Worm Breeder's Gazette 14(5): 18 (February 1, 1997)

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.

An Alternative Single Cell Marker for Mosaic Analysis: Visualization of Extrachromosomal Arrays by GFP

Aidyl Gonzalez-Serricchio, Paul Sternberg

Caltech, Pasadena, CA 91125

        Genetic mosaics in C. elegans are generated by the spontaneous
somatic loss of an extrachromosomal array or free duplication.  The free
duplications or extrachromosomal arrays contains a wild-type marker gene
(usually ncl-1) and a gene of interest in which the native genes are
mutant.  The loss of the free duplication or extrachromosomal array
results in a clone of cells lacking wild-type activity of the marker
gene and the gene of interest and thus allows determination of the cells
in which that gene acts.  Extrachromosomal arrays are often sufficiently
mitotically unstable that the mosaic pattern is complex, and thus a
facile method of scoring individual cells is important. 
        Green Fluorescent Protein (GFP) can be targeted to a specific
DNA sequence using a lac repressor/operator system developed by Andrew
Belmont.  We expressed a hybrid protein (GFP-LacI) with the DNA-binding
capability of LacI and the fluorescent properties of GFP under the
transcriptional control of an hsp16 promoter/enhancer, and included a
tandem repeat of 256 lac operators (lacO) in the injection mixture to
provide binding sites for GFP-LacI. ncl-1(e1865) worms were injected
with 50ng/ml of lacO repeat; 100ng/ml of GFP-LacI; 100ng/ml of C33C3
[ncl-1(+)]; and 40ng/ml of pRF4.  We found that after heat shock, cells
contained nuclear-localized bright spots ("dots") as well as diffuse
nuclear fluorescence ("haze").  The dots co-localize with DAPI-staining.
We can therefore directly visualize extrachromosmal arrays.  
        For use in mosaics, a dot indicates the presence of the
extrachromosomal array, while haze without a dot would indicate loss of
the array with perdurance of the GFP-LacI.  We followed vulval cell
lineages and observed mitotic loss of the extrachromosomal array. 
Typically, the segregation was 1:0, with the 1 being a dot and the 0
being haze.   
        To compare the efficacy of ncl-1 and GFP-LacI as a single cell
marker, we scored for Ncl phenotype in four different cells (e2, m2, m4,
and hyp10) of 51  roller worms (Table 1).  Some Ncl cells had dots,
indicating that the chromosomal array was indeed present, and that ncl-1
is not a completely reliable marker. Similarly, some non-Ncl cells did
not have dots.  Thus neither marker is perfect.  We suspect that the
efficiency of scoring each is cell dependent.   
        The comparison between ncl-1 and the GFP-LacI/lac indicates that
this new marker is comparable to ncl-1.  The main advantage of using the
fusion protein GFP-LacI and the 256 Lac operator repeat rather than Ncl
is that scoring the mutant Ncl phenotype is typically more difficult
than scoring cells with the bound GFP-LacI fusion protein.  This hybrid
protein  works well at 50ng/ml to 100ng/ml .    
        After integration of the array by X-ray irradiation, we were
able to visualize chromosomes.  Preliminary analysis of chromosomal
segregation in dividing vulval cells indicate mitotic stability and 2:2
segregation.  We have seen multiple dots in some polyploid cells are
examining the utility of this as a marker for polyploidy.  
        We are also currently testing whether mosaic results using
GFP-LacI plus lacO repeat as a marker are equivalent to those with Ncl. 
In addition, since we do not see early embryonic expression of GFP-LacI
expressed from hsp16, we are expressing GFP-LacI from the dpy-30
promoter (provided by Barbara Meyer).  We hope that this will allow
visualization of arrays can be seen ubiquitously throughout the worm,
including the germline and early embryo.  
        We find that the GFP-LacI method (dot or not dot) is much easier
than scoring nucleolus size.  Such ease of scoring may also be used for
more accurate mosaic analysis, especially if Ncl is used in conjunction.

Table 1  ncl-1 versus GFP-Lac/lacO256 as markers for mosaic analysis. 
Scored by cell: e2, m4, and hyp10.  Ncl+: normal nucleolus, rescue;
Ncl-: enlarged nucleolus, no rescue; +: GFP-LacI bound to lacO,
fluorescent dot; -: no fluorescence; haze: only GFP-LacI present,
presumably the array is lost; Ncl?: Ncl phenotype could not be
determined.  Rollers n=51  non-Rollers n=20    Of 20 non-roller animals,
none had GFP in e2, m4 or HSN, but there was haze in hyp10 in 7 animals.

Phenotype           e2        m4        hyp10     
Ncl       GFP
+         +         33        36        23             
-         +         6         12        7              
+         -         2         1 haze    8+1 haze       
-         -         6+1 haze  1+1 haze  5+1 haze       
?         +         0         0         1              
?         -         0         0         3              
nd                  3         0         2