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Connexin 43 is a gap junctional protein found predominantly in astrocytes. In the mammalian nervous system, it appears to play an organizational role during neural development. In the current study, conducted on the frog, Xenopus laevis, we found that connexin 43 occurs in glial cells during development of rhombomeres and that its expression is spatially and temporally regulated. We used neural (2G9) and cell proliferation (BrdU) markers to identify the overall organization of Xenopus rhombomeres and then tracked expression of connexin 43 and glial fibrillary acidic protein, an intermediate filament protein known to mark glia during rhombomeric development. 2G9 was expressed in rhombomeric centers (ventricular concavities) and outlying neuropil regions, whereas BrdU-labeled cells marked boundary regions (ventricular convexities), as early as stage 35/36. These labeling patterns persisted through premetamorphic stages of hindbrain development. At stage 47, 2G9-labeled profiles were highlighted by the presence of connexin 43, and at stage 49/50, connexin 43-labeled profiles, i.e., rhombomeric centers and neuropil, as well as rhombomeric boundaries, not labeled by connexin 43, became immunoreactive to glial fibrillary acidic protein. Cells of rhombomeric center regions and their processes in the outlying neuropil co-expressed glial fibrillary acidic protein and connexin 43 at a time that is characterized by the emergence of hindbrain auditory neural circuitry. Glial fibrillary acidic protein positive glial cells that appeared at rhombomeric boundaries never expressed connexin 43, but rather appeared to physically bisect ventricular convexities into adjacent rhombomeric regions. Thus, glial cells that express connexin 43 in developing rhombomeric centers may be similar to radial glia, assisting in formation of neural circuitry, while glial cells that do not express connexin 43, situated at rhombomeric boundaries, may be involved in demarcating adjacent rhombomeres.
Fig. 1.
Western blot analysis of protein loaded in the left lane shows a single 44 kDa band representing the putative Xenopus Cx43 protein. A molecular weight marker appears in the right lane.
Fig. 2.
Developmental changes in 2G9 expression showing: (A) emerging segmental organization of 2G9 stained neural tissue in the hindbrain of stage 35/36 tadpole; (B) definition of 2G9 clusters at the ventricular surface (arrows) and outlying neuropil regions (N) at stage 40/41 of hindbrain development; (C) localization of 2G9 clusters to rhombomere centers (ventricular concavities; arrows), but not boundary regions (ventricular convexities; arrow heads), and outlying neuropil (N) at stage 50; and (D) high magnification of the inbox in (C) depicting 2G9 stained cell processes emanating from the clusters (arrows) and reaching into the outlying neuropil (N). All figures show horizontal sections, where posterior is to the left. Magnification bars: 50 μm.
Fig. 3.
Results of double labeling portraying: (A) alternating 2G9 clusters (brown; arrows) and groups of dividing cells (red; arrow heads) at the ependymal surface in the hindbrain of a stage 40/41 tadpole; (B) high magnification image of the inbox in (A) demonstrating almost no overlap between the relatively quiescent cells of the 2G9 clusters (arrows) and the BrdU labeled proliferating cells (arrows heads). All figures show horizontal sections, where posterior is to the left. Magnification bars: 50 μm.
Fig. 4.
Developmental changes in Cx43 expression showing: (A) the appearance of Cx43 clusters (arrows) at the ependymal surface and in the outlying neuropil (N) of a stage 50tadpole; (B) high magnification view of the inbox in (A) depicting the punctate and fibrous characteristics of Cx43 immunostaining in the clusters (arrows) and the neuropil (N), as well as in cell processes emanating from the clusters and reaching into the neuropil; (C) similar Cx43 immunostaining viewed in the clusters (arrows) and the neuropil (N) of a transverse section at the level of the fourth rhombomere; and (D) high power image of the inbox in (C) displaying characteristic punctate Cx43 staining within clusters (arrows) and associated neuropil. (A) and (B) show horizontal sections, where posterior is to the left; (C) and (D) show transverse sections, where dorsal is up. Magnification bars: 50 μm.
Fig. 5.
Results of double labeling portraying: (A) co-localization of 2G9 (red) and Cx43 (brown) expression within clusters of cells at rhombomeric centers (arrows) and the outlying neuropil region (N) in the hindbrain of a stage 47 animal; (B) high magnification of the inbox in (A) demonstrating 2G9-Cx43 labeled profiles within ventricular clusters (arrows) and processes reaching into the outlying neuropil (N); rhombomeric boundary regions show no labeling above background (arrow heads); (C) BrdU-labeled S-phase nuclei (red; arrow heads) alternating with Cx43 clusters (brown; arrows) at the ependymal surface of the hindbrain seen 72 h post-BrdU exposure in stage 49tadpole; (D) high power view of the inbox in (C) portraying minimal overlap between the BrdU (ventricular convexities; arrow heads) and Cx43 labeled clusters (ventricular concavities; arrows). All figures show horizontal sections, where posterior is to the left. Magnification bars: 50 μm.
Fig. 6.
Results of single and double labeling in vibratome sections displaying: (A) GFAP immunoreactivity in rhombomeric centers (ventricular concavities; arrows), rhombomeric boundaries (ventricular convexities; arrow heads) and the outlying neuropil (N) of a stage 49/50 tadpole; (B) Cx43 expression in rhombomeric centers (ventricular concavities; arrows) and outlying neuropil regions (N); (C) an image of the same vibratome section that was labeled with GFAP (shown in A), now double stained with Cx43 and photographed with a combination filter (DAPI/FITC/TRITC) to highlight DAB-stained GFAP as described in (A), and subsequently rephotographed with a Texas red filter to show (D) red alkaline phosphatase labeled Cx43 as described in (B). All figures show horizontal sections, where posterior is to the right. Magnification bars: 100 μm.
