Transcriptional profiling reveals roles of intercellular Fgf9 signaling in astrocyte maturation and synaptic refinement during brainstem development.

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-Seq to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells for VEGF, TGFß, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional KO (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix that ensheaths principal neurons in the MNTB and the large calyx of Held terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in calyx of Held volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, perineuronal net structure, and synaptic refinement.

Environmental Elasticity Regulates Cell-type Specific RHOA Signaling and Neuritogenesis of Human Neurons.

The microenvironment of developing neurons is a dynamic landscape of both chemical and mechanical cues that regulate cell proliferation, differentiation, migration, and axon extension. While the regulatory roles of chemical ligands in neuronal morphogenesis have been described, little is known about how mechanical forces influence neurite development. Here, we tested how substratum elasticity regulates neurite development of human forebrain (hFB) neurons and human motor neurons (hMNs), two populations of neurons that naturally extend axons into distinct elastic environments. Using polyacrylamide and collagen hydrogels of varying compliance, we find that hMNs preferred rigid conditions that approximate the elasticity of muscle, whereas hFB neurons preferred softer conditions that approximate brain tissue elasticity. More stable leading-edge protrusions, increased peripheral adhesions, and elevated RHOA signaling of hMN growth cones contributed to faster neurite outgrowth on rigid substrata. Our data suggest that RHOA balances contractile and adhesive forces in response to substratum elasticity.

Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

UNLABELLED: Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. SIGNIFICANCE STATEMENT: Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidance in vivo. Consistent with this notion, we found that RF rates of spinal neuron growth cones were slower in vivo than what was observed in vitro. Together, our study provides the best evidence that growth cone-ECM adhesions clutch RF locally to guide axons in vivo.

Mechanochemical regulation of growth cone motility.

Neuronal growth cones are exquisite sensory-motor machines capable of transducing features contacted in their local extracellular environment into guided process extension during development. Extensive research has shown that chemical ligands activate cell surface receptors on growth cones leading to intracellular signals that direct cytoskeletal changes. However, the environment also provides mechanical support for growth cone adhesion and traction forces that stabilize leading edge protrusions. Interestingly, recent work suggests that both the mechanical properties of the environment and mechanical forces generated within growth cones influence axon guidance. In this review we discuss novel molecular mechanisms involved in growth cone force production and detection, and speculate how these processes may be necessary for the development of proper neuronal morphogenesis.

Regulation of ECM degradation and axon guidance by growth cone invadosomes.

Invadopodia and podosomes, collectively referred to as invadosomes, are F-actin-rich basal protrusions of cells that provide sites of attachment to and degradation of the extracellular matrix. Invadosomes promote the invasion of cells, ranging from metastatic cancer cells to immune cells, into tissue. Here, we show that neuronal growth cones form protrusions that share molecular, structural and functional characteristics of invadosomes. Growth cones from all neuron types and species examined, including a variety of human neurons, form invadosomes both in vitro and in vivo. Growth cone invadosomes contain dynamic F-actin and several actin regulatory proteins, as well as Tks5 and matrix metalloproteinases, which locally degrade the matrix. When viewed using three-dimensional super-resolution microscopy, F-actin foci often extended together with microtubules within orthogonal protrusions emanating from the growth cone central domain. Finally, inhibiting the function of Tks5 both reduced matrix degradation in vitro and disrupted motoneuron axons from exiting the spinal cord and extending into the periphery. Taken together, our results suggest that growth cones use invadosomes to target protease activity during axon guidance through tissues.