TY  - GEN
T1  - Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells
AU  - Lear, Sierra K.
AU  - Das, Alakananda
AU  - Goodman, Miriam B.
DO  - 10.17912/8FDA-CK77
UR  - http://beta.micropublication.org/journals/biology/8fda-ck77/
AB  - Four transgenes designed to drive expression of Cre recombinase and a nuclear-localized blue fluorescent protein (<Neuron-specific promoter>::Cre::F2A::BFP::AID::NLS::tbb-2 3’UTR) were expressed under the control of neuron-specific promoters. All transgenes were inserted into the ttTi5605 transposon insertion site in chromosome II of wild-type C. elegans animals (N2) using CRISPR/Cas9-mediated genome editing, equalizing any potential positional effects. We observed very faint BFP signal in live worms from the signal copy transgene insertions. Additionally, the signal was further masked by bright autofluorescence from the intestine. To observe the cells expressing these transgenes we performed immunofluorescence experiments using primary antibody against BFP. Using this assay, we studied the expression from touch receptor neuron (TRN)-specific promoters, mec-4p (A) and mec-17p (B) and pan-neuronal promoters, cmk-1p (C) and unc-119p (D). As expected, BFP expression driven by the mec-4promoter is observed exclusively in the six TRN nuclei (A). However, under mec-17 promoter, BFP expression is observed in two additional neuronal nuclei (B, green arrowheads) besides the six TRN nuclei (B). These two neurons are likely to be PVD or PDE neurons based upon the location of their nuclei relative to the PVM nucleus. In case of the pan-neuronal promoters, expression under cmk-1 and unc-119 promoters is observed in most, if not all neuronal nuclei, including in many head neurons (C and D top) and ventral nerve cord (VNC) neurons (C and D bottom). However, in a subpopulation of animals expressing BFP under the unc-119 promoter, intense fluorescence was observed in several additional larger nuclei along the lateral sides (D, blue arrowheads), which we identify as hypodermal cells and faint signals in even larger nuclei (D, pink arrowheads), which we identify as intestinal cells. We did not observe any fluorescence in our control animals (N2) which were treated similarly (data not shown). While this experiment confirmed expected expression patterns for mec-4p and cmk-1p, additional expression in other neurons for mec-17p and other tissue types in unc-119p promoters could reflect the specific promoter sequences used in this study or that low levels of expression in these unexpected cells were not readily detected from direct observation of fluorescent protein expression analyzed previously.
PY  - 2018
JO  - microPublication Biology
ER  -