The natural environment of a neuron is the three-dimensional (3D) tissue. In vivo, embryonic sensory neurons transiently express a bipolar morphology with two opposing neurites before undergoing cytoplasmic and cytoskeletal rearrangement to a more mature pseudo-unipolar axonal arbor before birth. The unipolar morphology is crucial in the adult for correct information transmission from the periphery to the central nervous system. On two-dimensional (2D) substrates this transformation is delayed significantly or absent. We report that a 3D culture platform can invoke the characteristic transformation to the unipolar axonal arbor within a time frame similar to in vivo, overcoming the loss of this essential milestone in 2D substrates. Additionally, 3D substrates alone provided an environment that promoted axonal branching features that reflect morphological patterns observed in vivo. We have also analyzed the involvement of soluble cues in these morphogenic processes by culturing the neurons in the presence and absence of nerve growth factor (NGF), a molecule that plays distinct roles in the development of the peripheral and central nervous systems. Without NGF, both 2D and 3D cultures had significant decreases in the relative population of unipolar neurons as well as shorter neurite lengths and fewer branch points compared to cultures with NGF. Interestingly, branching features of neurons cultured in 3D without NGF resemble those of neurons cultured in 2D with NGF. Therefore, neurons cultured in 3D without NGF lost the ability to differentiate into unipolar neurons, suggesting that this morphological hallmark requires not only presentation of soluble cues like NGF, but also the surrounding 3D presentation of adhesive ligands to allow for realization of the innate morphogenic program. We propose that in a 3D environment, various matrix and soluble cues are presented toward all surfaces of the cell; this optimized milieu allows neurons to elaborate their genuine phenotype and follow programmed instructions that are intrinsic to the neuron, but disrupted when cells were dissected from the embryo. Thus, this study presents quantitative data supporting that 3D substrates are critical for sustaining the in vivo ontogeny of neurons and deciphering signaling mechanisms necessary for designing biomaterial scaffolds for nerve generation and repair.

Neuronal differentiation, pathfinding and morphology are directed by biochemical cues that in vivo are presented in a complex scaffold of extracellular matrix (ECM). This microenvironment is three-dimensional (3D) and heterogeneous. Therefore, it is not surprising that more physiologically-relevant cellular responses are found in 3D culture environments rather than on two-dimensional (2D) flat substrates. One key difference between 2D and 3D environments is the spatial arrangement of cell-matrix interactions. Integrins and other receptor proteins link the various molecules presented in the extracellular environment to intracellular signaling cascades and thus influence a number of neuronal responses including the availability and activation of integrins themselves. We have previously reported that a 3D substrate induces an important morphological transformation of embryonic mouse dorsal root ganglion (DRG) neurons. Here, we investigate the hypothesis that β1-integrin signaling via focal adhesion kinase (FAK) and the RhoGTPases Rac and Rho influences neuronal morphology in 2D vs 3D environments. We report that β1-integrin activity and FAK phosphorylation at tyrosine 397 (FAKpY397) are linked to neuronal polarization as well as neurite outgrowth and branching. Rac and Rho expression are decreased in 3D vs 2D culture but not correlated with β1-integrin function. These results suggest that proper β1-integrin activity is required for elaboration of physiologic DRG morphology and that 3D culture provides a more appropriate milieu to the mimic in vivo scenario. We propose that neuronal morphology may be directed during development and regeneration by factors that influence how β1-integrin, FAK and RhoGTPase molecules integrate substrate signals in the 3D microenvironment.