Worm Breeder's Gazette 13(4): 30 (October 1, 1994)

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.

Vectorology II: Green Fluorescent Protein (or "It Isn't Easy Being Green").

Andrew Fire, Joohong Ahnn, Geraldine Seydoux, SiQun Xu

Carnegie Institution of Washington, Baltimore, Md. 21210

  Earlier this year, Marty Chalfie introduced the use of Aequorea victoria green
fluorescent protein (gfp) as a reporter for following gene expression in live animals. To
test the applicability of this reporter in screens for novel expression patterns during
embryogenesis, we constructed gfp fusion constructs using several promoters with
defined embryonic specificity. Results with lacZ and gfp fusions are compared in the
table below:
  myo-3 , unc-54 , ost-1 : lacZ fusions for these genes mimic expression patterns
for the endogenous myosin genes, with activity in body muscles beginning around the
comma stage of embryogenesis (1,2). Equivalent gfp fusions are also active (i.e., cause
fluorescence) in body muscles, but expression does not begin until the embryos have
reached three-fold elongation.
  hlh-1 :The endogenous gene and lacZ fusions express most strongly in body-wall
muscle precursors in cleaving and pre-comma embryos; expression continues in
differentiated body-wall muscles (3). Equivalent gfp fusions are active only in the
differentiated body-wall muscles.
  cey-1 :Somatic expression of the endogenous gene and of lacZ fusions is strongest
in early embryos (where most or all somatic cells express); later expression with lacZ
fusions has been seen most intensely in body muscles and pharynx (2). Equivalent gfp
fusions are active in differentiated body muscle and in pharynx, but no early embryonic
activity was observed.
  pes-10 :The endogenous gene and lacZ fusions express well in somatic lineages
in the early embryo, with occasional low-level postembryonic expression in gut (4).
Equivalent gfp fusions show only sporadic activity in differentiated gut.
  glp-1 :expression of the endogenous gene occurs in germ line as well as some
somatic tissues, most notably a set of embryonic cells at the 40-60 cell-stage (5).
glp-1 : lacZ fusions show strong expression in 40-60 cell embryos and in the
spermatheca; glp-1 :gfpfusions are active in spermatheca (weakly) but no activity is
seen in 40-60 cell embryos.
  Although the above data is encouraging for experiments that require GFP
fluorescence in terminally differentiated cells, the results suggest that activity might be
blocked in early and mid-stage embryos. The ability to make active GFP in embryos
appears to follow differentiation rather than morphogenesis (this was evident from an
EMS mutant screen looking for ost-1 ::gfpactivity in comma-stage embryos; the screen
yielded many fluorescent embryos in which morphogenesis had arrested while
differentiation continued).
  Activity of gfp constructs in pre-differentiation embryos could in principle be
blocked at several mechanistic levels. Analysis of RNA transcripts argues against a
transcriptional block: in situ hybridization was used to show that a pes-10 ::gfpfusion
transcript was produced at high levels in the early embryo, in a pattern indicative of the
pes-10 promoter. We hope to eventually examine the distribution of GFP protein in
these embryos using anti-GFP antibodies. In the interim, we have constructed a
hybrid gfp-lacZ fusion gene. This construct produces co-localized 337-gal activity and
green fluorescence when expressed in differentiated pharyngeal muscle (although the
fluorescence is somewhat less than with, gfp alone). Expression of the gfp-lacZ fusion
gene in early embryos using the pes-10 promoter results in a product which has
337-galactosidase activity but is not fluorescent.
  As a working hypothesis, we propose that the GFP protein might be made in early
embryos but fail to acquire fluorescent properties. In terms of gene expression studies,
this suggests some cautions. In particular, GFP fluorescence could appear or
disappear at a given point in development as a result of modulations in the formation of
the fluorochrome rather than changes in the level of GFP protein. Our results suggest
