Abstract
Retrograde BMP signaling and canonical pMad/Medea-mediated transcription regulate diverse target genes across subsets of Drosophila efferent neurons, to differentiate neuropeptidergic neurons and promote motor neuron terminal maturation. How a common BMP signal regulates diverse target genes across many neuronal subsets remains largely unresolved, although available evidence implicates subset-specific transcription factor codes rather than differences in BMP signaling. Here we examine the cis-regulatory mechanisms restricting BMP-induced FMRFa neuropeptide expression to Tv4-neurons. We find that pMad/Medea bind at an atypical, low affinity motif in the FMRFa enhancer. Converting this motif to high affinity caused ectopic enhancer activity and eliminated Tv4-neuron expression. In silico searches identified additional motif instances functional in other efferent neurons, implicating broader functions for this motif in BMP-dependent enhancer activity. Thus, differential interpretation of a common BMP signal, conferred by low affinity pMad/Medea binding motifs, can contribute to the specification of BMP target genes in efferent neuron subsets.
Highlights
Diverse neuronal subtypes are generated by the gene regulatory activities of subtype-specific combinations of transcription factors (Allan and Thor, 2015; da Silva and Wang, 2011; Hobert et al, 2010)
We describe our identification of a low affinity BMP-responsive DNA motif that instructively contributes to the differential specification of BMP target genes within efferent neuron subtypes
Examining the ability of each cold competitor to compete with tagged FMRFa BMP-response elements (BMP-REs), we reveal a necessary BMP-RE sequence of GGCGGGacaatGTaT, where capitalized nucleotides are found most necessary for pMad/Medea recruitment. (F,G) In these Electrophoretic Mobility Shift Assay (EMSA), we transfected S2 cell extracts with a 1Â, 5Â, and 10Â stoichiometric excess of untagged competitors. (F) Competition for the tagged BMP-low affinity (LA) by the untagged wildtype (BMP-LA) or the AE-like mutant
Summary
Diverse neuronal subtypes are generated by the gene regulatory activities of subtype-specific combinations of transcription factors (Allan and Thor, 2015; da Silva and Wang, 2011; Hobert et al, 2010). In Drosophila, retrograde BMP signaling occurs within most efferent neurons, regulating a diverse suite of target genes in a subtype-specific manner; for example, the neuropeptide genes unique to each neurosecretory subtype, and the partially overlapping battery of genes that support the growth and strengthening of motor neuron neuromuscular junction synapses (Veverytsa and Allan, 2011; Miguel-Aliaga et al, 2008; Allan et al, 2003; Kim and Marques, 2010; Vuilleumier et al, 2019; Ball et al, 2010; Marques et al, 2003).
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