J Cell Biol
August 21, 2000;
Gap junctional communication in the early Xenopus embryo.
In the Xenopus embryo
, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral
patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16-128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral
blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral
and dorsal aspects of the embryo
, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8
did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral
J Cell Biol
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Figure 1. Whole-mount photographs of 16–32-cell stage embryos, coinjected with LY (Mr = 457) and dextran-rhodamine (Mr = 10,000). Dextran is too large to pass through gap junctional channels and serves as a marker for the injected cell and all cells joined to the injected cell by cytoplasmic bridges. A and B show examples of undetectable dye transfer, while C and D show a sample of embryos where LY appears to spread away from the injected cell. Notice, however, that the nonpermeant dextran shows an identical pattern to the LY. Bar, 0.5 mm.
Figure 2. No evidence for junctional transfer of LY was detected in sectioned embryos coinjected with LY and dextran-rhodamine. An embryo in which LY appeared to transfer to adjacent cells (Fig. 2A and Fig. B) was serially sectioned. ∼70 cross sections along the vegetal to animal axis were analyzed. Colocalization of the dyes was always seen, indicating that there was no junctional transfer. (C–F) The dyes were unevenly distributed between the injected cell (very bright green or red cell) and a second cell, which is marked by an asterisk. (G–H) Both dyes remained colocalized in one cell in a plane of section that did not include the injected cell. The presence of dextran-rhodamine in asterisked cells indicates the presence of persistent cytoplasmic bridges with the more brightly stained injected cells. Bars: (A) 250 μm; (C) 200 μm.
Figure 3. Intraembryonic reflection and selective masking of fluorescent signal by pigmented cells in whole-mount embryos. 32-cell stage embryos were injected with dextran-fluorescein (DF, mol wt = 10,000) in one dorsal (less pigmented, A) or one ventral (highly pigmented, B) animal cell in tier 1. The lens effect is demonstrated in whole-mount A where fluorescence is seen in many surrounding cells. In contrast, only two less intensely fluorescent cells are seen in whole-mount B. However, sections of embryo A in C and of embryo B in D confirm that after either injection, the junction-impermeable dextran-fluorescein is confined to the injected cell and to those cells that are joined by cytoplasmic bridges. Bars: (A) 250 μm; (C) 200 μM.
Figure 4. The pattern of neurobiotin (NB) transfer among dorsal blastomeres of normal embryos at the 16-, 32-, 64-, and 128-cell stages. One dorsal blastomere of either 16- (A and B), 32- (C and D), 64- (E and F), and 128- (G–K) cell stage embryos was coinjected with NB and dextran-fluorescein. Sectioned embryos show NB (A, C, E, G, and I) and dextran-fluorescein (B, D, F, H, and K) fluorescence. The 16-cell stage embryo in A, B shows NB transfer into two neighboring animal cells, marked 1 and 2. The injected blastomere of the 32-cell embryo in (C and D) transfers into two animal cells, marked 1 and 2 in C, and is still connected by a cytoplasmic bridge with another animal cell marked with a star in D. The 64-cell stage embryo in E and F shows neurobiotin transfer into at least six neighboring animal cells. The injected blastomere of the 128-cell embryo transfers neurobiotin into >10 animal cells (G and H). Serial sections reveal transfer into more vegetal cells, seen in I, where the injected cell is no longer in the plane of section (K). Bar, 200 μm.
Figure 5. An example of neurobiotin transfer in ventral blastomeres. One ventral blastomere of a normal 64-cell stage embryo was coinjected with neurobiotin and dextran-fluorescein (A–D). The sectioned embryo shows neurobiotin (A and C) and dextran-fluorescein (B and D). More than 10 cells receiving neurobiotin are seen in the animal section (A and B). At least three additional cells that received NB are seen in a different section, vegetal from the injected cell, which is not in this plane of section (D). Bar, 200 μm.
Cao, A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. 1998, Pubmed