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    • At this point it is worthwhile mentioning that the

    2018-10-20

    At this point it is worthwhile mentioning, that the B27 supplement can be purchased containing 0.3μM retinyl acetate which is a biologically inactive storage form of vitamin A. However, only dietary retinol bound to the retinol binding protein 4 (RBP4) is taken up into caspofungin where it can then be stored either as retinyl esters or oxidized in two steps to the bioactive metabolite RA (Chen and Reese, 2011; Napoli, 2012; Orsolits et al., 2013). Interestingly, only some cell types seem to have the ability to take up retinol/RBP4 and to metabolize retinol to RA inside the cell. Newer data suggest that RA synthesized in one cell type can act on adjacent cells. This paracrine signalling is a common theme in development and the differentiation of cells in vitro (Gudas and Wagner, 2011). In the present study we aimed to analyze the effect of early retinoid signalling already during the neural induction phase on the development of the neuronal culture. Therefore, two NPC lines were generated (NPC and NPC) one of which was treated with RA during the initial neural induction phase of EB formation (NPC). Thereafter both lines were treated equally, i.e. in the absence of biologically active RA in the culture medium. In the early phase of differentiation no difference in number or size of formed EBs was observed and also the onset of rosette formation was comparable. Both NPC lines were morphologically similar and did not differ within the expression of neural precursor marker proteins such as Pax6, nestin or Sox2, indicating that RA treatment during the neurulation phase did not influence NPC properties so far. A comparative transcriptome analysis of the two cell lines revealed a RA-induced expression of the transcription factor FoxG1 which is required for early telencephalon patterning (Hebert and Fishell, 2008) and, on the other hand, also of HoxA1 which is involved in hindbrain patterning (Chambers et al., 2009a). These findings suggest that early RA treatment induces a more general neuronal commitment of the NPCs whereas RA activity is needed also during later phases of development for hindbrain/spinal cord differentiation. To elucidate these interactions in more detail, a gene expression analysis of terminally differentiated neurons in the absence versus presence of RA would be needed. In this regard, Paschaki and colleagues have performed a comparative transcriptome analysis of anterior and posterior tissues of murine embryos lacking the endogenous RA synthesizing enzyme retinaldehyde dehydrogenase 2 (Paschaki et al., 2013). These studies conducted at early stages of embryonic development revealed a down-regulation of the anterior FoxG1 as well as of the posterior HoxA1 in the RA-deficient mice. In the transcriptome analysis of our NPC lines, the expression of several neuronal transcripts including various proteins involved in synaptic transmission was significantly up-regulated in the NPC sample. Interestingly, these included GABAergic genes, such as the vesicular GABA transporter and glutamic acid decarboxylase 2, as well as transcripts involved in glutamatergic neurotransmission like the vesicular glutamate transporter vGLUT3. Glia-specific transcripts, however, were not up-regulated in the NPC samples again suggesting that early RA treatment favours neuronal versus glial differentiation. In order to investigate properties of terminally differentiated neurons we analyzed morphological aspects and the cellular composition of the NPC-derived cultures. Interestingly, the quantification of neuronal outgrowth revealed that early RA treatment promotes axonal growth which led to the development of neurons with axons twice as long as the dendrites. This corresponds to Dotti\'s model of a functional neuron (Dotti et al., 1988) and was only found in differentiated neurons of the NPC line. In addition, after 50days of terminal differentiation neural cultures derived from NPC contained significantly more neurons as compared to NPC which contained more glial cells. In this context it has been described that a passage number of >20 of a NPC line raises the glial proportion within the neuronal culture (Tang et al., 2013), however, all here described experiments with both of the NPC lines (NPC and NPC) were performed during passages #10–20 of the NPCs. Therefore, we conclude from our observations and the transcriptomic analysis that the RA treatment during early neurulation has a beneficial effect on the neurodevelopment since more neurons survive during terminal differentiation stages in the NPC line compared to the NPC line. Since terminal differentiation is induced by bFGF withdrawal it should be mentioned that RA and FGF have an interesting regulatory relationship in morphogenesis (Casci, 2008; White et al., 2007). In this study it was suggested that removal of FGF may effectively up-regulate RA signalling. This, however, should not play a role in our study since we apply RA during EB formation in the absence of bFGF, and apply bFGF only in later phases.