Substrate three-dimensionality induces elemental morphological transformation of sensory neurons on a physiologic timescale

Abstract

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.

FIG. 1.

Morphological transition of mouse DRG neurons during embryonic development. Before E14 DRG neurons are bipolar, extending one axon each toward the peripheral and central nervous systems. Between E14 and E15, the neurons transform into the canonical pseudo-unipolar morphology found in mature animals. Redrawn by A.R. from Ref.¹³

FIG. 2.

Sensory neuron morphology is affected by environment dimensionality. Representative confocal images of DRG neurons cultured on two-dimensional (2D) collagen-coated coverslips (a) and within three-dimensional (3D) collagen gels (b). Growth cones in 2D (c) were larger, richer in filopodia, and lamellipodia and more flattened than those in 3D (d). Neurons were co-labeled for βIII-tubulin (red) and NF160 (green). Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (blue). (e) Quantitative analysis of growth cone projected area (n_2D=n_3D=40, p<0.01). Boxes enclose 25th (black) and 75th (white) percentiles of each distribution and are bisected by the median; white diamonds represent the mean; whiskers indicate the 5th and 95th percentiles. Neurolucida reconstructions of representative DRG neurons cultured on 2D (f) and within 3D (g) collagen substrates. Scale bars, 10 μm in (a–d), 20 μm in (f, g). Color images available online at www.liebertonline.com/tea

FIG. 3.

Effect of environment dimensionality on neurite outgrowth and branching. DRG neurons cultured within 3D substrates (n=90) are predominately unipolar with longer neurites and more branching than neurons cultured on 2D substrates (n_glass=87; n_gel=65). (A) Number of primary neurites, (B) number of branch points per neurite, (C) length of the longest neurite, and (D) total neurite length. Boxes enclose 25th (black) and 75th (white) percentiles of each distribution and are bisected by the median; white diamonds represent the mean; whiskers indicate the 5th and 95th percentiles. Symbols denote significant differences (p<0.05): *from 2D gels, ⁺from 2D, ^#between all the culture conditions. (E) A direct comparison was made between the individual effects of substrate stiffness (2D collagen coated glass vs. 2D collagen gel) and environment dimensionality (2D collagen gel vs. 3D collagen gel). Phase contrast images of the DRG neurons cultured for 2 days on 2D collagen-coated glass (left), on top of 2D collagen gel (middle) and within 3D collagen gel (right). Scale bars, 20 μm.

FIG. 4.

Effect of environment dimensionality on DRG neuron response to NGF withdrawal. Without NGF, both 2D and 3D cultures had decreases in the relative population of unipolar neurons (A) and number branch points per neurite (B) compared to cultures with NGF. The neurons exhibited decreased total and individual neurite lengths as a response to NGF withdrawal compared to cultures containing NGF; these data are plotted as % change versus cultures with NGF (C, D). Notably, branching features of neurons cultured in 3D without NGF resemble those of neurons cultured in 2D with NGF [middle two bars in (B)]. Symbols denote significant differences (p<0.05): *between 2D and 3D culture within the same conditions, ⁺from culture with NGF. Representative confocal images of DRG neurons cultured on 2D (E) and within 3D collagen substrates (F) in the absence of NGF. Cultures were stained for βIII-tubulin (red). Scale bars, 10 μm. NGF, nerve growth factor. Color images available online at www.liebertonline.com/tea

FIG. 5.

Model of cell response to culture dimensionality. On a 2D substrate, matrix and soluble cues are segregated and provide an artificial environment that alters the integration of extracellular cues and results in a delayed polarization program. When exposed to a 3D environment, matrix ligands and soluble cues completely surround the cell, and in response, membrane receptors are arranged in an unrestricted configuration. In this setting, signaling events are regulated according to intrinsic programming and the neurons develop into the proper pseudo-unipolar morphology with long and branched axons as found in vivo. Color images available online at www.liebertonline.com/tea