Abstract
Tenascin C (Tnc) is an alternatively spliced, multimodular extracellular matrix glycoprotein present in the ventricular zone of the developing brain. Pax6-deficient small eye (sey) mouse mutants show an altered Tnc expression pattern. Here, we investigated the expression of Tnc isoforms in neural stem/progenitor cells and their regulation by the paired-box transcription factor Pax6. Neural stem/progenitor cells cultured as neurospheres strongly expressed Tnc on the protein level. The Tnc isoform expression in neural stem/progenitor cells was analyzed by reverse transcriptase-PCR and dot blot Southern hybridization. In total, 20 different Tnc isoforms were detected in neurospheres derived from embryonic fore-brain cell suspensions. The Tnc isoform containing the fibronectin type III domains A1A4BD is novel and might be neural stem/progenitor cell-specific. Transient overexpression of Pax6 in neurospheres of the medial ganglionic eminence did not alter the total Tnc mRNA expression level but showed a pronounced regulative effect on different Tnc isoforms. The larger Tnc isoforms containing four, five, and six additional alternatively spliced fibronectin type III domains were up-regulated, whereas the small Tnc isoforms without any or with one additional domain were down-regulated. Thus, Pax6 is a homeodomain protein that also modulates the splicing machinery. We conclude that the combinatorial code of Tnc isoform expression in the neural stem/progenitor cell is complex and regulated by Pax6. These findings suggest a functional significance for individual Tnc isoforms in neural stem/progenitor cells.
Highlights
Tenascin C (Tnc) consists of several serially arranged protein domains
Tenascin C is a multimodular extracellular matrix glycoprotein that is expressed in neural progenitor cells during early CNS development
Serum-free conditions neural stem/progenitor cells can be cultivated in the presence of the growth factors EGF or bFGF, which leads to the formation of free-floating cellular aggregates called neurospheres [22]
Summary
Tissue Culture—Neurosphere cultures were performed as described previously [14]. Briefly, timed pregnancy NMRI mice were sacrificed and staged according to Theiler’s criteria [23]. 20 l of transfection solution was mixed with 2.5 g of plasmid DNA and incubated for 20 min at room temperature before addition to 70 –90% confluent cultures for 4.5 h in DMEM. Immunocytochemistry—Electroporated neurosphere-derived cells were plated 1 day after transfection on PORN-coated (10 g/ml for 1 h at 37 °C) dishes for 3 h These cultures were washed once in PBS and fixed for 15 min in 4% paraformaldehyde. Detection was performed by the standard ECL method according to the manufacturer’s instructions (Amersham Biosciences) after incubation with anti-rabbit horseradish peroxidase-coupled secondary antibodies (1:5000, Dianova) for 2 h at ambient temperature
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