Worm Breeder's Gazette 11(3): 78

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.

Fluorescent Detection of mRNA by Whole-Mount in situ Hybridization: Only Ten Points of Light

David Greenstein and Gary Ruvkun

We set out to develop non-radioactive in situ hybridization methods 
with the goal of eventually obtaining single-cell revolution.  
Fluorescent detection (FD) was chosen because this method is sensitive 
enough to detect single-copy DNA sequences and has allowed detection 
of mRNA in cultured cells with subcellular resolution.  We adapted 
fixation protocols that were developed for the detection of the lin-14 
protein for detection of mRNA using modifications of procedures for 
Drosophila.  In order to detect hybridized probe, we used a monoclonal 
antibody specific for digoxigenin-labeled DNA (described by L.  Avery 
WBG 11(1) 68) and fluoresceinated secondary and tertiary antibodies.  
At present, our protocol is best used for detecting abundant mRNAs in 
embryos and L1 and L2 larvae.  We will send the protocol on request.
With this protocol, we are able to detect pharyngeal myo-3 in mRNA 
in embryos, L1 larvae, and L2 larvae.  Identical results are obtained 
using either myo-1 or myo-2 probes.  Our results indicate that 
pharyngeal myosin mRNA is exclusively synthesized in pharyngeal muscle 
cells, not a surprising finding.  However, we were surprised that the 
level of resolution was equal to that obtained by using antibodies to 
pharyngeal myosin.  The results indicate that pharyngeal myosin mRNA 
is first expressed late in embryogenesis at about the three-fold stage.
Partially staining pharynges are never seen, indicating that the 
pharyngeal muscle cells must all synthesize pharyngeal myosin mRNA at 
about the same time.  Pharyngeal myosin mRNA could also be detected 
using immunocytochemistry (Genius Kit).  Comparison of FD with 
alkaline phosphatase based immunochemistry indicated that FD had 
greater resolution and lower background, yet had comparable 
sensitivity to the enzymatic method.  An advantage of FD was that 
staining of interior regions could be visualized in the presence of 
hypodermal staining.  Moreover, the results could also be viewed with 
the confocal microscope.
The current protocol, though still not as sensitive as we would like,
has allowed the detection of mRNA for the heterochronic gene lin-14, 
the nuclear hormone receptor gene crf-2, and the POU-homeobox gene ceh-
18.  A major problem is that the signals for each of these three lower 
abundance mRNA's are speckled.  Possibly, the speckling is an artifact,
due to a failure to detect all of the mRNA or because the antibodies 
promote the formation of aggregates.  Another difficulty is that the 
conditions for detection of lower abundance mRNA distort the 
morphology (e.g., swelling of the nuclei), making it hard to assign 
hybridization to specific cells.  Despite these caveats, we have been 
able to obtain some useful information.  Our results indicate that 
most cells of three-fold stage embryos contain lin-14 mRNA, whereas no 
staining is seen prior to the two-fold stage, in agreement with the 
antibody studies.  In contrast, crf-2 mRNA is detected only during the 
first half of embryogenesis.  ceh-18 mRNA is first detected at the 
three-fold stage in the pharynx, ventral cord neurons, and numerous 
but unidentifiable cells in the head.  Our method does not allow 
detection of mRNA'a for genes that are expressed at an even lower 
level (e.g., unc-86, lin-11, ced-4, or mab-5).