The NK-2 homeodomain transcription factor CEH-28 is specifically expressed in the M4 pharyngeal motor neuron, where it regulates synaptic assembly and pharyngeal peristalsis. We are interested in how CEH-28 controls multiple M4 functions, and we are identifying CEH-28 targets.
dbl-1 encodes a TGF-b family protein expressed in M4 and a subset of other pharyngeal and non-pharyngeal neurons, and we have found
dbl-1 expression is specifically lost in M4 in
ceh-28 mutants. The
dbl-1 promoter contains separable regulatory elements controlling expression in M4 and other neurons. We identified an M4-specific enhancer in the
dbl-1 promoter and found mutation of predicted CEH-28 binding sites strongly reduced enhancer activity. We asked if DBL-1 functions downstream of CEH-28 to regulate M4 synaptic assembly, but
dbl-1 mutants exhibited normal M4 synapses and pharyngeal peristalses, suggesting
dbl-1 mediates a distinct, non-autonomous activity of M4. To test this hypothesis, we examined cells located nearby M4 in
ceh-28 and
dbl-1 mutants. The pharyngeal
g1 gland cells extend processes contacting M4, and both
ceh-28 and
dbl-1 mutants exhibit morphological abnormalities in these processes. Similar defects were also observed in
sma-6 and
daf-4 mutants affecting receptors in the Sma/Mab pathway. However,
sma-2 and
sma-3 mutants affecting R-Smads in this pathway have normal gland cells, suggesting DBL-1 affects gland cells through Smad-independent function of the Sma/Mab pathway. We can partially rescue the gland cell defects in
dbl-1 and
ceh-28 mutants by expressing
dbl-1 only in M4. M4 contains dense core vesicles (DCVs) that mediate neuropeptide and protein secretion, but these DCVs do not regulate DBL-1 secretion as
unc-31 mutants defective in DCV release exhibit normal
g1 gland cells. Based on these findings, we hypothesize that CEH-28 is a direct activator of
dbl-1 expression in M4, and that DBL-1 secreted from M4 affects gland cell morphology by Smad-independent signaling through the Sma/Mab TGF-b signaling pathway. M4 is a multi-functional cell that stimulates pharyngeal peristalsis and regulates sensory perception under hypoxic conditions, and our work identifies another novel function for this neuron.