Comparative transcriptomic profiling of peripheral efferent and afferent nerve fibres at different developmental stages in mice
SCIENtIfIC RepoRts |
Comparative transcriptomic profiling of peripheral efferent and afferent nerve fibres at different developmental stages in mice
OPEN Peripheral nerve injury impairs motor and sensory function in humans, and its functional recovery largely depends on the axonal outgrowth required for the accurate reinnervation of appropriate targets. To better understand how motor and sensory nerve fibres select their terminal pathways, an unbiased cDNA microarray analysis was conducted to examine differential gene expression patterns in peripheral efferent and afferent fibres at different developmental stages in mice. Gene ontology (GO) and Kyoto Enrichment of Genes and Genomes (KEGG) analyses revealed common and distinct features of enrichment for differentially expressed genes during motor and sensory nerve fibre development. Ingenuity Pathway Analysis (IPA) further indicated that the key differentially expressed genes were associated with trans-synaptic neurexin-neuroligin signalling components and a variety of gammaaminobutyric acid (GABA) receptors. The aim of this study was to generate a framework of gene networks regulated during motor and sensory neuron differentiation/maturation. These data may provide new clues regarding the underlying cellular and molecular mechanisms that determine the intrinsic capacity of neurons to regenerate after peripheral nerve injury. Our findings may thus facilitate further development of a potential intervention to manipulate the therapeutic efficiency of peripheral nerve repair in the clinic.
Injured axons in the adult mammalian peripheral nervous system (PNS), unlike those in the central nervous
system (CNS), were considered to have the intrinsic ability to initiate regeneration in the appropriate context1?4.
However, numerous studies have indicated that this endogenous regenerative capacity is still limited since
functional recovery is usually poor in the context of complete axonal transection with relatively large gap distances5.
Peripheral nerve injury has a deleterious impact on daily life, as it significantly impairs motor and sensory
function in patients. Therefore, a better understanding of how efferent and afferent nerve fibres select their terminal
pathways, and further, how proximal axonal sprouts reach and reinnervate their topographically appropriate
targets6, is of fundamental interest to both clinical and basic neuroscientists7?9.
Generally, functional recovery after peripheral nerve injury largely depends on the accuracy of axonal
pathfinding and the efficiency of reinnervation of distal target organs10,11. A central issue in neural regeneration is the
manner in which various neurons reestablish their distinct and predictable phenotypes by forming functionally
appropriate synaptic connections10. Previous studies have indicated that regenerating motor axons are guided
through the motor pathways to reestablish nerve-muscle contact in a process termed ?preferential motor
reinnervation?12,13. However, the molecular mechanism underlying this phenomenon is not yet fully understood14,15.
The sciatic nerve is the largest single nerve in humans. The rodent sciatic nerve injury model is the most
commonly used model for studying peripheral nerve regeneration. The spinal segments associated with the sciatic
nerve correspond to the lumbar dorsal root ganglia (L4-L6 DRGs)16,17. Efferent nerve fibres are axons originating
from motor neurons, while afferent nerve fibres are processes extending from sensory neurons. In the spinal cord,
motor neuron cell bodies are present in the ventral horn of the grey matter, and receive inputs from interneurons
transmitting signals via sensory integration feedback18. Efferent nerve fibres conduct signals away from the CNS
to their target peripheral organs, and form neuromuscular junctions with innervated muscles. Unlike motor
neuron somas, sensory neuron somas are located in the DRGs of the PNS. Axonal processes from groups of afferent
neuron somas extend in two opposite directions: the peripheral branch runs into the spinal nerve to innervate the
peripheral target organs, while the central branch runs to the dorsal horn of the spinal cord, ultimately conveying
sensory information from the PNS to the CNS19. During the development of selective sensory-motor circuits,
fundamental biological processes, such as axon guidance, selective target reinnervation, and synaptic plasticity in
neural networks, must be regulated properly to establish the appropriate neural circuits20.
