Timothy S. McClintock

Olfactory Receptor Trafficking Involves Conserved Regulatory Steps

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiatedodora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 inodora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.

Differentially expressed transcripts from phenotypically identified olfactory sensory neurons.

In comparing purified mouse olfactory sensory neurons (OSNs) with neighboring cells, we identified 54 differentially expressed transcripts. One‐third of the transcripts encode proteins with no known function, but the others have functions that correlate with challenges faced by OSNs. The OSNs expressed a diversity of signaling protein genes, including stomatin (Epb7.2), S100A5, Ddit3, Sirt2, CD81, Sdc2, Omp, and Ptpla. The elaboration of dendrites, cilia, and axons that places OSNs in contact with diverse cell types and signals presumably also requires large investments in cytoskeletal‐associated proteins, lipid biosynthesis, and energy production. Several of the genes encode proteins that participate in these biological processes, including ATP5g3, Ndufa9, Sqrdl, Mdh1, Got1, β‐2 tubulin, Capza1, Bin3, Tom1, Acl6, and similar to O‐MACS. Three transcripts had restricted expression patterns. Similar to O‐MACS and Gstm2 had zonally restricted expression patterns in OSNs and sustentacular cells but not in Bowmans glands, suggesting that zonality can be differentially regulated by cell type. The mosaic expression pattern of S100A5 in approximately 70% of OSNs predicts that it is coexpressed with a subset of odorant receptors. We captured four abundant transcripts, Cyp2a4, similar to Cyp2g1, Gstm2, and Cbr2, that encode xenobiotic metabolizing enzymes expressed by sustentacular cells or Bowmans glands, reinforcing the interpretation that clearance of xenobiotic compounds is a major function of these cells. Within the olfactory epithelium, Cbr2 is a new anatomical marker for sustentacular cells. We also discovered that Reg3g is a marker for respiratory epithelium. J. Comp. Neurol. 483:251–262, 2005.

Mouse olfactory sensory neurons express 10,000 genes.

Olfactory epithelial cells from olfactory marker protein‐green fluorescent protein (OMP‐GFP) mice were separated by fluorescence‐activated cell sorting into a GFP+ sample enriched in mature olfactory sensory neurons (OSNs) and a GFP– sample enriched in all other cells. GeneChip expression profiling of these samples provided a predictive measure of expression in OSNs. Validation tests comparing the ratio of GFP+/GFP– signal intensity against expression patterns from in situ hybridization for 189 mRNAs proved statistically significant and provided probabilities of expression in OSNs scaled according to the signal intensity ratios. These probabilities predict that, among 11,596 mRNAs detected in the GFP+ sample, more than 10,000 are expressed in OSNs. Transcripts and overrepresented categories of mRNAs detected in the GFP+ sample agreed with known properties of OSNs and predict additional properties. For example, ciliogenesis and spermatogenesis were overrepresented, consistent with similarities between OSN cilia and sperm flagella. Chromatin assembly mRNAs were expressed throughout the OSN cell lineage, consistent with the hypothesis that chromatin remodeling plays a role in OSN differentiation. We detected numerous signaling proteins and receptors, such as 30 nonchemosensory G‐protein‐coupled receptors, including the presynaptic glutamate receptor mGlur4 and the Wnt receptor Fzd3. The largest group of mRNAs, however, was the hundreds of transcriptional regulators that presumably determine the OSN phenotype. The absence of OMP protein in OMP‐GFP mice had no detectable effect on mRNA abundance. Within limits prescribed by the nature of microarray data and the in situ hybridization validation, these data should be useful in directing further experiments on OSN function. J. Comp. Neurol. 502:1138–1156, 2007.

Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells.

Abstract: Olfactory receptors are difficult to express functionally in heterologous cells. We found that olfactory receptors traffic poorly to the plasma membrane even in cells with neuronal phenotypes, including cell lines derived from the olfactory epithelium. Other than mature olfactory receptor neurons, few cells appear able to traffic olfactory receptors to the plasma membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co‐expression of the missing C‐terminal fragment with these truncated receptors prvented their expression in the plasma membrane. Intramolecular interactions between N‐ and C‐terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.

