Worm Breeder's Gazette 14(1): 70 (October 1, 1995)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

ama-1 antisense experiments: Embryonic transcription is required for initiation of gastrulation

Jo Anne Powell-Coffman, Jennifer Knight, Bill Wood

Department of Molecular, Cellular, and Developmental Biology University of Colorado Boulder, CO 80309-0347

        We are examining the requirements for maternally provided versus
embryonically transcribed gene products in early developmental
decisions.  Past studies have shown that C. elegans embryos initiate
transcription prior to gastrulation (1, 2).  Indeed, the embryonic
pes-10 transcript is detectably expressed by the 4-cell stage (3).
Devitellinized embryos cultured in the presence of a-amanitin, which
inhibits RNA polymerase II, cease dividing at approximately 100 cells.
The lineage-specific timing of early cleavages in these embryos is not
dramatically altered (2).  a-amanitin experiments cannot address the
potential role of embryonic transcription in directing gastrulation,
since perturbations of the vitelline membrane disrupt gastrulation (4).

        To block the translation of maternally provided RNA polymerase
II large subunit in otherwise intact embryos, we injected capped
antisense ama-1 RNA (50 ng / ml) into the gonads of wild-type
hermaphrodites.  (The ama-1 cDNA clones were generously provided by
David Bird.)  We scored the progeny of these animals 20 hrs. to 48 hrs.
postinjection, and 95% of the embryos arrested early in development (n =
850).  We examined 40 of these embryos under Nomarski optics, and all
arrested at 80 to 100 cells and showed no signs of morphogenesis or
differentiation.  To further assay embryonic transcription, we injected
antisense ama-1 into a strain carrying vet-6::lacZ, which is strongly
expressed by the 28-cell stage (5).  The resulting arrested embryos did
not detectably express the reporter gene, indicating that the antisense
ama-1 RNA had effectively blocked the function of RNA polymerase II.
Moreover, preliminary results show that the cleavage pattern of ama-1
antisense embryos is normal through the first four cell cycles, and
P-granules are segregated appropriately; thus, these
maternally-controlled events are not detectably disrupted.  To control
for possible artifacts caused by RNA injection, we injected
hermaphrodites with RNA antisense to her-1, an embryonically transcribed
sex-determining gene.  Of 2609 progeny examined, 1.1% failed to hatch,
and only 0.3% arrested with fewer than 300 cells.  Further controls for
ama-1 antisense specificity are underway.

        We have recorded the development of ama-1 antisense embryos past
the point of cell division arrest using the 4-D microscope, and all of
the embryos (n = 6) failed to gastrulate.  The Ea and Ep cells and their
daughters divided precociously and did not migrate into the blastocoel,
reminiscent of the maternal-effect emb-16 mutant phenotype (6).  These
data indicate that embryonically transcribed gene products are necessary
for initiating gastrulation.  Further analysis of ama-1 antisense
embryos will be instructive as to the role of embryonically transcribed
genes in early development.

1.  Schauer, I. and W. B.Wood (1990) Development 113: 1303-1317

2.  Edgar, L. G., Wolf, N. and W. B. Wood (1994) Development 120: 443 -

3.  Seydoux, G. and A. Fire (1994) Development 120: 2823 - 2834

4.  Schierenberg, E. and B. Junkersdorf (1992) Roux's Arch. Dev. Biol.
202: 10-16

5.  Pettitt, J. and W. B. Wood. (1993) Worm Breeder's Gazette. 13 : 53

6.  Denich, K.T.R., Schierenberg, E., Isnenghi, E. and R. Cassada.
(1984) Roux's Arch. Dev. Biol.  193:    164 - 179