A-type motor neurons (DA, VA, SAB) are innervated by specific command interneurons (AVA, AVD, AVE). The UNC-4 homeoprotein is expressed in all A-type motor neurons where it functions to maintain normal levels of synaptic vesicles. UNC-4 is also required in VA motor neurons to regulate presynaptic specificity. In
unc-4 mutants, inputs to VA motor neurons from AVA, AVD, and AVE are replaced with connections from AVB. In an effort to define the mechanism whereby UNC-4 regulates A-type motor neuron synaptic differentiation, we have established methods for culturing
unc-4 -expressing motor neurons in vitro .
unc-4 ::GFP is initially detected midway through embryonic development (~400 min) and is ultimately expressed in 13 embryonically-derived motor neurons (9 DAs, 3 SABs, 1 I5). GFP-positive cells are rarely seen among blastomeres of freshly dissociated
unc-4 ::GFP embryos but are easily detected after several hours in culture. The delay in
unc-4 ::GFP expression is consistent with the observation that older embryos are not dissociated in our preparations and that early blastomeres are capable of differentiating in vitro.
unc-4 ::GFP expressing cells exhibit neuronal-like morphology with elongated processes. Direct counting in the microscope as well as quantitation by FACS shows that
unc-4 ::GFP neurons comprise 2-4% of all cells after 1-2 days in culture. This proportion is comparable to the fraction of
unc-4 ::GFP cells present in the mature embryo (i.e. 13 out of 550 total cells). Antibody staining detects synaptic vesicle proteins Synaptotagmin and UNC-17 which indicates that
unc-4 ::GFP cells express specific differentiated traits of cholinergic motor neurons. VA motor neurons, which are postembryonically derived, do not appear to differentiate in culture, however, as evidenced by the lack of
del-1 ::GFP expression in vitro in a genetic background in which
del-1 ::GFP is expressed in VA motor neurons in vivo . The nAChR subunit gene,
acr-5 , is negatively regulated by UNC-4 in DA motor neurons in L1 larvae. We will determine if UNC-4 regulates this known target gene in vitro by asking if
acr-5 ::YFP is co-expressed with
unc-4 ::CFP in cells isolated from an
unc-4 mutant. In that event, it is reasonable to assume that UNC-4 also controls other downstream genes in these cultures and that these UNC-4-regulated targets could be detected by microarray experiments with FACS sorted
unc-4 ::GFP cells from wildtype vs
unc-4 mutant backgrounds. Blastomeres prepared from embryos expressing the AVA-specific reporter,
opt-3:: GFP, give rise to GFP-positive neurons with elongated processes. It will be interesting to determine if these presumptive AVA-like interneurons retain synaptic specificity for A-type motor neurons in culture.