulation, Developmental , Genes, Reporter , Mesoderm/cytology , Mesoderm/physiology , Mice , Mice, Transgenic , Olfactory Bulb/cytology , Olfactory Bulb/embryology , Olfactory Receptor Neurons/ultrastructure , Organ Culture Techniques , Prosencephalon/embryology , Signal Transduction/physiology , Wnt Proteins/geneticsABSTRACT
Development of the olfactory pathway requires interaction between cells and signals of different origin. Olfactory receptor neurons (ORN) in the olfactory placodes (OP) extend axons towards the forebrain (FB); with innervation taking place at a later time following degradation of the basement membrane. Cells from the OP migrate along ORN axons and differentiate into various elements, including ensheathing and Gonadotropin Releasing Hormone (GnRH)+ cells. The importance of the olfactory connection and migration is highlighted by the severe endocrine phenotype in Kallmann's patients who lack this migratory pathway. Little is known about the genetic control of intrinsic ORN properties. Inactivation of the distalless-related Dlx5 prevents connections between ORNs and FB. Using a grafting approach we show that misguidance and lack of connectivity is due to intrinsic defects in ORN neurites and migratory cells (MgC), and not to environmental factors. These data point to a cell-autonomous function of Dlx5 in providing ORN axons with their connectivity properties. Dlx5 also marks a population of early MgC that partly overlaps with the GnRH+ population. In the absence of Dlx5 MgCs of the Dlx5+ lineage migrate, associated with PSA-NCAM+ axons, but fail to reach the FB as a consequence of the lack of axonal connection and not an inability to migrate. These data suggests that Dlx5 is not required to initiate migration and differentiation of MgCs.
