Axon growth and guidance genes identify nascent, immature, and mature olfactory sensory neurons

Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1 , two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1–4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs ( Plxna1, Plxna4, Nrp2, Efna5 ) or specifically in mature OSNs ( Plxna3, Unc5b, Efna3, Epha5, Epha7 ), although some were specific to immature OSNs ( Plxnb1, Plxnb2, Plxdc2, Nrp1 ). Cell adhesion molecules were expressed either by both immature and mature OSNs ( Dscam, Ncam1, Ncam2, Nrxn1 ) or solely by immature OSNs ( Chl1, Nfasc1, Dscaml1 ). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus. © 2010 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106698/1/22497_ftp.pd

ROLES OF EMX2 IN ODORANT RECEPTOR GENE EXPRESSION AND OLFACTORY SENSORY NEURON AXON GROWTH

The sense of smell relies upon the detection of odorants by neurons located in the nasal cavity. These neurons, referred to as olfactory sensory neurons (OSNs), line the olfactory epithelium and extend axons that make synaptic connections with mitral/tufted cells in the olfactory bulb. The mechanisms by which these synaptic connections form remain largely unknown. The development of these synaptic connections relies on the axons of immature OSNs innervating the olfactory bulb. The primary goal of this dissertation was to identify components of the mechanisms used by immature OSN axons to innervate the olfactory bulb. To accomplish this goal, a knockout mouse model was used. OSN axons, of Emx2 knockout mice fail to innervate the olfactory bulb. As EMX2 is a transcription factor, this model was used investigate the possible causes of the defective OSN axon growth. To gain a better understanding of OSN axon growth, differences in expression of axon growth and guidance genes in immature and mature OSNs was investigated. This analysis revealed that many axon growth and guidance genes are differential expressed, and helped to identify immature OSN specific genes. The data also revealed a previously unrecognized developmental stage, termed nascent OSNs, identified by the expression of Cxcr4. Analysis of Emx2-/- mice revealed that EMX2 is necessary for OSN survival, odorant receptor expression and expression of the axonogenesis related gene Ablim1. EMX2 is necessary for the expression of many odorant receptor genes; however the loss of odorant receptor expression does not explain the axon growth defects. Apoptosis is increased in Emx2-/- mice, an outcome that may be due to the failed axon growth. Analysis of axon guidance gene expression identified a large reduction in Ablim1 expression in Emx2-/- mice. Ablim1 is expressed by immature OSNs, placing it in the proper cell type to regulate OSN axon growth. The loss of Ablim1 expression in Emx2-/- mice indicates defective signaling in the axon growth cone and a possible mechanism regulating OSN axon growth into the olfactory bulb. The data presented in this dissertation provide new insight into the regulation of odorant receptor gene expression and OSN axon growth

Molecular Events in the Cell Types of the Olfactory Epithelium during Adult Neurogenesis

BACKGROUND: Adult neurogenesis, fundamental for cellular homeostasis in the mammalian olfactory epithelium, requires major shifts in gene expression to produce mature olfactory sensory neurons (OSNs) from multipotent progenitor cells. To understand these dynamic events requires identifying not only the genes involved but also the cell types that express each gene. Only then can the interrelationships of the encoded proteins reveal the sequences of molecular events that control the plasticity of the adult olfactory epithelium. RESULTS: Of 4,057 differentially abundant mRNAs at 5 days after lesion-induced OSN replacement in adult mice, 2,334 were decreased mRNAs expressed by mature OSNs. Of the 1,723 increased mRNAs, many were expressed by cell types other than OSNs and encoded proteins involved in cell proliferation and transcriptional regulation, consistent with increased basal cell proliferation. Others encoded fatty acid metabolism and lysosomal proteins expressed by infiltrating macrophages that help scavenge debris from the apoptosis of mature OSNs. The mRNAs of immature OSNs behaved dichotomously, increasing if they supported early events in OSN differentiation (axon initiation, vesicular trafficking, cytoskeletal organization and focal adhesions) but decreasing if they supported homeostatic processes that carry over into mature OSNs (energy production, axon maintenance and protein catabolism). The complexity of shifts in gene expression responsible for converting basal cells into neurons was evident in the increased abundance of 203 transcriptional regulators expressed by basal cells and immature OSNs. CONCLUSIONS: Many of the molecular changes evoked during adult neurogenesis can now be ascribed to specific cellular events in the OSN cell lineage, thereby defining new stages in the development of these neurons. Most notably, the patterns of gene expression in immature OSNs changed in a characteristic fashion as these neurons differentiated. Initial patterns were consistent with the transition into a neuronal morphology (neuritogenesis) and later patterns with neuronal homeostasis. Overall, gene expression patterns during adult olfactory neurogenesis showed substantial similarity to those of embryonic brain

Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae

BACKGROUND: Two sibling members of the Anopheles gambiae species complex display notable differences in female blood meal preferences. An. gambiae s.s. has a well-documented preference for feeding upon human hosts, whereas An. quadriannulatus feeds on vertebrate/mammalian hosts, with only opportunistic feeding upon humans. Because mosquito host-seeking behaviors are largely driven by the sensory modality of olfaction, we hypothesized that hallmarks of these divergent host seeking phenotypes will be in evidence within the transcriptome profiles of the antennae, the mosquito's principal chemosensory appendage. RESULTS: To test this hypothesis, we have sequenced antennal mRNA of non-bloodfed females from each species and observed a number of distinct quantitative and qualitative differences in their chemosensory gene repertoires. In both species, these gene families show higher rates of sequence polymorphisms than the overall rates in their respective transcriptomes, with potentially important divergences between the two species. Moreover, quantitative differences in odorant receptor transcript abundances have been used to model potential distinctions in volatile odor receptivity between the two sibling species of anophelines. CONCLUSION: This analysis suggests that the anthropophagic behavior of An. gambiae s.s. reflects the differential distribution of olfactory receptors in the antenna, likely resulting from a co-option and refinement of molecular components common to both species. This study improves our understanding of the molecular evolution of chemoreceptors in closely related anophelines and suggests possible mechanisms that underlie the behavioral distinctions in host seeking that, in part, account for the differential vectorial capacity of these mosquitoes

Genomics of Mature and Immature Olfactory Sensory Neurons

The continuous replacement of neurons in the olfactory epithelium provides an advantageous model for investigating neuronal differentiation and maturation. By calculating the relative enrichment of every mRNA detected in samples of mature mouse olfactory sensory neurons (OSNs), immature OSNs, and the residual population of neighboring cell types, and then comparing these ratios against the known expression patterns of \u3e300 genes, enrichment criteria that accurately predicted the OSN expression patterns of nearly all genes were determined. We identified 847 immature OSN-specific and 691 mature OSN-specific genes. The control of gene expression by chromatin modification and transcription factors, and neurite growth, protein transport, RNA processing, cholesterol biosynthesis, and apoptosis via death domain receptors, were overrepresented biological processes in immature OSNs. Ion transport (ion channels), presynaptic functions, and cilia-specific processes were overrepresented in mature OSNs. Processes overrepresented among the genes expressed by all OSNs were protein and ion transport, ER overload response, protein catabolism, and the electron transport chain. To more accurately represent gradations in mRNA abundance and identify all genes expressed in each cell type, classification methods were used to produce probabilities of expression in each cell type for every gene. These probabilities, which identified 9,300 genes expressed in OSNs, were 96% accurate at identifying genes expressed in OSNs and 86% accurate at discriminating genes specific to mature and immature OSNs. This OSN gene database not only predicts the genes responsible for the major biological processes active in OSNs, but also identifies thousands of never before studied genes that support OSN phenotypes

Expression pattern of Stomatin-domain proteins in the peripheral olfactory system

Recent data show that Stomatin-like protein 3 (STOML3), a member of the stomatin-domain family, is expressed in the olfactory sensory neurons (OSNs) where it modulates both spontaneous and evoked action potential firing. The protein family is constituted by other 4 members (besides STOML3): STOM, STOML1, STOML2 and podocin. Interestingly, STOML3 with STOM and STOML1 are expressed in other peripheral sensory neurons: dorsal root ganglia. In here, they functionally interact and modulate the activity of the mechanosensitive Piezo channels and members of the ASIC family. Therefore, we investigated whether STOM and STOML1 are expressed together with STOML3 in the OSNs and whether they could interact. We found that all three are indeed expressed in ONSs, although STOML1 at very low level. STOM and STOML3 share a similar expression pattern and STOML3 is necessary for STOM to properly localize to OSN cilia. In addition, we extended our investigation to podocin and STOML2, and while the former is not expressed in the olfactory system, the latter showed a peculiar expression pattern in multiple cell types. In summary, we provided a first complete description of stomatin-domain protein family in the olfactory system, highlighting the precise compartmentalization, possible interactions and, finally, their functional implications

Assessment of the olfactory function in Italian patients with type 3 von Willebrand disease caused by a homozygous 253 Kb deletion involving VWF and TMEM16B/ANO2.

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability