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Generation of diverse neuronal subtypes in cloned populations of stem-like cells.

Varga BV, Hádinger N, Gócza E, Dulberg V, Demeter K, Madarász E, Herberth B - BMC Dev. Biol. (2008)

Bottom Line: Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes.Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons.Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Developmental Neurobiology, Institute of Experimental Medicine of Hungarian Academy of Sciences, Budapest, Hungary. vargab@koki.hu

ABSTRACT

Background: The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material.

Results: In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons.

Conclusion: The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

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Neuron formation and gene expression after short- or long-term exposure to RA. Differentiation of NE-4C cells was induced by exposure to 10-6 M RA for 12, 72 or 168 hours. A: The number of neurons was determined by counting NeuN-positive nuclei after a 168-hour period of differentiation (stage 4). In comparison to longer (72-hour or 168-hour) treatments, short term (12-hour) induction resulted in less NeuN-positive neurons. (Averages and standard deviation values were calculated from 4 identically treated sister-cultures (n = 4)). B: Short-term (12-hour) or long-term (72 or 168 hours) presence of RA did not cause significant changes in the expression of hox2b, gbx2 and pax6 genes. The continuous presence of RA did not inhibit the expression of the dorsal forebrain specific emx2 gene, but was sufficient to reduce the RA-sensitive otx2 mRNA level. The results of a representative RT-PCR assay on non-induced (stage 1; left column) and differentiated, neuron-rich (stage 4; three right columns) cultures are shown.
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Figure 8: Neuron formation and gene expression after short- or long-term exposure to RA. Differentiation of NE-4C cells was induced by exposure to 10-6 M RA for 12, 72 or 168 hours. A: The number of neurons was determined by counting NeuN-positive nuclei after a 168-hour period of differentiation (stage 4). In comparison to longer (72-hour or 168-hour) treatments, short term (12-hour) induction resulted in less NeuN-positive neurons. (Averages and standard deviation values were calculated from 4 identically treated sister-cultures (n = 4)). B: Short-term (12-hour) or long-term (72 or 168 hours) presence of RA did not cause significant changes in the expression of hox2b, gbx2 and pax6 genes. The continuous presence of RA did not inhibit the expression of the dorsal forebrain specific emx2 gene, but was sufficient to reduce the RA-sensitive otx2 mRNA level. The results of a representative RT-PCR assay on non-induced (stage 1; left column) and differentiated, neuron-rich (stage 4; three right columns) cultures are shown.

Mentions: Bearing in mind that RA may also serve as a "posteriorizing and ventralizing" agent [38], we compared the effects of short-and long-term presence of RA. In addition to the standard 48-hour treatment, NE-4C cells were exposed to 10-6 M RA for 12, 72 and 168 hours and the expression of several region-specific genes was investigated on the 7th day (stage 4) of induction (Fig. 8). Each exposure to RA resulted in neuron formation (Fig. 8A), but the proportion of neurons was smaller after the shortest (12 hour) induction. Long-term exposure to RA (168 h for NE-4C and 96 hours for R1 ES) effectively reduced the otx2 transcript level (Fig. 8B), but failed to cause relevant changes in the expression of many positional genes including the anterior-dorsal emx2.


Generation of diverse neuronal subtypes in cloned populations of stem-like cells.

Varga BV, Hádinger N, Gócza E, Dulberg V, Demeter K, Madarász E, Herberth B - BMC Dev. Biol. (2008)

Neuron formation and gene expression after short- or long-term exposure to RA. Differentiation of NE-4C cells was induced by exposure to 10-6 M RA for 12, 72 or 168 hours. A: The number of neurons was determined by counting NeuN-positive nuclei after a 168-hour period of differentiation (stage 4). In comparison to longer (72-hour or 168-hour) treatments, short term (12-hour) induction resulted in less NeuN-positive neurons. (Averages and standard deviation values were calculated from 4 identically treated sister-cultures (n = 4)). B: Short-term (12-hour) or long-term (72 or 168 hours) presence of RA did not cause significant changes in the expression of hox2b, gbx2 and pax6 genes. The continuous presence of RA did not inhibit the expression of the dorsal forebrain specific emx2 gene, but was sufficient to reduce the RA-sensitive otx2 mRNA level. The results of a representative RT-PCR assay on non-induced (stage 1; left column) and differentiated, neuron-rich (stage 4; three right columns) cultures are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2556672&req=5

Figure 8: Neuron formation and gene expression after short- or long-term exposure to RA. Differentiation of NE-4C cells was induced by exposure to 10-6 M RA for 12, 72 or 168 hours. A: The number of neurons was determined by counting NeuN-positive nuclei after a 168-hour period of differentiation (stage 4). In comparison to longer (72-hour or 168-hour) treatments, short term (12-hour) induction resulted in less NeuN-positive neurons. (Averages and standard deviation values were calculated from 4 identically treated sister-cultures (n = 4)). B: Short-term (12-hour) or long-term (72 or 168 hours) presence of RA did not cause significant changes in the expression of hox2b, gbx2 and pax6 genes. The continuous presence of RA did not inhibit the expression of the dorsal forebrain specific emx2 gene, but was sufficient to reduce the RA-sensitive otx2 mRNA level. The results of a representative RT-PCR assay on non-induced (stage 1; left column) and differentiated, neuron-rich (stage 4; three right columns) cultures are shown.
Mentions: Bearing in mind that RA may also serve as a "posteriorizing and ventralizing" agent [38], we compared the effects of short-and long-term presence of RA. In addition to the standard 48-hour treatment, NE-4C cells were exposed to 10-6 M RA for 12, 72 and 168 hours and the expression of several region-specific genes was investigated on the 7th day (stage 4) of induction (Fig. 8). Each exposure to RA resulted in neuron formation (Fig. 8A), but the proportion of neurons was smaller after the shortest (12 hour) induction. Long-term exposure to RA (168 h for NE-4C and 96 hours for R1 ES) effectively reduced the otx2 transcript level (Fig. 8B), but failed to cause relevant changes in the expression of many positional genes including the anterior-dorsal emx2.

Bottom Line: Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes.Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons.Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Developmental Neurobiology, Institute of Experimental Medicine of Hungarian Academy of Sciences, Budapest, Hungary. vargab@koki.hu

ABSTRACT

Background: The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material.

Results: In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons.

Conclusion: The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

Show MeSH
Related in: MedlinePlus