The innervation of the mammary gland is controlled by brain-derived neurotrophic factor (BDNF), and sexually dimorphic sequestering of BDNF by the truncated form of TrkB (TrkB.T1) directs male-specific axonal pruning in mice. It is unknown whether other cues modulate these processes. We detected specific, non-dimorphic, expression of Semaphorin family members in the mouse mammary gland, which signal through PlexinA4. PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland, which could be reduced by genetic co-reduction of BDNF. Moreover, in males, PlexinA4 ablation delayed axonal pruning, independently of the initial levels of innervation. In support of this, in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling. Overall, our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling. Upon inhibition of trophic signaling, these repulsive factors may promote axonal pruning.

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Apoptotic cells expose Phosphatidylserine (PS), that serves as an "eat me" signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.