Comparing transcriptome profiles of neurons interfacing adjacent cells

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Comparing transcriptome profiles of neurons interfacing adjacent cells and nano-patterned substrates reveal fundamental neuronal interactions Koby Baranes, Dror Hibsh, Sharon Cohen, Tony Yamin, Sol Efroni, Amos Sharoni, and Orit Shefi Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03879 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Comparing transcriptome profiles of neurons interfacing adjacent cells and nano-patterned substrates reveal fundamental neuronal interactions Koby Baranes†‡¶#, Dror Hibsh‡§#, Sharon Cohen†‡∥, Tony Yamin‡⊥, Sol Efroni‡§, Amos Sharoni‡⊥* and Orit Shefi†‡* †Faculty of Engineering, Bar-Ilan University, Ramat-Gan, 5290002, Israel ‡Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, 5290002, Israel §Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel ∥Gonda Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan, 5290002, Israel ⊥Department of Physics, Bar-Ilan University, Ramat-Gan, 5290002, Israel ¶Current address: Department of Clinical Neurosciences, University of Cambridge, Cambridge, CB2 0QQ, United Kingdom *Corresponding

author: Orit Shefi (E-mail: [email protected]) and Amos Sharoni (E-mail:

[email protected]) #These

authors contributed equally to this work

Developing neuronal axons are directed by chemical and physical signals towards a myriad of target cells. According to current dogma the resulting network architecture is critically shaped by electrical interconnections, the synapses. However, key mechanisms translating neuronal interactions into neuronal growth behavior during network formation are still unresolved. To elucidate these mechanisms, we examined neurons interfacing nano-patterned substrates and compared them to natural inter-neuron interactions. We grew similar neuronal populations under three connectivity conditions: (1) the neurons are isolated, (2) the neurons are interconnected, and (3) the neurons are connected only to artificial substrates, then quantitatively compared both the cell morphologies and the transcriptome-expression profiles. Our analysis shows that while axon-guidance signaling pathways in isolated neurons are predominant, in isolated neurons interfacing nanotopography these pathways are downregulated, similarly to the interconnected neurons. Moreover, in nanotopography interfacing neurons genes related to synaptogenesis and synaptic regulation are highly expressed, i.e. again resembling behavior of interconnected neurons. These molecular findings demonstrate that interactions with nanotopographies, although not leading to electrical coupling, play a comparable functional role in two major routes; neuronal guidance and network formation, with high relevance to the design of regenerative interfaces.

Keywords: Neuronal growth; nano-topography; gene expression; RNA-sequencing

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The study of elemental processes in neural network formation is fundamental to a better understanding of brain function and the development of post-trauma therapeutics. Neurons form functional networks via interconnected axons and dendrites. Studies have identified neuronal growth processes, but have not yet revealed how a single neuron acquires a specific structure for a specific function. Here we study the interactions of neurons with artificial nano-patterned substrates, as a method to identify fundamental pathways in neuronal growth and network formation, important to physiological and pathological conditions. During network organization, neurites target and make contacts with other neurons. A key mechanism is the ability of the sensory-motile growth cones at the tips of growing processes to measure long- and short-range environmental cues and use them to develop accordingly.1-3 Well-studied guidance cues include spatial concentration gradients of target-recognizing chemorepellent and chemoattractive molecules, many of which have been identified.3, 4 Other potent environmental cues are physical. Micro- and nanoscale physical cues are known to direct and promote neuronal growth.5-9 A recently developed paradigm is the ‘substratecytoskeletal coupling’ model.10, 11 According to this model, growth cones can move forward if they can couple intracellular motility signals to a fixed extracellular translocation substrate via cell-surface adhesion receptors. In such a case, topography is a key factor. According to current dogma, physical connections between neurons eventually lead to the formation of electrochemically active synapses and network circuitry.12 The nature and extent of neuronal connectivity with neighboring cells critically influences neuronal development and morphogenesis.13-15 We have shown that neurons in culture significantly change their growth strategy after recognizing and targeting neighboring cells.16-19 Isolated neurons tend to elaborate their dendritic tree, putting out more branches to achieve as many interconnections as possible in minimal time.16 But, once contact between neurons is established, the branching strategy alters to favor greater efficiency in neurite length and volume. Notably, such modification of the growth strategy following intercellular contact has also been observed in vivo.12, 20, 21 With the development of fabrication technologies and the need for engineered scaffolds and replacements, artificial substrates have been designed and examined.22 Interactions with these substrates significantly alter neuronal growth patterns.6, 19, 22-25 In a previous study we found that, remarkably, the development of isolated neurons which interact with nanotopographical cues differs from that of isolated neurons on substrates with no such topography. Using descriptive morphometric parameters, we showed that interactions between neurons and artificial nanotopographies trigger modifications in growth behaviors similar to those of neighboring cells.19 Specifically, neurons anchored to the topography seem to shift from a strategy of elaborated growth to one of economic growth, reminiscent of that 2 ACS Paragon Plus Environment

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we observed after neuron-neuron interaction. Moreover, a study by the group of D. R. Colman showed that micro-beads that interact with neurons in culture trigger the formation of functional presynaptic boutons, even though no neuronal partner is involved.26 Characteristic features of presynaptic structural elements, such as presynaptic vesicles and microtubular structures, were visualized at these artificial synapse sites.27 These effects of neuron-substrate interactions, which are reminiscent of those observed after neuron-neuron interaction, raise the question what are the functional interactions fundamental to neuronal networks. To answer this question, we grew identical neuronal populations in three distinct connectivity states: isolated neurons, interconnected neurons, and isolated neurons in contact with artificial nanotopographical cues. We analyzed the morphology and connectivity status at the single-cell level in each of the three states. We then deep sequenced the three populations and studied their gene-expression profiles, looking for differentially expressed genes (DEGs). High expression of genes related to synaptogenesis and synaptic regulation was observed in the two connected conditions, even though in one of them the interaction was with artificial nanotopographical elements. Furthermore, we found a link between physical guidance and chemical guidance, as receptors for axon guidance molecules had lower expression levels when neurons interacted with the topographical cues. This unique approach provided insights into the impact of neuronal connectivity vs. nanotopography on biological functions and pathways, shedding light on fundamental mechanisms that govern neuronal growth and circuitry. To investigate alterations in gene-expression profiling during processes of neuronal connectivity, we designed a triple model system of single neurons in culture (Figure 1a and Figure S1a): (i) 'Non-contact' - isolated cultured neurons with no interactions between them on non-patterned (flat) substrates (4 cells/mm2), (ii) ‘Contact’ - interconnected cultured neurons on similar non-pattered substrates (40 cells/mm2), and (iii) ‘Lines’ - isolated neurons that interact with artificial nano-cues but not with other neurons (8 cells/mm2). The ‘Lines’ substrates consisted of continuous oxide lines , 3µm width and 75nm height (Figure 1b and Figure S1b). An invertebrate model was used which enabled us to carry out detailed quantitative analysis of neuronal morphology at the single-cell level in parallel to genetic profiling. In all three populations, neurons first lost their original processes, remaining only with the soma, and then began to regenerate them back. By 1 day after plating the outgrowth and development of their dendritic trees had already started. After a minimum of 6 days in vitro (6-DIV) nearly all of the neurons (over 80%) were found to interconnect and to establish the 'Contact' neuronal population (Figure 1a, middle panel). 'Non-contact' and 'Lines' neurons elongated and branched without forming a network within this time period (Figure 1a, left and right panels, respectively). The 'Lines' neurons showed substantial alignment with the 3 ACS Paragon Plus Environment

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nanotopography, as we previously demonstrated.19 By this stage the ‘Contact’ neurons were forming synapses, detectable by FM1-43-staining of synaptic vesicles,23 thus providing evidence that the developed neuronal networks were functional, as corroborated by electrophysiological recordings (data not shown). First, we quantitatively analyzed the morphology of the three neuronal groups, using the Sholl method.28 ‘Contact’ and ‘Lines’ neurons had significantly fewer branches and fewer neurites originating from their soma compared to the 'Non-contact' neurons (Figure 1c). The analysis of the differences between the ‘Non-contact’ to the ‘Contact’ and ‘Lines’ neurons revealed a shift towards a more economical morphological arrangement (36 vs. 14 and 16 branching points and 7-8 vs. 4 and 4 originating neurites, respectively) (Figure 2b-c), resulting in a more simplified dendritic tree. To note, neurons with no connections showed neurite growth rates (Figure S1c), in agreement with our previously published results.19,

23

This data clearly shows that the morphological alterations triggered by interactions with the artificial nanostructures resemble the growth dynamics following neuron-neuron interaction. Further analysis of neuronal interactions with nanotopographical elements of a few different shapes (Figure 2a) led to similar growth statistics. The average number of branching points, number of neurites originating from the soma and total neurite length were similar to neurons grown on the long line-patterned substrates (Figure 2b-d). Moreover, to rule out any material composition effect, the same populations of neurons were grown on top flat substrates coated with the same oxide material as the nanotopographical elements and compared to neurons on top flat control substrates. It can be seen that the neurons developed dendritic trees with similar characteristics showing no material preference (Figure 2b-d). Taken together, these results demonstrate that a contact to topography is a major factor in neuronal maturation similarly to neuron-neuron interaction. In order to analyze the relationships between the 3 culture conditions and to reveal mechanisms underlying interactions of neurons with artificial nanostructures or with other neurons we used a gene-expression-profiling approach. Total RNA was extracted from the three 6-DIV neuronal populations and deep sequenced, followed by reconstructing the denovo transcriptome assembly (see flow diagram, Figure S2a). We used a 3-dimensional (3D) analysis of differential expression to classify our transcriptome data into patterns, based on a previous study.29 From this classification, we selected three that represent the most relevant settings for this study (Figure S3a, c). We distinguished between 5 cases: upregulated genes for isolated neurons, the ‘Non-contact’ and ‘Lines’, and not for the ‘Contact’ neurons (referred to as NC-C-L), upregulated genes after neuron-neuron contact (NC-C-L); downregulated genes (NC-C-L) or upregulated genes (NC-C-L) after any contact (neuronneuron or neuron-artificial nano-cue), and upregulated genes after contact with the nano-lines only (NC-C-L). We established functional and evolutionary relationships of the differentially 4 ACS Paragon Plus Environment

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expressed genes which are presented as gene ontology (GO) terms using DAVID (see supporting information). To gain a better knowledge of which genes in each category are the most prominent, we looked at the top 50 DEGs per category. In addition, relationships among the 3 culture conditions were investigated by pairwise enrichment analysis, in which all possible pairs of conditions were compared and screened for DEGs that were significantly enriched relative to the prominent clusters obtained. In previous studies that examined specific genes it has been shown that changes in cell morphology are followed by upregulation of cytoskeletal reorganization genes.11, effect of nanotopography has been extensively studied.5-9,

19, 22, 34, 35

30-33

The

Data suggests that the

modifications are mechanically driven by forces between cells and between cells and topographical

cues,

mechanotransductive

regulating

gene

pathways.22, 35, 40

expression

in

the

neurons,22,

36-39

and

Here, we focus on dominantly expressed genes in two

main statuses where ‘Lines’ are more similar to the ‘Contact’ and ‘Lines’ more similar to the ‘Non-contact’. First, we present the transcriptome data showing that neurons with interconnections to other neurons (category

NC-C-L)

exhibited enrichment of protein kinase activity and RNA

metabolic processes such as processing and helicase activity (Figure 3b), which contain genes that were top upregulated (Figure S5). These clusters (see Table S1) are related to translation and protein synthesis processes, that characterize interconnected neurons during development, and whose relevant genes are especially concentrated at synaptic zones.41, 42 In addition, these neurons exhibited upregulation of a gene, suggested to inhibit neurite outgrowth during axon regeneration in vitro (CSPG4) (Figure 4).43-45 Further support comes from previous reports on the upregulation of proteins which are involved in ECM remodeling and degradation, as the family of matrix metalloproteases (MMPs),46 as MMP8 and MMP16 in our database. These NC-C-L

findings indicate that following the establishment of neuronal interconnections there is

downregulation of processes related to synapses that are no longer needed, explaining the morphological changes of 'Contact' neuron of the dendritic tree remodeling. Following, we present the main findings for which the ‘Lines’ population exhibits similar expression levels to the ‘Contact’ population. We start with cytoskeleton components that together with cell-adhesion molecules control numerous extracellular cues that coordinate the guidance of axons to their targets. The 'Non-contact' neurons (NC-C-L), with no connections to other neurons or to nano-topographies were enriched in GO-terms which may be related to cytoskeleton and axon guidance (such as cell projection organization GO:0030030 and cell projection part GO:0044463) (Figure 3c).47, 48 The ‘axon guidance’ GO-term (GO:0007411) was also, though less significantly, enriched (Figure S5 and Table S1). Interestingly, the lack of enrichment of axon guidance genes in the ‘Lines’ condition suggests that although these


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neurons are not connected to other neurons, they rely less on chemical guidance cues for signaling but rather base their navigation on topographical cues from the nano-line patterns. Cell-adhesion processes are important for cell-surface interactions, whereas outside-in signals between the ECM and the cells are transmitted by focal adhesions.49 In comparison to the ‘Non-contact’ group, ‘Lines’ neurons interacting with nanotopography upregulated genes related to cell adhesion and synapses but downregulated genes related to cytoskeleton remodeling. Nonetheless, the ‘Lines’ group showed upregulation in two key genes involved in the control of actin cytoskeleton, which were also upregulated in ‘Contact’ neurons (RhoV and ROCK1). One well-known pathway of cell-cell communication is via Notch signaling. Here, we found that Notch1 and an activator of the Notch signaling pathway called TACE (also known as ADAM17), are upregulated after contact with the nanotopographies (Figure 4), suggesting a functional interaction with the artificial elements. Another member that mediates cell adhesion, of the superfamily of cadherins and is dependent on calcium ions50 was upregulated (FAT1). A different cell-adhesion family of proteins, integrins, is even more highly upregulated both in ‘Contact’ and in ‘Lines’ conditions. For example, the integrin ILKAP that modulates cell adhesion and growth factor signaling.51 Overall, interactions with nanotopography were associated here with a significant upregulation of genes related to cell adhesion (Figure 4, Figure S5 and Table S1). Focal adhesion formation demonstrates a mechanical linkage between the cells and the substrate.52-55 The mechanism by which focal adhesions mediate the adhesion between integrin and the actin cytoskeleton is influenced by physical forces present in the extracellular environment, in this case the nanotopography.49, 56 These focal adhesion points have been shown to be in the nano-scale range.57 It has been shown that following the establishment of neuronal interconnections electrical and chemical synapses are activated by gap-junction channels or by distinct receptors to allow the control of signal transduction.58-60 Interestingly, neurons after any contact (category NC-CL) were enriched in GO-terms related to synapse processes (Figure 3d and Table S1). Genes related to synaptic processes in the ‘Contact’ conditions were upregulated, for example that are known to regulate synaptogenesis,61-63 and synaptic organization (Figure 4 and Table S1). Surprisingly, neurons after contact with the nanotopographies (‘Lines’), that weren’t expected to express functional related genes, showed upregulation of the receptor GRIA2 and the gap junctional protein Inx11 (Figure 4). In our model system gap junctions are particularly abundant and are the foundation of chemical synapse formation.64 Of the 21 reported innexin genes,65 we detected 17 genes including in the ‘Lines’ neurons where we found also CDK5, a protein kinase which regulates synaptogenesis and neurite outgrowth,66 and Nf1 and CASK.67 Other genes that were upregulated after the interaction with the artificial nano-elements included SNAP29 (synaptosomal-associated protein 29) and CadN (neuronal cadherin) which in part mediate pre- and post-synaptic adhesion. These findings may relate those genes 6 ACS Paragon Plus Environment

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exclusively to interactions with the artificial nanostructures suggesting that contact with artificial cues triggers the formation of both chemical and electrical synapses. Since synaptic transmission requires energy, we were intrigued to find that the top 50 DEGs for the ‘Lines’ neurons included genes related to mitochondrial processes as well as mitochondrion-related GO-terms, possibly inferring synaptic activity (Figure 3e and Figure S5). Support for that notion comes from our clustering analysis for the ‘Lines’ condition, where we observed GO-terms related to the ubiquitin-proteasome system (Figure 3e) found to regulate synaptic transmission at both pre- and postsynaptic terminals.68 In the ‘Lines’ group more signal transduction related genes were upregulated, genes that participate in the fibroblast group factor (FGF) pathway and are considered to be Ras-specific guanine nucleotide exchange factors. SOS2 and GrB2 were upregulated more strongly (data not shown) and are acting together in response to calcium, leading to activation of the Ras and Raf/Erk kinase cascades.69 Voltage-gated ion channels trigger processes such as neurite outgrowth, neurotransmitter release and gene expression.47, 48, 70 A subunit of these channels, Cav1.2 (CACNA1C), was significantly upregulated in ‘Lines’ neurons (Figure S6). This protein was reported to be localized in the postsynaptic zone together with neurofibromin-1 (Nf1),70-72 which was also upregulated in the ‘Lines’ condition (Figure 4). In addition, Nf1 was found to regulate the Ras signal-transduction pathway, as well as to bind with the NMDA-receptor complex and interact with CASK (upregulated in the ‘Lines’) (Figure 4). Several calcium signaling related genes were upregulated in ‘Non-contact’ neurons (NC-C-L), and some were also regulated in the 'Lines' neurons (NC-C-L) (Figure S5 and Figure S6). The 'Non-contact' condition also showed upregulation of ion channels Cav2.2 (CACNA1B). According to the literature, calmodulin (CaM) is associated with high voltage-activated (HVA) channels70 such as those discussed above. It also mediates the control of ion channels by calcium, and regulates voltage-gated sodium channels.73 To

complement

the

gene

expression

profiling

results,

we

performed

immunocytochemistry studies labeling two markers of dominant clusters, synapsin-1 and actin (Figure 5 and Figure S7). Our results, in agreement with,26,

27, 74

that based on

morphological observations demonstrate similar levels of expression for both contact populations (‘Lines’ and ‘Contact’). Higher density of synapsin-1 could be detected in proximity to the lines and especially line edges. To summarize, two common paradigms may define the complex network formation process. In the first, traditionally functional connections between neurons serve as the key factors in neuronal structure formation, with active synapses strengthening connected branches while unneeded connections are eliminated. This economic process demonstrates a ‘form-follows-function’ mode12. The second paradigm posits that the morphology is a major 7 ACS Paragon Plus Environment

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effector in determining the role of neurons within a network, in a ‘function-follows-form’ manner.75 Furthermore, mechanical tension in axons was found to play a key role in network formation by regulating neuronal interconnections37 and increasing the accumulation of neurotransmitters.76 We and others have demonstrated that interactions with artificial cues can also alter cell growth patterns similarly to the interactions of neurons with other neurons.19, 26, 27

In this study we were able to compare distinct morphological parameters together with wide

genetic profiles of neurons without any contact, interconnected, or in contact with nanostructures. In a previous study we emphasized the critical role of physical interactions in neuronal growth and guidance on similar nanostructures.19 In the present study we were able to recognize the genetic modifications leading to these phenotypes. By recognizing the DEGs that commonly predominate in cell-cell and cell-substrate interactions, we revealed key functional players in neuronal network formation. Our results indicate that the mechanotransduction play a role in regulation of the modifications triggered by topography, possibly transmitting their signal to the nucleus and ultimately leading to differential gene expression. We show that electrically active synaptic interactions are not the sole factor in neuronal maturation and establishment. Moreover, our results highlight the importance of the extracellular environment of cells, as other neighbor cells and ECM in the brain. Our results also have important implications towards the design of regenerative platforms, i.e., using the interactions with the external environment as a mean for an effective biomimetic regeneration process.

Acknowledgment This work was supported by the Israel Science Foundation Individual grant #1053/15 (O.S.) and by the Levi Eshkol Fellowship from The Israeli Ministry of Science, Technology and Space

(K.B.).

The

authors thank the FIB/SEM center in the Bar Ilan Institute of Nanotechnologies and Advanced Materials (BINA) for assistance with the high-resolution imaging. The authors thank Ganit Eran Indech for the fabrication of some of the nano-patterned substrates.

Competing interests The authors declare no competing interests

Supporting information Supporting information is available for this paper Raw RNA-seq reads / The array data from this study is available upon request

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Figure legends: Figure 1. Model for neuronal cultures of controlled densities. (a) A standard photolithography process was used to fabricate 20-mm long line patterns, 75 nm in height and 3 µm width. (b) Phase (upper panels) and Scanning Electron Microscopy (lower panel) images of the different patterns. White arrow marks the 75nm height nano-line. Scale bars, upper panel (clockwise): 50 µm, 50 µm, 100 µm; lower panel: 500 nm. (c) Experimental procedure: neurons were extracted from ganglia 8 to 15 of the leech CNS and plated on top of the different substrates at high density ('Contact', middle) and at low densities ('Non-contact', left, and 'Lines', right) as represented in the schematic drawing. Seeding area is represented in mm2 below each scheme. (d) Phase images of 6-DIV neuronal cultures of the 3 culture conditions: isolated (‘Non-contact’) neurons, interconnected (‘Contact’) neurons and isolated neurons on top of topographic nano-cues (‘Lines’). Scale bars, 50 µm. Figure 2. Morphological analyses of neuronal cultures with controllable densities. (a) Impact on neuronal architecture as reflected in a Sholl analysis. The number of intersecting neurites at a distance of each radius from the cell body (10-µm spacing) was measured (illustrated on the right). Significant differences between the 3 culture conditions were seen up to the 70-µm radius ring (means ± SE, ANOVA). (b) Total neurite growth rate (in microns per day) on top of the nano-lines was significantly higher than in either of the other two culture conditions (means ± SD, ANOVA). (c) Analysis of distinct morphological parameters for the control substrates (flat and vanadium oxide) compared to the different patterned substrates (dashed lines, long lines and ‘S’ patterns) (means ± SE, t-test). Figure 3. Functional annotation analysis for differentially expressed genes. DEGs from each of the five groups were clustered via David according to their function. The graph represents the most significantly enriched GO-terms for each condition: NC-C-L (a), NC-C-L (b), NC-C-L (c), NC-C-L (d), and NC-C-L (e) using a p ≤ 0.05 (Bonferroni). The fold enrichment value is reported in the x-axis. Numbers in bold on the right of each bar indicates the number of genes enriched in the analysis. Figure 4. Pairwise enrichment analysis. Volcano plots of the posterior probability of differential expression (PPDE) versus log2Fold change. Upper panels represent “Non-contact” vs. “Contact” comparison, middle panels represent “Non-contact” vs. “Lines” comparison and lower panels represent “Contact” vs. “Lines” comparison. The threshold was set to PPDE  0.95 and Log2Fold change of -2 and +2. Genes enriched in ‘Non-contact’ neurons are marked in orange, those enriched in ‘Contact’ neurons in blue, and those enriched in ‘Lines’ neurons in green. Specific enriched genes related to cytoskeleton remodeling, synapse processes and cell adhesion, were highlighted for each condition. All of the represented genes are uniquely expressed in each comparison. Figure 5. Immunostaining for synaptic related markers. Confocal microscopy images for the three culture conditions at 6 DIV, for the synaptic marker synapsin1 (green), showing upregulation for both ‘Contact’ and ‘Lines’ neurons. In red -tubulin. Scale bars 75 m.


