Modulation of TGF-beta signaling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Bone morphogenetic proteins (BMPs) are potent secreted signaling factors that function at long- and short-range and elicit critical cellular responses during development and homeostasis. Long-range signaling is key to the formation and function of morphogen gradients which control cell fate and tissue allocation. Short-range signaling sculpts cellular junctions and has been implicated in the growth, development and homeostasis of synaptic junctions, such as Drosophila neuromuscular junction (NMJ). The fly NMJ is a glutamatergic synapse similar in composition and physiology to mammalian central synapses. The fact that individual NMJs can be reproducibly identified and are easily accessible for electrophysiological and optical analysis makes this genetic model system uniquely suited for in vivo studies on NMJ growth and synapse assembly, maturation and plasticity. BMP signaling is critical for NMJ growth, neurotransmitter release and synapse homeostasis. Developmental growth is known to be mediated by retrograde canonical BMP pathway, but the targets of BMP signaling in motor neurons are poorly understood. The mechanisms by which neurotransmitter release and synapse homeostasis are regulated by BMP signaling are largely unknown. Several BMP signaling pathways have been implicated. At synaptic terminals, Gbb/ BMP7 binding to BMP receptors (BMPRs) on the motor neuron membrane triggers canonical and non-canonical BMP signaling. During canonical signaling, Gbb forms high-order BMP/BMPR (Gbb/Wit/Tkv/Sax) complexes that are endocytosed and transported to the motor neuron soma where they regulate various transcriptional programs required for NMJ growth and function. During non-canonical signaling, the BMP/BMPR complexes remain at synaptic terminals where they bind to LIMK1 to regulate synapse stability. In addition, we discovered a local, synaptic BMP signaling pathway that does not require Gbb. Instead, active postsynaptic type-A glutamate receptors appear to form trans-synaptic columns with presynaptic BMP/BMPR complexes and induce phosphorylation and accumulation of the BMP effector, Mad/ SMAD1,5,8 at the active zone. We think that such trans-synaptic columns offer a versatile means for (1) relaying synapse activity status to the presynaptic motor neurons via fast conformational modifications, and (2) facilitating interactions that stabilize the type-A glutamate receptors at postsynaptic densities. Motor neurons (MNs) must coordinate all these genetically distinct BMP pathways to build appropriate NMJs, then mature the synapses and tune their activity. This coordination is accomplished through limited and tightly controlled levels of various pathway components that are shared among different BMP signaling modalities; mutations that favor (or disfavor) one of these signaling pathways appear do so at the expense of the others. Thus, MNs may use the local BMP signaling to monitor synapse activity then coordinate NMJ growth with synapse maturation and stabilization. To examine how MNs respond to different BMP signaling modalities, we first used high-quality scRNA-seq to assemble a comprehensive atlas of the entire larval ventral nerve cord (VNC). The Drosophila VNC shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. We utilized sequences from more than 30,000 single cells (from multiple experimental sets), un-supervised algorithms, reiterative processes and known specific transcripts to cluster all VNC cells and identify different cell types. Our atlas includes several glia subtypes, neuroblast and newborn neurons organized in a developmental trajectory, motor neurons and interneurons. We showed that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. We described multiple populations of larval glia and endorsed the general view that glia cells perform consistent activities throughout development. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separated all MN bundles and built a dataset from which it is possible to identify individual MNs and link individual neuron clusters to morphologically characterized known neuron types. We examined the expression of various neurotransmitter receptors and showed that multiple neurotransmitter inputs converge onto primary larval MNs. We discovered a novel glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. Throughout this work, we used a battery of antibodies and selective drivers to identify various cell populations and validate new metabolic and signaling pathways connecting cells within the nervous system. We are now in the process of examining how VNCs, in particular MNs, respond to synaptic perturbations or disruptions of various BMP signaling modalities. Our scRNAseq atlas provides a high-resolution characterization of larval VNC, capturing primary neurons, glia, and the functional landscape that coordinates larval behavior. At the same time, this dataset offers unique insights into neurogenesis and into the strategies and signaling networks utilized for the generation of the adult VNC. This atlas represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.