Worm Breeder's Gazette 11(3): 15

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.

The C. elegans MyoD Homologue as an Early Marker for Body Wall Muscle Lineages

Andrew Fire, Susan White Harrison, Harold Weintraub and Mike Krause

Experiments in vertebrate systems have identified several genes 
which can apparently function as switches to regulate myogenesis.  In 
the last gazette, cloning and characterization of a C.  elegans 
homologue of one of these genes, 'MyoD', was reported (MK and HW, WBG 
11#2p40).  We have been using transformation to analyze the expression 
and activities of the C.  elegans MyoD homologue (the gene is 
currently designated 'CeMyoD').A restriction fragment starting 3.3 kb 
upstream of the CeMyoD coding region and extending 2.1 kb into the 
gene was used to make translational fusions to  -galactosidase with 3' 
ends either from unc-54 or from CeMyoD.  When these constructs were 
injected into wild type C.  elegans and the first generation stained 
with Xgal, strong staining in body wall type muscles was observed.  No 
staining in pharyngeal muscles was seen.  In order to analyze 
embryonic expression we made transformed lines by coinjecting the 
CeMyoD- Gal-unc54 (3') fusion pPD37.48 with either the unc-22 
antisense plasmid pPD10.46 (WBG10#2 p89) or the rol-6(su1006) plasmid 
pRF4 (Mello et al WBG 11#1 p18).  Essentially identical embryonic 
patterns are seen in several independent lines transformed with each 
of the two co-selection markers, so that we attribute the pattern to 
properties of the pPD37.48 fusion construct.
No staining is seen earlier than the 28 cell stage.  At that stage 
staining is seen in just four cells that are apparently the four MS 
derived blastomeres MSaa, MSap, MSpa, and MSpp.  One or two cell 
divisions later, staining begins to appear in posterior cells which 
are presumably C and D derived myoblasts.  At approximately 256 cells 
distinct staining is seen in many body wall muscle cell precursors, 
but staining is absent in the region of the pharyngeal precursors.  
Very strongly staining cells are seen in young larvae in a position 
consistent with these being the M myoblast and immediate descendents.
Several conclusions and speculations derive from the staining 
patterns.  First it is clear from the limitation of staining at the 28 
cell stage to the MS granddaughters that cells at this stage are 
already different in their gene expression.  Second, the lack of stain 
in the MS derived pharyngeal cells suggests that expression of the 
fusion construct pPD37.48 must be turned off in these cells and the 
fusion protein lost.  An intriguing speculation is that the MS 
sublineage has some sort of intrinsic muscle identity, with additional 
muscle cells recruited from other lineages and subtracted from the MS 
lineage as 'afterthoughts'.
Sequences upstream of the myoD coding region are apparently required 
for the body wall muscle staining pattern.  A deletion which removes 
all but 300 b of flanking DNA 5' to the coding region was co-
transformed with rol-6 and produces a pattern of larval gut staining 
but no staining of muscles or their precursors.
Using expression vectors based on the unc-54 (body wall muscle 
myosin) and myo-2 (pharyngeal myosin genes) we have made fusions to 
the CeMyoD cDNA which should express intact CeMyoD product in either 
of the two mature muscle cell types.  Constructs designed to express 
CeMyoD product in pharyngeal muscle lead to arrest of worms as young 
larvae.  Although these animals have a superficially normal pharynx, 
it is conceivable that expression of CeMyoD in pharyngeal muscle cells 
interferes with normal function by causing a transformation in fate 
from pharyngeal to body wall muscle types.  The construct designed to 
overexpress CeMyoD in body wall muscle produces a much more subtle 
phenotype: animals carrying this construct in an extrachromosomal 
array are viable but are abnormally pale.  Although occasional defects 
in egg laying or slightly sluggish movement in these strains are seen, 
most of the animals appear to move and behave normally.  A parallel 
construct with the CeMyoD cDNA in antisense orientation produces no 
evident phenotype upon injection, consistent with the possibility that 
CeMyoD normally plays an early role in body wall muscle cell 
determination or differentiation.