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Figure 1. Model for neuronal cultures of controlled densities. (a) A standard photolithography process was used to fabricate 20-mm long line patterns, 75 nm in height and 3 µm width. (b) Phase (upper panels) and Scanning Electron Microscopy (lower panel) images of the different patterns. White arrow marks the 75nm height nano-line. Scale bars, upper panel (clockwise): 50 µm, 50 µm, 100 µm; lower panel: 500 nm. (c) Experimental procedure: neurons were extracted from ganglia 8 to 15 of the leech CNS and plated on top of the different substrates at high density ('Contact', middle) and at low densities ('Non-contact', left, and 'Lines', right) as represented in the schematic drawing. Seeding area is represented in mm2 below each scheme. (d) Phase images of 6-DIV neuronal cultures of the 3 culture conditions: isolated (‘Non-contact’) neurons, interconnected (‘Contact’) neurons and isolated neurons on top of topographic nano-cues (‘Lines’). Scale bars, 50 µm. 177x195mm (300 x 300 DPI)

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Figure 2. Morphological analyses of neuronal cultures with controllable densities. (a) Impact on neuronal architecture as reflected in a Sholl analysis. The number of intersecting neurites at a distance of each radius from the cell body (10-µm spacing) was measured (illustrated on the right). Significant differences between the 3 culture conditions were seen up to the 70-µm radius ring (means ± SE, ANOVA). (b) Total neurite growth rate (in microns per day) on top of the nano-lines was significantly higher than in either of the other two culture conditions (means ± SD, ANOVA). (c) Analysis of distinct morphological parameters for the control substrates (flat and vanadium oxide) compared to the different patterned substrates (dashed lines, long lines and ‘S’ patterns) (means ± SE, t-test). 177x124mm (300 x 300 DPI)


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Figure 3. Functional annotation analysis for differentially expressed genes. DEGs from each of the five groups were clustered via David according to their function. The graph represents the most significantly enriched GO-terms for each condition: NC-C-L (a), NC-C-L (b), NC-C-L (c), NC-C-L (d), and NC-C-L (e) using a p ≤ 0.05 (Bonferroni). The fold enrichment value is reported in the x-axis. Numbers in bold on the right of each bar indicates the number of genes enriched in the analysis. 177x230mm (300 x 300 DPI)


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Figure 4. Pairwise enrichment analysis. Volcano plots of the posterior probability of differential expression (PPDE) versus log2Fold change. Upper panels represent “Non-contact” vs. “Contact” comparison, middle panels represent “Non-contact” vs. “Lines” comparison and lower panels represent “Contact” vs. “Lines” comparison. The threshold was set to PPDE  0.95 and Log2Fold change of -2 and +2. Genes enriched in ‘Non-contact’ neurons are marked in orange, those enriched in ‘Contact’ neurons in blue, and those enriched in ‘Lines’ neurons in green. Specific enriched genes related to cytoskeleton remodeling, synapse processes and cell adhesion, were highlighted for each condition. All of the represented genes are uniquely expressed in each comparison. 177x111mm (300 x 300 DPI)


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Figure 5. Immunostaining for synaptic related markers. Confocal microscopy images for the three culture conditions at 6 DIV, for the synaptic marker synapsin1 (green), showing upregulation for both ‘Contact’ and ‘Lines’ neurons. In red -tubulin. Scale bars 75 m.

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TOC Title: Comparing transcriptome profiles of neurons interfacing adjacent cells and nano-patterned substrates reveal fundamental neuronal interactions 82x42mm (600 x 600 DPI)


Comparing transcriptome profiles of neurons interfacing adjacent cells

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