Neuronal expression in cleavage-arrested ascidian blastomeres requires gap junctional uncoupling from neighbouring cells.
1. When anterior-animal (a4-2) blastomeres isolated from 8-cell ascidian embryos were cultured under cleavage-arrested conditions in contact with anterior-vegetal (A4-1) blastomeres (a-A blastomere pairs), the a4-2 blastomeres differentiated into neuronal cells that expressed Na+ and delayed K+ channels at a time when normal sister embryos became tadpole larvae (after 40 developmental hours at 9 degrees C). When a4-2 blastomeres were cultured in contact with posterior-animal (b4-2) blastomeres (a-b blastomere pairs), the a4-2 blastomeres differentiated into epidermal cells expressing Ca2+ channels and tunic on their exterior surface. In these blastomere pairs, we analysed changes in gap junctional communication during neural and epidermal differentiation by using both dye transfer and double voltage clamp. 2. In both types of blastomere pairs, gap junctional communication was detectable at 5 h by double voltage clamp and at 7 h by dye transfer. Gap junctional communication in both types gradually increased until 25 h (equivalent to the neurula stage). However, during 25-35 h (late neurula or tailbud) in the a-A pair it decreased and finally disappeared, while it increased steeply in the a-A pair. When blastomere pairs were treated with a transcription inhibitor, actinomycin D, gap junctional communication also appeared at around 7 h but remained at a plateau level, showing neither a steep increase in a-b pairs nor a disappearance in a-A pairs. 3. In blastomere triplets in which an epidermally committed a4-2 was in contact with both blastomeres of an a-A pair, the epidermally committed a4-2 blastomere did, but the neurally committed a4-2 blastomere did not, communicate through gap junctions with the A4-1 blastomere, indicating that gap junctional communication is restricted when a4-2 blastomeres are neurally committed. 4. When a kinase inhibitor, K252a (0.5-1.0 microM), was applied at 20 h (prior to the disappearance of gap junctional communication), gap junctional communication was maintained in the a-A pair for more than 40 h. The persistence of gap junctional communication delayed the expression of Na+ and K+ channels in the a4-2 blastomere. However, channel expression followed an almost normal time sequence in single neuronally committed a4-2 blastomeres separated from A-a pairs and treated with K252a. 5. We conclude that the persistence of gap junctions causes a delay in expression of neuronal characteristics, and suggest that one of the functional roles of embryonic gap junctions is to time the expression of neuron-specific ion channels and other markers in preneuronal cells.
Saitoe
M
M
Department of Behavioral Physiology, Faculty of Medicine, University of Gumma, Japan.
Inazawa
T
T
Takahashi
K
K
D000818:Animals; D001757:Blastomeres / Q000187:drug effects / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D003609:Dactinomycin / Q000494:pharmacology; D004594:Electrophysiology; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D026121:Indole Alkaloids; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014561:Urochordata / Q000378:metabolism*
D002227:Carbazoles; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D026121:Indole Alkaloids; D007473:Ion Channels; D007546:Isoquinolines; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; C017475:lucifer yellow; C049985:staurosporine aglycone; D011493:Protein Kinase C