Worm Breeder's Gazette 13(5): 59 (February 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.

Genetic Mosaic and Heterochronic Analyses of Inductions Occurring in Early Embryogenesis

Christopher A. Shelton and Bruce Bowerman

Institute of Molecular Biology, University of Oregon, Eugene OR 97403-1229

Prior to the 28-cell stage of the C. elegans embryo, differences in the
lineages of several AB daughters with equivalent potential are generated
by two inductive interactions (Hutter and Schnabel 1994; Mango et al.,
1994; Mello et al., 1994; Moskowitz et al., 1994).  The P2 blastomere
affects the lineage pattern of the ABp blastomere, breaking the
equivalence of ABa and ABp and leading to the production of ABp specific
cell types such as intestinal/rectal valve cells.  Later, at the 12-cell
stage, the MS blastomere induces lineage differences in the ABara and
ABalp blastomeres, resulting in, for example, the production of pharyngeal
cells.  Although the two signals occur at different times, both inductive
signals require the same transmembrane protein, GLP-1, to alter the AB
lineage.  Two simple models can be considered:  first, the response to the
signal may be specified by qualitative differences in the ligands used by
the P2 and MS blastomeres; second, a time-dependent cell-intrinsic change
may take place within the AB daughters from the four-cell stage to the 12-
cell stage such that inductive cues received at these two different times
are "interpreted" differently.

By removing the eggshell of embryos and manipulating early blastomeres
(Goldstein, 1992), we are examining where the specificity lies regarding
the response of the AB blastomeres to the inductive cues emanating from
the MS and P2 blastomeres.  First, we reproduced both interactions.  In
15/21 cases, we have been able to place a P2 blastomere in contact with
ABa and ABp blastomeres and obtained production of intestinal valve cells,
as assayed by staining with monoclonal antibody (MAb) J126.  AB
blastomeres alone did not produce J126 positive cells, in 9/9 cases.  By
combining laser ablation with physical manipulations, we also have
reproduced the MS inductive event; in 4/4 cases, cells staining positive
with MAb 3NB12, a marker of pharyngeal muscle cells, have been obtained
when we placed the MS and E blastomeres in contact with the 8 grand-
daughter cells of ABa and ABp and, after waiting one cell division,
ablated the MS and E blastomeres (eliminating MS derived pharyngeal
cells).  To test the models outlined above, we performed "heterochronic"
blastomere recombination experiments; we placed a P2 blastomere next to
the 8 granddaughter cells of ABa and ABp obtained from a separate,
older, embryo.  Using double labeling, 6/9 cases produced 3NB12 positive
cells and 0/9 produced J126 positive cells.  Thus, contact by the two
daughters of AB with a P2 blastomere induces the production of intestinal
valve cells while contact between the 8 grand-daughters of ABa and ABp
with a P2 blastomere induces the production of pharyngeal muscle cells. 
This result indicates that the response by the AB daughters to inductive
cues depends not on the nature of the signal, but on time-dependent
molecular changes within the AB descendants.

By recombining blastomeres from different genetic backgrounds, we also are
addressing which blastomeres require the function of two previously
identified maternal effect loci to allow these inductions to occur. 
First, the apx-1 gene is required for the P2 signal and has been proposed
to encode a ligand for the GLP-1 protein (Mello et al., 1994).  Consistent
with this idea, we have found that apx-1 function is required in the P2
blastomere both for induction of intestinal valve cells from the two
daughters of AB and for heterochronic induction of pharyngeal cells from
the eight daughters of AB.  Second, the maternal gene skn-1 encodes a
transciption factor that is required to specify the identity of the 4-cell
stage blastomere EMS (Bowerman et al., 1992).  In addition, ABa fails to
produce pharyngeal cells in skn-1 mutants, either because skn-1 is
required in ABa descendants or because skn-1 is required for MS to induce
ABa descendants.  By mosaic analysis using isolated blastomeres from wild-
type and skn-1 mutant embryos, we have found that skn-1 function is
required in MS, but not in the daughters of AB, for the MS-mediated
induction of pharyngeal cells from the AB daughters.  This result suggests
that SKN-1 may control the expression of the signaling molecule(s)
responsible for this induction.  By continuing these blastomere
manipulation experiments, we hope to define more precisely how embryonic
inductions distinguish the fates of initially equivalent blastomeres.