Journal:

Article Title: Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension

doi: 10.1038/sj.emboj.7600202

Figure Lengend Snippet: Axon-extending activity of Meteorin depends on satellite glial cells. (A–H) Dissociated cells of the E14.5 mouse DRG were cultured for 48 h at a high density in the absence (A, C, E, F) or presence (B, D, G, H) of Meteorin (80 ng/ml). They were then stained with the 2H3 antibody to identify neurons (A, B); the same fields were observed with Nomarski optics in (C) and (D), respectively. A dense axonal network and large neuronal cell bodies are apparent in the presence of Meteorin (B). Meteorin also changed the morphology of 2H3-negative, non-neuronal cells from flat and polygonal (C) to bipolar (D) (79.5±5.8% of cells (N=81–112)). Cells were alternatively stained with antibodies to SOX10 (E, G) or to BFABP (F, H). Meteorin-responsive non-neuronal cells were positive for SOX10 and BFABP, indicating that they were satellite glial cells. Scale bar (shown in (A)): 100 μm (A–D) or 60 μm (E–H). (I–K) DRG cells cultured at a high density with Meteorin were stained with antibodies to CGRP (I), to Ret (J), or to parvalbumin (K). CGRP-positive small and Ret-positive intermediate neurons, but not parvalbumin-positive large neurons, are apparent. Scale bar, 60 μm. (L–N) DRG cells were cultured for 48 h at a low density in the absence (L) or presence of either Meteorin (80 ng/ml) (M) or NGF (20 ng/ml) (N). They were then stained with the 2H3 antibody. The morphology of satellite glial cells is shown in the lower panels. 2H3-positive neurons remained flat and fibroblast-like in appearance in the absence or presence of Meteorin, but they manifested a large soma and extended long axons in the presence of NGF. In contrast, satellite glial cells adopted a bipolar morphology in response to Meteorin (81.5±3.8% of cells (N=31–47)), but remained flat and polygonal in the presence of NGF. Scale bar: 150 μm (main panels) or 40 μm (insets). (O, P) The satellite glia (∼90% pure) and neurons (>90% pure) were purified from the mouse DRG at E14.5 by sequential plating on tissue grade dishes. Satellite glia-enriched cells were conditioned for 48 h in D/F12-N2 without (O) or with (P) Meteorin (80 ng/ml). CM was prepared from each culture. Fresh Meteorin (80 ng/ml) was then added to the former CM. DRG neurons were cultured with each CM for 48 h and were stained with the 2H3 antibody. With the CM in which Meteorin was supplemented later, neurons remained flat and fibroblast-like in appearance (O). In contrast, with the CM prepared from Meteorin-treated satellite glia (P), neurons had large soma and elongated axons. Scale bar, 100 μm. (Q) Satellite glia-enriched cells were treated for 20 min with or without Meteorin. Cell lysates were prepared and were subjected to immunoblot analysis with antibodies to phosphorylated ERK1/2 (P-Erk1/2) or ERK1/2. Meteorin activated phosphorylation of ERK1/2 in satellite glia. (R) Total RNA prepared from DRG cells that had been cultured with or without Meteorin was subjected to RT–PCR analysis with primers specific for NGF, BDNF, or G3PDH (internal control) cDNAs.

Article Snippet: RT–PCR analysis was performed with a Cell-to-cDNA kit (Ambion) and the following primers: NGF-S (5′-TCAGCATTCCCTTGACACAGC-3′); NGF-AS (5′-TCCAGTGTTTGGAGTCGATGC-3′); BDNF-S (5′-GAGCTGAGCGTGTGTGACAG-3′); BDNF-AS (5′-GGTCAGTGTACATACACAGG-3′); G3PDH-S (5′-GGCCGGTGCTGAGTATGTCG-3′); and G3PDH-AS (5′-GCACGTCAGATCCACGACGG-3′).

Techniques: Activity Assay, Cell Culture, Staining, Purification, Western Blot, Reverse Transcription Polymerase Chain Reaction