that this might be less problematic in postembryonic differentiated tissue, although it
should be noted that we have not looked at any postembryonic non-differentiated
  We have taken several approaches toward producing a gfp vector for use in early
embryos. A multiple-intron gfp gene was constructed (see Fire and Xu article), but
did not relieve the inhibition in early embryos. Mutagenesis of gfp fusion containing
lines are in progress (in principle it might be possible to find a mutant version of gfp or
a mutant worm strain in which early acquisition of GFP fluorescence was not blocked).
  For some applications of gfp reporters, labeling of a specific molecule in its
normal context is required, while for other applications the critical aim is to label the
cells expressing the fusion. We thought that it might be possible to circumvent
problems with gfp in the latter cases by targeting the produced protein to different
intracellular compartments, potentially allowing a more amenable environment for
fluorescence. In lines with gfp expressed with no other protein sequences attached,
the fluorescent signal is uniformly distributed through the cytoplasm. We've
examined the effects of several different targeting signals on GFP localization and
embryonic fluorescence:
  Nuclear localization signal: Attachment of the SV40 nuclear localization signal
(NLS) from our older lacZ vectors (6) results in fluorescence activity which is distinctly
stronger in the nucleus than the cytoplasm. Curiously, the degree of nuclear
localization is greatly improved in fusions which contain gfp, the nuclear localization
signal, and an appended mass of protein ( lacZ and pieces of myosin and HLH-1 are
variably sufficient for this). The incomplete nuclear localization of the simple NLS-gfp
protein may be due to the ability of this relatively small protein to diffuse out of the
  Secretion signal: We have previously used a synthetic secretion signal to drive
secretion of several different expressed molecules (e.g., 7). Secreted gfp constructs
driven by the myo-2 , pes-10 and mec-7 promoters have been tested. The resulting
fluorescence accumulation is both intracellular and extracellular. The intracellular
staining with the myo-2 promoter appeared reticular and might represent the internal
secretion apparatus of the cell. Interestingly, coeloemocytes appeared to scavenge
secreted gfp expressed postembryonically from pes-10 or mec-7 ,storing the fluorescent
material in internal vacuoles or droplets.
  Mitochondrial matrix localization signal: An N-terminal mitochondrial matrix
localization signal from chicken mitochondrial aspartate aminotransferase (8) was
synthesized and incorporated into the gfp expression cassette. This signal was
sufficient to localize gfp to mitochondrial structures in body wall muscle. (This signal
might also be useful for other non-GFP experiments; ced-9 anyone?).
  Preliminary results in pre-morphogenesis embryos have been most encouraging
with mitochondrial-localized gfp (Mgfp). Fluorescence activity from hlh-1 :Mgfphas
been seen in lima bean-stage embryos that would not be fluorescent with the
cytoplasmic GFP. So far, no fluorescent signals have been seen in premorphogenesis
embryos with the nuclear or secreted GFP's.
  Other GFP notes: a) As reported by Marty, anesthesia with azide or propylene
phenoxitol produces rapid fading of fluorescent signal. We have been mounting
animals in 1mM levamisole, which yields much more stable fluorescence in the
immobilized animals. b) We've now made a large number of protein fusions with GFP
on the C-terminus. All of these have worked at some level, suggesting that gfp, like
lacZ , is relatively tolerant to being appended to the C-terminus of other proteins. One
fusion with GFP on the N-terminus (the gfp::lacZfusion) functioned, but activity may
be less than simple GFP.
 (1) J. Schwartzbauer, pers comm.
 (2) V. Plunger, pers. comm.
 (3) Krause et al., Cell 63, 907.
 (4) Seydoux & Fire, wbg 13#3, 33.
 (5) Seydoux & Fire, Development, in press.
 (6) Fire et al., Gene 93, 189
 (7) Perry et al., Genes & Dev. 7, 216
 (8) Jausi et al., J. Biol. Chem. 260, 16060.
 Thanks to Marty Chalfie (gfp), Verena Plunger ( cey-1 ),Jim Henson (Kermit).