Basic FGF increases communication between cells of the developing neocortex - PubMed (original) (raw)
Basic FGF increases communication between cells of the developing neocortex
B Nadarajah et al. J Neurosci. 1998.
Abstract
We have found that basic fibroblast growth factor (bFGF), applied to cortical progenitor cells in vitro, produces an increase in the expression of the gap junction protein connexin (Cx) 43 and in the mRNA encoding Cx 43. This effect was evident in both proliferating and nonproliferating cells. The elevated levels of mRNA suggest that bFGF is likely to exert its effect by upregulating the rate of transcription of the Cx 43 gene. We have further shown that the increase in Cx 43 expression is mediated through the receptor tyrosine kinase pathway and is associated with enhanced intercellular dye-coupling mediated by gap junctions. These results suggest that gap junction channels provide a direct conduit for mitogens released in response to bFGF to effectively regulate proliferation during corticogenesis.
Figures
Fig. 1.
Immunocytochemical analysis of cells maintained in 10% FCS for 24 hr before exposure to bFGF in serum-free medium for 4 hr. Connexin staining is shown in green, and propidium iodide labeling of nuclei is shown in red.a–c, Images taken from control (a) and from cultures treated with 10 and 20 ng of bFGF (b, c), all stained for Cx 43 with a polyclonal antibody raised against the intracellular loop of the protein.d, e, g, h, Images collected from control (d, g) and from cultures treated with 20 ng of bFGF (e, h) stained for Cx 43 with polyclonal antibodies raised against the amino and C termini, respectively. Note the intracellular labeling of Cx 43 in all treated cultures. f, Absence of labeling in cells when the primary antibody was omitted. (i, j) Examples of images taken from control and from cultures treated with 20 ng of bFGF and labeled for Cx 26. Scale bar, 100 μm.
Fig. 2.
Localization of Cx 43 in Western blots of cultures maintained in 10% FCS before bFGF treatment. a, Coomassie blue-stained gel containing the same samples as in_b_. b, Localization of 45 and 76 kDa bands using an anti-Cx 43 polyclonal antibody raised against the intracellular loop of the protein. c, d, Immunolocalization of 45 and 76 kDa bands with polyclonal (c) and monoclonal (d) antibodies raised against the C terminus of the protein.a–d, Lanes 1–3 correspond to samples from control and from cultures treated with 10 and 20 ng of bFGF, respectively. e, Detection of 45 and 76 kDa bands in different fractions of heart extracts. Lane 1_corresponds to whole heart extract, and lanes 2 and_3 correspond to supernatant and pellet of the membrane fractions, respectively.
Fig. 3.
Immunocytochemical analyses of cultures after different treatments. Connexin staining is shown in_green_, and propidium iodide labeling of nuclei is shown in red. a–c, Images taken from cultures maintained initially in 10% FCS and later changed to medium containing 0.5% serum before exposure to bFGF in serum-free medium for 4 hr.d–f, Images of cultures that were transferred to medium containing cycloheximide for a period up to 6 hr after bFGF treatment.g–i, Images of cultures treated with MTA and bFGF.a, d, and g are images of control cultures, whereas b, e, and_h_ and c, f, and_i_ are from cultures treated with 10 and 20 ng of bFGF, respectively. Scale bar: a–i, 100 μm. j, k, Images of bFGF-treated cultures stained for BrdU (k) or double-labeled for BrdU and Cx 43 (j). (l) Image of a culture double-labeled for PCNA and Cx 43. Connexin staining is shown in_green_, and BrdU/PCNA-labeled nuclei are shown in_red_. Scale bar: j–l, 80 μm.
Fig. 4.
Western blot analyses of cultures after different treatments. a, Detection of a 45 kDa band in cultures that were transferred to a medium containing cycloheximide for a period up to 6 hr after treatment with bFGF. b, Detection of 45 kDa bands in cultures treated with MTA and bFGF. Lanes 1–4 correspond to samples from control, and from cultures treated with 10, 20, and 30 ng of bFGF. The position of the molecular markers is given on the right. c, Northern blot of Cx 43 from cultures exposed to bFGF. Lanes 1–3 correspond to control and to cultures treated with 10 and 20 ng of bFGF; note the increased abundance of Cx 43 transcripts in the sample from a culture treated with 20 ng of bFGF.
Fig. 5.
Localization of a higher molecular weight protein corresponding to a 76 kDa band in cultures immunoprecipitated using Cx 43 antibodies and detected with an anti-bFGF serum. Lanes 1 and 2 correspond to control and cultures treated with 20 ng of bFGF; note the absence of this band in the control cultures.
Fig. 6.
Lucifer yellow injections in cells in control (a, b) and bFGF-treated (c, d) cultures showed transfer of dye to neighboring cells. Note that the larger clusters of dye-coupled cells in treated cultures are brightly labeled, in contrast to the cells from control cultures. _Arrows_point to the injected cells.
Fig. 7.
Frequency of cell coupling in control and bFGF-treated cultures. Treatment with 20 ng of the growth factor resulted in the frequent presence of large clusters of coupled cells.NC, Noncoupled.
References
- Acevedo P, Bertram JS. Liarozole potentiates the cancer chemopreventive activity of and the up-regulation of gap junctional communication and connexin43 expression by retinoic acid and beta-carotene in 10T1/2 cells. Carcinogenesis. 1995;16:2215–2222. -PubMed
- Becker DL, Evans WH, Green CR, Warner A. Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions. J Cell Sci. 1995;108:1455–1467. -PubMed
- Bennett MVL, Barrio LC, Bargiello TA, Spray DC, Hertzberg E, Saez JC. Gap junctions: new tools, new answers, new questions. Neuron. 1991;6:305–320. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous