Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.
Bibliographical noteFunding Information:
We thank Drs. M. Halpern, C. Kimmel, and M. Wester®eld (University of Oregon) for ®sh stocks and Drs. K. Helde, R. Riggle-man, and D. Grunwald (University of Utah) for the gift of the zebra-®sh cDNA library. We thank Dr. Truus te Kronnie, M. McGrail, S. Fahrenkrug, Dr. H. J. Yost, and Dr. M. Halpern for useful comments and discussions on the manuscript and the helpful discussions of Dr. R. Riggleman on in situ hybridization in zebra®sh. We are grateful to M. McGrail for her assistance with confocal microscopy and R. Essner for his help in the establishment and maintenance of our zebra®sh facility. J.J.E. was supported by Minnesota Sea Grant USDOC/NA 46RG0101-02 (Paper JR 416) and J.G.L. was supported by a fellowship from the American Heart Association (AHA), MO af®liation. E.C.B. is an Established Investigator of the AHA. This work was supported by NIH Grant R01-RR06625 to P.B.H., NIH Grant HL45466 to E.C.B., and NIH Grant R01-GM46277 to R.G.J.