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Presence of a Neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development

ABSTRACT: During development of the nervous system, neurons connect to one another in a precisely organized manner. Sensory systems provide a good example of this organization, whereby the composition of the outside world is represented in the brain by neuronal maps. Establishing correct patterns of neural circuitry is crucial, as inaccurate map formation can lead to severe disruptions in sensory processing. In rodents, olfactory stimuli modulate a wide variety of behaviours essential for survival. The formation of the olfactory glomerular map is dependent on molecular cues that guide olfactory receptor neuron axons to broad regions of the olfactory bulb and on cell adhesion molecules that promote axonal sorting into specific synaptic units in this structure. Here, we demonstrate that the cell adhesion molecule Amigo1 is expressed in a subpopulation of olfactory receptor neurons, and we investigate its role in the precise targeting of olfactory receptor neuron axons to the olfactory bulb using a genetic loss-of-function approach in mice. While ablation of Amigo1 did not lead to alterations in olfactory sensory neuron axonal targeting, our experiments revealed that the presence of a neomycin resistance selection cassette in the Amigo1 locus can lead to off-target effects that are not due to loss of Amigo1 expression, including unexpected altered gene expression in olfactory receptor neurons and reduced size of glomeruli in the ventral region of the olfactory bulb. Our results demonstrate that insertion of a neomycin selection cassette into the mouse genome can have specific deleterious effects on the development of the olfactory system and highlight the importance of removing antibiotic resistance cassettes from genetic loss-of-function mouse models when studying olfactory system development.

ORGANISM(S): Mus musculus

PROVIDER: GSE249314 | GEO | 2024/02/26

REPOSITORIES: GEO

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Project description:Olfactory sensory neurons distinguish a large variety of odor molecules and direct the information through their axons to the olfactory bulb, the first site for the processing of olfactory information in the brain. Olfaction is very important for most mammals for the maintenance of a good quality of life. Accumulating evidences endorse that olfactory sensory decline is connected with neurodegenerative disorders including schizophrenia, depression, multiple sclerosis, Huntington's, Alzheimer's and Parkinson's diseases. For several decades, neuroanatomical, volumetric, and histological approaches have been the gold standard techniques employed to characterize the olfactory bulb functionality. Diagnosis and treatment of olfactory dysfunction remain significant health care challenges to society. Novel strategies and clues that assist in the identification of biomarker and drug development for aid in the prevention and cure of neurological diseases are necessary. However, little attention has been focused specifically on the molecular composition of the olfactory bulb from the perspective of proteomics. To this end, an in-depth mapping of the olfactory bulb proteome was carried out using high resolution tandem mass spectrometry, revealing a repertoire of 7,754 proteins. A large proportion of the identified proteins were predicted to be involved in diverse biological processes including signal transduction, metabolism, transport, olfaction and protein synthesis. Pathway analysis of the identified proteins shows that, these proteins are predominantly involved in metabolic and neural processes, chromatin modeling, and synaptic vesicle transport associated with neuronal transmission. In total, our study offers valuable understandings into the molecular composition of the human olfactory bulb proteome that could possibly help neuroscience community to understand the olfactory bulb better and open avenues for intervention strategies for olfactory dysfunction in the future.

Project description:The olfactory sensory system is formed by the coordinated morphogenesis and differentiation of the peripheral olfactory epithelium (OE) and the anterior forebrain. At early stages, immature olfactory receptor neurons (ORN) elongate their axons to penetrate the brain basement membrane, contact and form synapses with projection neurons of the olfactory bulb primordium. Axonal elongation is accompanied by migration of the GnRH+ neurons, followed by their ingression in the septo-hypothalamic area of the forebrain. This process is specifically impaired in the KallmannM-bM-^@M-^Ys syndrome (KS), a disorder characterized by anosmia and central hypogonadism. A set of transcription factors are master regulators of olfactory connectivity and GnRH neuron migration. We explored the transcriptional network underlying this process, by profiling the OE and adjacent mesenchyme at distinct embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene essential for olfactory development, that causes a KS-like phenotype when deleted. We also applied analysis of conserved co-expression to integrate the obtained data with information on KS disease genes. The prevalent categories of genes differentially expressed during development are neuronal differentiation, extracellular remodelling and cell adhesion. From the analysis of Dlx5 mutant tissues we identify about 120 genes with a prevalence of intermediate filaments, cell signalling, epithelial and neuronal differentiation. Filtering for true OE expression and for the presence of Dlx5 binding sites, yielded twenty genes, of the following categories: 1) transmembrane adhesion/receptor molecules, 2) axon-glia interaction molecules, 3) synaptic proteins, 4) scaffold/adapter for signalling molecules. To functionally analyze these genes in vivo, we used three zebrafish fluorescent reporter zebrafish strains, in which we monitored early phases of olfactory/GnRH development upon gene downmodulation. The depletion of three (of five) Dlx5 targets affected axonal extension and targeting, while two (of two) altered GnRH neuron position and neurite organization. In one experiment we compare the olfactrory sensory epithelium from wild-type embryos, at three times of development, i.e. E11.5, E12.5 and E14.5. In a second experiment we compare the olfactory sensory epithelium from wild-type embryo with that from Dlx5 knock-out embryos, at the age E12.5

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Presence of a Neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development

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