Dear George (and whoever else is interested),
Having spent a good portion of the last twenty years looking for the "ideal"
marker, I suppose I should have something to say. :-)
Though I am responsible to a large extent for the methods used to make
triploids, it was never our intent to use them as lineage labels. However,
Sue Bryant and her coworkers used them with moderate success in their limb
regeneration work.
A lot of our work has involved ectodermal derivatives, and pigmentation has
served us well. See:
Graveson, A.C., B.K. Hall, and J.B. Armstrong. (1995) The relationship
between migration and chondrogenic potential of trunk neural crest cells in
Ambystoma mexicanum. Roux's Archives, 204: 477-483.
Smith, S.C., M.J. Lannoo, and J.B. Armstrong. 1990. Development of the
mechanoreceptive lateral-line system in the axolotl: placode specification,
guidance of migration, and the origin of neuromast polarity. Anat. Embryol.
182: 171-180.
We have also used the fluorescent dyes DiI and DiO with good results. The
methods are essential those used in other organisms, but I can prepare a
detailed protocol for anyone who is interested. We had earlier tried the
fluorescently labeled dextrans, but never had good success with them. We
also spent some time looking at isozyme variants. Though we found a couple
(and I can dig up the references for anyone who may be interested), these
require electrophoresis of fairly large tissue samples, and are therefore
not practical for most purposes. We were hoping to find some non-essential
enzyme with a null allele; then we could have used histochemistry and
presumably would have been able to pick out individual cells.
About the time we were getting frustrated with that search, molecular
techniques had advanced to the point where we felt it would be worthwhile to
try introducing an easily detectable foreign gene. As beta-galactosidase is
not supposed to be present in vertebrates, we tried introducing a plasmid
carrying the E. coli lac Z gene. See:
Whiteley, M., and J.B. Armstrong. 1991. On the origin of the mesoderm in the
Mexican axolotl, Ambystoma mexicanum. Can. J. Zool. 69: 1221-1225.
Whiteley, M., W.S. Fletcher, and J.B. Armstrong. 1990. The use of lacZ
fusion constructs as a cell lineage marker in the axolotl. Axolotl
Newsletter 19: 22-27.
(Please don't ask me for the plasmid. It was a gift from another lab, and
not ours to pass on. In any event, we haven't used it for several years,
and lost our stock when a freezer broke down.)
Though we thought we had stable integration, we were not able to find second
generation animals carrying the marker. Perhaps it wasn't integrated into
the germ line. I still think this is the way to go, though it will require
a concerted effort.
Good luck,
John
John B. Armstrong jbarm at uottawa.ca
Department of Biology
University of Ottawa
Ottawa, Ontario K1N 6N5
CANADA
