Evidence that pioneer olfactory axons regulate telencephalon cell cycle kinetics to induce the formation of the olfactory bulb

AbstractEarly olfactory axons follow a specific pathway to reach the developing telencephalon. We observed that a subpopulation of these axons, the pioneer olfactory axons, penetrate into the ventricular zone of a highly restricted region of the telencephalon at E13 and E14. At E15, this same telencephalic region evaginates to form the olfactory bulb. To investigate the possibility that the pioneer olfactory axons induce the olfactory bulb by influencing precursor cell populations, we compared cell cycle kinetics and differentiation in the olfactory bulb primordium and the adjacent neocortex using cumulative bromdeoxyurldine labeling. The results showed that, 24 hr after the arrival of the first pioneer axons, the duration of the cell cycle is prolonged significantly in the olfactory bulb primordium. In addition, twice as many cells have exited the mitotic cycle in the olfactory bulb primordium versus the adjacent cortex. These findings suggest that pioneer olfactory axons play a role in the induction of the olfactory bulb by selectively modulating cell cycle kinetics in the olfactory bulb primordium. Afferent axons may influence target morphogenesis by modulating target precursor cell proliferation in other developing neural structures

Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

<p>Abstract</p> <p>Background</p> <p>Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an <it>in vitro </it>system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.</p> <p>Results</p> <p>We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days <it>in vitro</it>.</p> <p>Conclusion</p> <p>This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed <it>in vivo</it>. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.</p

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC

A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration

Thrombospondins are multimeric extracellular matrix glycoproteins that play important roles in development, synaptogenesis and wound healing in mammals. We previously identified four putative thrombospondins in the genome of the starlet sea anemone Nematostella vectensis. This study presents the first analysis of these thrombospondins, with the goals of understanding fundamental roles of thrombospondins in the Eumetazoa. Reverse transcriptase PCR showed that each of the N. vectensis thrombospondins (Nv85341, Nv22035, Nv168100 and Nv30790) is transcribed. Three of the four thrombospondins include an RGD or KGD motif in their thrombospondin type 3 repeats at sites equivalent to mammalian thrombospondins, suggesting ancient roles as RGD integrin ligands. Phylogenetic analysis based on the C-terminal regions demonstrated a high level of sequence diversity between N. vectensis thrombospondins. A full-length cDNA sequence was obtained for Nv168100 (NvTSP168100), which has an unusual domain organization. Immunohistochemistry with an antibody to NvTSP168100 revealed labeling of neuron-like cells in the mesoglea of the retractor muscles and the pharynx. In situ hybridization and quantitative PCR showed that NvTSP168100 is upregulated during regeneration. Immunohistochemistry of the area of regeneration identified strong immunostaining of the glycocalyx, the carbohydrate-rich matrix coating the epidermis, and electron microscopy identified changes in glycocalyx organization during regeneration. Thus, N. vectensis thrombospondins share structural features with thrombospondins from mammals and may have roles in the nervous system and in matrix reorganization during regeneration

Influence of Olfactory Epithelium on Mitral/Tufted Cell Dendritic Outgrowth

Stereotypical connections between olfactory sensory neuron axons and mitral cell dendrites in the olfactory bulb establish the first synaptic relay for olfactory perception. While mechanisms of olfactory sensory axon targeting are reported, molecular regulation of mitral cell dendritic growth and refinement are unclear. During embryonic development, mitral cell dendritic distribution overlaps with olfactory sensory axon terminals in the olfactory bulb. In this study, we investigate whether olfactory sensory neurons in the olfactory epithelium influence mitral cell dendritic outgrowth in vitro. We report a soluble trophic activity in the olfactory epithelium conditioned medium which promotes mitral/tufted cell neurite outgrowth. While the trophic activity is present in both embryonic and postnatal olfactory epithelia, only embryonic but not postnatal mitral/tufted cells respond to this activity. We show that BMP2, 5 and 7 promote mitral/tufted cells neurite outgrowth. However, the BMP antagonist, Noggin, fails to neutralize the olfactory epithelium derived neurite growth promoting activity. We provide evidence that olfactory epithelium derived activity is a protein factor with molecular weight between 50–100 kD. We also observed that Follistatin can effectively neutralize the olfactory epithelium derived activity, suggesting that TGF-beta family proteins are involved to promote mitral/tufted dendritic elaboration

Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons.

BackgroundOlfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.ResultsWe demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro.ConclusionThis culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment

Making autotutor agents smarter: Autotutor answer clustering and iterative script authoring

AutoTutor uses conversational intelligent agents in learning environments. One of the major challenges in developing AutoTutor applications is to assess students’ natural language answers to AutoTutor questions. We investigated an AutoTutor dataset with 3358 student answers to 49 AutoTutor questions. In comparisons with human ratings, we found that semantic matching works well for some questions but poor for others. This variation can be predicted by a measure called “question uncertainty”, an entropy value on semantic cluster probabilities. Based on these findings, we propose an iterative AutoTutor script authoring process that can make AutoTutor agents smarter and improve assessment models by iteratively adding and modifying both questions and ideal answers