transgenic axolotls & cleavage pacemaker

Richard Gordon gordonr at cc.UManitoba.CA
Mon Jul 7 09:32:07 EST 1997

Dr. George M. Malacinski, Director, Axolotl Colony
Department of Biology, Indiana University
Bloomington IN 47405, USA
Phone: (812) 855-1131, Fax: (812) 855-6705
E-mail: malacins at indiana.edu (George Malacinski)

Dear George,

Cleavage involves a "pacemaker" area at the animal pole that somehow sets
off the synchronous cleavage waves (refs below). May I guess that the
pacemaker itself isn't operating? If so, this suggests three tacts:

1) treat the animal pole as if you were trying to restart a human heart:
pulse it with electricity (or Ca, or something else involved in the
pacemaker "clock"), maybe with a period equal to that of cleavage;

2) don't mess up the cells electrically in the first place: try, for
example, ballistic injection of DNA into naturally fertilized eggs, right
after laying;

3) inject either at the animal or vegetal pole, on the assumption that
there is an ionic current between the two poles, and these points might be
least vulnerable; i.e., a hole in the "side", no matter how small, may
induce a current that adds to or short circuits the normal A/V current,
disrupting the embryo's symmetry, and preventing the A/V current from
starting off the pacemaker.

Cleavage waves could probably be classed along with differentiation waves
as "ultraslow" calcium waves (Jaffe, 1995). If their slower nature is
considered, much of the literature on calcium oscillations might be
valuable in perhaps finding a molecular way of restarting the animal pole
pacemaker (some refs below).

Yours, -Dick Gordon

Berridge, M.J. (1991). Caffeine inhibits inositol-trisphosphate-induced
membrane potential oscillations in Xenopus oocytes. Proc. R. Soc. Lond.
Biol.  244(1309), 57-62.

Dabauvalle, M.C., M. Doree, R. Bravo & E. Karsenti (1988). Role of nuclear
material in the early cell cycle of Xenopus  embryos. Cell  52(4), 525-533.

Dupont, G. & A. Goldbeter (1992). Oscillations and waves of cystolic
calcium: insights from theoretical models. BioEssays  14(7), 485-493.

Eichwald, C. & F. Kaiser (1995). Model for external influences on cellular
signal transduction pathways including cytosolic calcium oscillations.
Bioelectromagnetics  16(2), 75-85.

Gillis, A.M. (1991). Calcium keeps the cellular clock ticking. BioScience
41(6), 377-378.

Goldbeter, A. (1996). Biochemical Oscillations and Cellular Rhythms: The
Molecular Bases of Periodic and Chaotic Behaviour. New York: Cambridge
University Press.

Jaffe, L.F. (1995). Calcium waves and development. Ciba Found. Symp.  188,

Jafri, M.S., S. Vajda, P. Pasik & B. Gillo (1992). A membrane model for
cytosolic calcium oscillations. A study using Xenopus  oocytes. Biophys. J.
63(1), 235-246.

Keating, T.J., R.J. Cork & K.R. Robinson (1994). Intracellular free calcium
oscillations in normal and cleavage-blocked embryos and artificially
activated eggs of Xenopus laevis. J. Cell Sci.  107, 2229-2237.

Keizer, J. & G.W. De Young (1992). Two roles for Ca2+ in agonist stimulated
Ca2+ oscillations. Biophys. J.  61, 649-660.

Kirschner, M.W., J.C. Gerhart, K. Hara & G.A. Ubbels (1980). Initiation of
the cell cycle and establishment of bilateral symmetry in Xenopus  eggs.
In: S. Subtelny & N.K. Wessells (eds.), The Cell Surface: Mediator of
Developmental Processes, New York: Academic Press, p. 187-215.

Kubota, H.Y., Y. Yoshimoto & Y. Hiramoto (1993). Oscillation of
intracellular free calcium in cleaving and cleavage-arrested embryos of
Xenopus laevis. Dev. Biol.  160(2), 512-518.

Parekh, A.B., M. Foguet, H. Lubbert & W. Stuhmer (1993). Ca2+ oscillations
and Ca2+ influx in Xenopus  oocytes expressing a novel 5-hydroxytryptamine
receptor. J. Physiol. Lond.  469, 653-671.

Speksnijder, J.E. (1992). The repetitive calcium waves in the fertilized
ascidian egg are initiated near the vegetal pole by a cortical pacemaker.
Dev. Biol.  153(2), 259-271.

>	Worked out a protocol for electrically activating freshly spawned
>	unfertilized eggs. Once "shocked" the animal hemispere pigmentation,
>	which begins looking like a tiny donut is stamped onto the animal pole,
>	evens out and the donut disappears as pigment blankets the animal pole.
>	Developed a scheme for swelling axolotl sperm heads (a prerequisite,
>	apparently, for successful uptake of a foreign gene).
>HOWEVER, virtually NONE of the eggs (electrically activated)--injected
>with either fresh (crude extract) sperm, swollen head sperm, or
>swollen/construct-treated sperm--cleave!
>What have we done wrong?  What do we need to do to prepare axolotl eggs
>so that they will respond to injected sperm by cleaving?
>I believe we are working on a FALSE assumption: The way to get cleavage
>is to inject sperm directly into electrically activated eggs.

Dr. Richard Gordon, Department of Radiology
University of Manitoba, Health Sciences Centre
820 Sherbrook Street, Winnipeg, MB R3A 1R9 Canada
Phone: (204) 789-3828,  Fax: (204) 787-2080, Home: (204) 589-0411
E-mail: GordonR at cc.UManitoba.ca

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