Characterization and isolation of immature neurons of the adult mouse piriform cortex

Physiological studies indicate that the piriform or primary olfactory cortex of adult mammals exhibits a high degree of synaptic plasticity. Interestingly, a subpopulation of cells in the layer II of the adult piriform cortex expresses neurodevelopmental markers, such as the polysialylated form of neural cell adhesion molecule (PSA‐NCAM) or doublecortin (DCX). This study analyzes the nature, origin, and potential function of these poorly understood cells in mice. As previously described in rats, most of the PSA‐NCAM expressing cells in layer II could be morphologically classified as tangled cells and only a small proportion of larger cells could be considered semilunar‐pyramidal transitional neurons. Most were also immunoreactive for DCX, confirming their immature nature. In agreement with this, detection of PSA‐NCAM combined with that of different cell lineage‐specific antigens revealed that most PSA‐NCAM positive cells did not co‐express markers of glial cells or mature neurons. Their time of origin was evaluated by birthdating experiments with halogenated nucleosides performed at different developmental stages and in adulthood. We found that virtually all cells in this paleocortical region, including PSA‐NCAM‐positive cells, are born during fetal development. In addition, proliferation analyses in adult mice revealed that very few cells were cycling in layer II of the piriform cortex and that none of them was PSA‐NCAM‐positive. Moreover, we have established conditions to isolate and culture these immature neurons in the adult piriform cortex layer II. We find that although they can survive under certain conditions, they do not proliferate in vitro either. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 748–763, 2016     

Improved method for visualizing cells revealed dynamic morphological changes of ventral neuroblasts during ventral cleft closure of Caenorhabditis elegans

The formation of intricate and functional biological structures depends on the dynamic changes of cellular morphology. Confocal laser scanning microscopy (CLSM) is a widely used method to reveal the three-dimensional (3-D) structure of cells during the development of Caenorhabditis elegans (C. elegans) and other model organisms. Improving the efficiency and image quality of CLSM would benefit studies using this method. We found that CED-10::GFP::CED-10, a green fluorescent protein (GFP) marker, is intensely expressed beneath the cell surface, facilitating visualization of cellular morphology in C. elegans embryos. By combining the unique properties of this marker, and with the help of direct 3-D rendering of images obtained by CLSM, we developed a simple but powerful method for investigating cellular morphology in developing embryos. Using this method we, for the first time, document the dynamic changes in the morphology of ventral neuroblasts in vivo during ventral cleft closure.     

A Switch in Tissue Stem Cell Identity Causes Neuroendocrine Tumors in Drosophila Gut

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Dividing neuron precursors express neuron-specific tubulin

Neuronal differentiation involves specific molecular and morphological changes in precursors and results in mature, postmitotic neurons. The expression of neuron-specific β tubulin, as detected by the monoclonal antibody TuJ1, begins during the period of neurogenesis. Indeed, TuJ1 expression precedes that of the 160 kD neurofilament protein in both the central and peripheral nervous systems. In the embryonic rat spinal cord, bipolar cells and some mitotic cells in the ventricular zone were TuJ1 immunoreactive (IR). Sensory ganglia also contained cells with TuJ1-IR mitotic spindles in situ. In embryonic rat sensory and sympathetic ganglion cell cultures pulsed with the thymidine analog bromodeoxyuridine (BrdU), TuJ1 label was detected in the spindle of mitotic cells and in the midbody of cells joined at cytokinesis, indicating that neuron-specific tubulin expression was initiated during or before the final mitosis of neuronal progenitors. Dorsal root ganglion cultures included TuJ1-IR cells with several shapes that may reflect morphological transitions, from flattened stellate neural crest-like cells to differentiated bipolar neurons. Indeed, the presence of flattened TuJ1-IR cells was correlated with neurogenesis. Some sympathetic neuron precursors possessed long TuJ1-IR neurites, as well as TuJ1-IR spindle microtubules and BrdU-labeled chromosomes, indicating that these precursors can possess long processes during metaphase. These results support the hypothesis that neuron-specific tubulin expression represents an early molecular event in neuronal differentiation exhibited by a wide range of neuronal precursors. The cessation of proliferation can occur at different points during neuronal differentiation, as TuJ1-IR was detected in cells undergoing mitosis. Future studies directed toward understanding the molecules that initiate neuron-specific tubulin expression may lead to the factors that control the initial phases of neuronal differentiation. © 1995 John Wiley & Sons, Inc.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Delta expression in post-mitotic neurons identifies distinct subsets of adult-specific lineages in Drosophila

The Drosophila ventral nerve cord is comprised of numerous neuronal lineages, each derived from a stereotypically positioned neuroblast (NB). At the embryonic stage the unique identities of each NB, and several of their neuronal progeny, are well characterized by spatial and temporal expression patterns of molecular markers. These patterns of expression are not preserved at the larval stage and thus the identity of adult-specific lineages remains obscure. Recent clonal analysis using MARCM has identified 24 adult-specific lineages arising from thoracic NBs at the larval stage. In this study, we have explored a role for the Delta protein in development of the post-embryonic Drosophila ventral nerve cord. We find that Delta expression identifies 7 of the 24 adult-specific lineages of the thoracic ganglia by being highly enriched in clusters of newly born post-mitotic neurons and their neurite bundles. The Delta lineages constitute the majority of bundles projecting to the ventral neuropil, consistent with a role in processing leg sensory information. Targeted knockdown of Delta in neurons using RNAi results in significantly decreased leg chemosensory response and a relatively unaffected leg mechanosensory response. Delta RNAi knockdown in Delta lineages also gives a more diffuse bundle terminal morphology while the overall path-finding of neurite bundles is unaffected. We also identify a male-specific Delta lineage in the terminal abdominal ganglia, implicating a role for Delta in development of sexually dimorphic neural networks. Examples of Delta-expressing neurites contacting Notch-expressing glia are also seen, but are not common to all Delta lineages. Altogether, these data reveal a fundamental pattern of Delta expression that is indicative of an underlying developmental program that confers identity to adult lineage neurons.     

Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence in vivo

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