Roles of Drosophila Beadex and CG9650 in the development and functioning of the larval neuromuscular junctions

Abstract

In eukaryotes, all voluntary and involuntary actions like, cognition, learning & memory, voluntary movements, feeding, etc., are coordinated by the employment of neuronal circuitry that transmits the signal from the source (in the central nervous system) to an effector (another neuron, tissue or organ). Neurotransmission, a process in which neurotransmitters released by axon terminals of a neuron binds to receptors on dendrites of another neuron, or other effector tissue or organ, is indispensably responsible for these actions. Many voluntary actions, like locomotion, result from chemical synapses that are formed between a motor neuron and a skeletal muscle, which are also known as neuromuscular junctions (NMJ). Along with appropriate growth and accurate organization, a functional NMJ demands a well balanced expression of molecular effectors for robust synaptic transmission. Several signaling pathways, including the Wnt pathway, BMP pathway, MAPK pathway, and Syt4 underly the formation and maintenance of a functional NMJ. Many of the signaling molecules involved in these pathways regulate various morphological features of the NMJ like the span area, branch length, bouton numbers & size, as well as the physiology at the synapse. Drosophila larval neurons have been used extensively as a model to identify new molecular players and decode neuronal circuits involved. Extensive work in Drosophila larval NMJ led to the identification of major molecular players and their developmental and functional roles, like endocytosis e.g. by studies on shibire (dynamin), exocytosis by studies on cacophony (calcium ion channel), SNARE proteins (for synaptic vesicle fusion), etc., regulators of NMJ morphology, like highwire, futsch, TDP-43, Rae1, Dishelved, LIMK1, etc., active zone assembly players BRP, Syd-1 (RhoGAP100F), Lipirin-α., etc. Though these studies have proven to be valuable paradigms to study the mammalian synapses, many new molecular candidates whose function and interactions at the NMJ remain uncovered. Additionally, even though several pathways have been elucidated, the mechanism of action and genetic interaction between different molecular players is yet not clear. Our lab had previously identified two such players - Beadex (Bx), the Drosophila homolog of Human LMOs; and CG9650, the Drosophila homolog of Human BCL11A and BCL11B, that affect the NMJ morphology of the Drosophila third instar larvae. The mutant of Bx (Bx7 ) and RNAi-mediated neuronal knockdown of both Bx and CG9650 exhibited defects in larval locomotion. In the present study, using a combination of techniques from behavioral assays, Drosophila genetics, to imaging studies, we show that while Bx regulates NMJ span area, CG9650 governs the bouton morphology. Subsequent electrophysiological recordings revealed that both were important for maintaining the normal spontaneous firing of the neurons. To deduce their mechanism of action at NMJs, we performed a microarray analysis, in the case of Bx and discovered its plausible involvement in the retrograde BMP pathway via the LIMK signaling. On the other hand, an mRNA-sequencing analysis for CG9650 suggested its roles in the regulation of c-fos, the component of the AP1 transcription complex, that works along the JNK pathway at the NMJ.