Transcriptional changes during neuronal death and replacement in the olfactory epithelium

The olfactory epithelium has the unusual ability to replace its neurons. We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5--7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlh 1, Hes 6, Lmyc 1, c-Myc, Mxd 4, Id 1, Nmyc 1, Cited 2, c-Myb, Mybl 1, Tead 2, Dp 1, Gata 2, Lmo 1, and Sox1 1. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

Effects of shell condition and size upon the shell choice behavior of a hermit crab

Abstract Hermit crabs attracted to simulated sites of predation upon gastropods, a source of empty shells, were more likely to inhabit shells of worse condition than crabs collected by visual searches, suggesting that shell condition affects shell choice behavior. Laboratory tests showed that Pagurus pollicaris Say inhabiting shells with lip damage, hydroid colonies, and pitting by boring organisms exchanged shells more frequently than crabs in unmodified shells. Shell fit also affected laboratory shell exchange; i.e., crabs in relatively small shells exchanged significantly more than crabs in relatively large shells. The shell conditions inducing the highest observed rates of shell exchange were those potentially increasing the risk of predation: lip damage, relatively small shells, and shells weakened by boring organisms. The active shell exchange behavior induced in crabs inhabiting damaged, pitted, and fouled shells explains the overrepresentation of these shell conditions at predation events where the probability of shell exchange is high.

Emx2 Stimulates Odorant Receptor Gene Expression

The mechanisms selecting a single odorant receptor (OR) gene for expression in each olfactory sensory neuron (OSN) establish an OR expression pattern critical for odor discrimination. These mechanisms are largely unknown, but putative OR promoters contain homeodomain-like sites, implicating homeobox transcription factors such as Emx2. At embryonic day 18.5, expression of 49-76% of ORs was decreased in mice lacking Emx2, depending on the metric used. The decreases were due to fewer OSNs expressing each OR. Affected ORs showed changes that were disproportionately greater than the 42% reduction in mature neurons and similar decreases in unrelated olfactory neuron-enriched messenger RNAs in Emx2(-/-) mice. Both Class I and Class II ORs decreased, as did ORs expressed in both the dorsal and ventral regions of the epithelium. Conversely, 7% of Class II ORs tested were expressed more frequently, suggesting that some ORs are independent of Emx2. Emx2 helps stimulate transcription for many OR genes, which we hypothesize is through direct action at OR promoters, but Emx2 appears to have no significant role in regulating other aspects of OR gene expression, including the zonal patterns, OR gene cluster selection mechanisms, and singularity of OR gene choice.

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.

Molecular, mass spectral, and physiological analyses of orcokinins and orcokinin precursor-related peptides in the lobster Homarus americanus and the crayfish Procambarus clarkii

Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY((SO3))DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.

Olfactory-enriched transcripts are cell-specific markers in the lobster olfactory organ

Genes expressed specifically in a tissue are often involved in the defining functions of that tissue. We used representational difference analysis of cDNA to amplify 20 cDNA fragments representing transcripts that were more abundant in the lobster olfactory organ than in brain, eye/eyestalk, dactyl, pereiopod, or second antenna. We then independently confirmed that the transcripts represented by these clones were enriched in the olfactory organ. The 20 cDNA fragments represent between 6 and 15 different genes. Six of the cDNAs contained sequences highly similar to known gene families. We performed in situ hybridization with these six and found that all were expressed in subsets of cells associated with the aesthetasc sensilla in the olfactory organ. Clones OET‐07, an ionotropic receptor, and OET‐10, an α tubulin, were specific to the olfactory receptor neurons. OET‐02, a monooxygenase, was expressed only in the outer auxiliary cells. OET‐03, a serine protease, was specific to the collar cells. OET‐11, an α2 macroglobulin, was expressed by the receptor neurons and the collar cells. OET‐17, a calcyphosine, was expressed in the receptor neurons, inner auxiliary cells, and collar cells. The identities and expression patterns of these six transcripts predict involvement in both known and novel properties of the lobster olfactory organ. J. Comp. Neurol. 455:125–138, 2003.