In the present study, an unbiased cDNA microarray analysis was performed to examine differential gene
expression patterns in both efferent and afferent nerve fibres at different developmental stages in mice. The
detection window was from the postnatal day 0 (P0) to 5 weeks (5 W), as the de novo axonal sprouting and synaptic
formation and remodelling during the regenerative processes is analogous with those in the early developmental
stages2,21. The aim of this study was to provide a framework for the integrated analysis of gene regulatory networks
during peripheral motor and sensory neuronal differentiation/maturation. We also aimed to further elucidate the
biological processes and molecular mechanisms underlying the activation of the intrinsic neuronal regenerative
capacity after peripheral nerve injury. These data provide new insights for the development of potential
interventions designed to manipulate the therapeutic efficiency of peripheral nerve repair in the clinic.
Tissue Collection Process and Microarray Data Quality Assessment. We performed cDNA
microarray analysis using RNA samples extracted from efferent and afferent nerve fibres originating from the
L4?L6 segments of the spinal cord. The dissection range is illustrated using circles in the spinal cord diagram
(Fig.?1A). At different developmental stages (P0, 1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W]), the efferent
and afferent nerve fibres (also known as the ventral and dorsal root fibres, respectively) were carefully dissected
and collected using an anatomy microscope.
The sample correlation matrices indicated that all the 24 samples (8 groups in triplicate) could be
approximately clustered into four sets corresponding to the four experimental time points (Fig.?1B). Additionally, the
three-dimensional principal component analysis (PCA) plot indicates that the samples could be generally
classified based on the time points and the category of efferent vs. afferent nerve fibre (Fig.?1C).
General Overview of Differentially Expressed Genes in Efferent and Afferent Nerve Fibres at
Different Developmental Stages. The histograms in Fig.?2A show the results of the differential
expression analysis in efferent vs. afferent nerve fibres at different time points. These data indicate that the maximum
differential expression occurred at 1 W. This implies that the time period from P0 to 1W is the time window for
motor and sensory neuronal cell fate determination. The differential gene expression declined with time during
the postnatal period from 1 W through 5 W.
Figure?2B indicates that very large numbers of genes were differentially expressed in the efferent and afferent
nerve fibres throughout development. It is noteworthy that both types of nerve fibres displayed the maximum
number of differentially expressed genes at the 3 W time point.
The temporal expression profiles of the significantly differentially expressed genes in efferent and afferent
nerve fibres were visualized using Short Time-series Expression Miner (STEM) software. A comparison of the
five distinct expression patterns of the genes differentially expressed between the efferent and afferent nerve fibres
is presented in Fig.?2C. The total numbers of differentially expressed genes in the efferent and afferent nerve
fibres were 12,897 and 11,817, respectively. Of these genes, 2,440 genes in efferent nerve fibres and 1,776 genes in
afferent nerve fibres were first up-regulated and reached a peak in expression at 3W. The expression levels of these
genes then recovered at 5 W following an inverted U-shaped curve. Additionally, 3,131 genes in the efferent nerve
fibres and 3,344 genes in afferent nerve fibres were linearly up-regulated at all experimental time points; 2,923
genes in the efferent nerve fibres and 2,789 genes in afferent nerve fibres were linearly down-regulated during
development. Moreover, 1,364 genes in the efferent nerve fibres and 1,066 genes in afferent nerve fibres were first
down-regulated from P0 to 3 W and then recovered at 5 W following a U-shaped curve. Lastly, 813 genes in the
efferent nerve fibres and 829 genes in afferent nerve fibres had unchanged expression levels at P0 and 1W, were
down-regulated at 3 W, and were then up-regulated at 5 W (Fig.?2C and Table?S2).
Hierarchical Clustering Analysis of Differentially Expressed Genes. Hierarchical clustering analysis
was performed on differentially expressed genes in efferent and afferent nerve fibres at each experimental time
point. The results were visualized using heat map images. The heat maps indicated that nearly 1/4th of the
differentially expressed genes in the efferent and afferent nerve fibres were first up-regulated and then down-regulated,
while a little less than 1/3rd of the differentially expressed genes were first down-regulated and then up-regulated
at all developmental stages. The gene expression patterns in the motor and sensory nerve fibres during
development are shown in Fig.?3. All the differentially expressed genes involved in efferent and afferent nerve fibre
development are listed in Table?S3.
GO Analysis of Biological Processes, Cellular Components, and Molecular Functions Involved
in Efferent and Afferent Nerve Fibre Development. In order to analyse the patterns of differential
gene expression in the efferent and afferent nerve fibres during the different developmental stages, the GO
analysis database integrated with DAVID?s toolkit was used to identify the most significant differentially expressed
genes associated with neuronal development and outgrowth. There genes were categorised based on the following
defined terms: Biological Processes, Cellular Components, and Molecular Functions (Fig.?4).
The enriched subcategories under Biological Processes in the efferent and afferent nerve fibres during
development are presented in Fig.?4A. Among them, four biological processes (axon guidance, cell differentiation, cell
adhesion, and chloride transport) were differentially expressed in the efferent and afferent nerve fibres at all time
points. It is noteworthy that the anterior commissure morphogenesis, roundabout signalling pathway, cell
chemotaxis, and neuron projection guidance categories only exhibited significant differential expression at P0. In these
categories, there were no statistical differences between the two types of nerve fibres after P0.
In the Cellular Components category, the GO analysis indicated that the excitatory synapse and inhibitory
synapse subcategories had no statistically significant differences between the efferent and afferent nerve fibres at
P0, although they had significant differential expression starting at 1W. In addition, the differential expression
patterns were similar to those in the presynaptic and postsynaptic membrane subcategories (Fig.?4B).
The enrichment of Molecular Function in the different developmental stages is summarized in Fig.?4C.
Intriguingly, gamma-aminobutyric acid A (GABAA) receptor activity, calcium ion binding, chloride channel
activity, clathrin binding, and extracellular ligand-gated ion channel activity subcategories were found to have
differential expression at all four experimental time points. In contrast, the ionotropic glutamate receptor activity
and transmembrane receptor protein tyrosine kinase activity subcategories displayed no statistically significant
differences at P0, but had significant differential expression at 1W and thereafter.
Critical Signalling Pathways Involved in Efferent and Afferent Nerve Fibre Development. The
GO analysis provided overall insight into the cellular and molecular regulation of motor and sensory neuronal cell
fate determination. Kyoto Enrichment of Genes and Genomes (KEGG) analysis was further performed to identify
critical signalling pathways during motor and sensory neuronal differentiation/maturation (Fig.?5). It is
noteworthy that two signalling pathways, namely axon guidance and hedgehog signalling, were significantly differentially
expressed between the efferent and afferent nerve fibres during development as early as P0. In contrast, significant
differential expression of proteins involved in SNARE interactions during vesicular transport in the efferent and
afferent nerve fibres only occurred at 1W. In addition to these pathways, synaptic plasticity-associated signalling
pathways and proteins involved in GABAergic and cholinergic synapses were significantly differentially expressed
at 1 W. In contrast, glutamatergic and dopaminergic synapses were significantly differentially expressed at 3W
The Z-scores calculated from the average expression profiles of the differentially expressed genes involved in
the six most critical signalling pathways during development are listed in Fig.?5B. The Z-score curves indicate
that the changes in the axon guidance and GABAergic synapse pathways at different developmental stages were
relatively similar between the efferent and afferent nerve fibres. In contrast, the expression changes for mRNAs
involved in neuroactive ligand-receptor interactions, cholinergic synapses, glutamatergic synapses, and
dopaminergic synapse pathways were different between the two types of nerve fibres. This implies that the fundamental
patterns of gene expression inherent in motor and sensory neuronal cell fate determination have distinct features.
Although the differential expression signatures in the efferent and afferent nerve fibres during development
had common characteristics, there were demonstrable differences between the two types of nerve fibres. Such
differences were observed in the glutamatergic and dopaminergic synapse pathways. For instance, the significant
differential expression of proteins in glutamatergic synapses was first detected at 1W in efferent nerve fibres. In
contrast, this distinctive change in expression was first observed at 3W in the afferent nerve fibres. The significant
differential expression of proteins at dopaminergic synapses was first detected at 1W in efferent nerve fibres, and
at 5 W in the afferent nerve fibres (Fig.?5C,D).
Cascade Regulation of Differentially Expressed Genes Involved in Efferent and Afferent Nerve
Fibre Development. To further identify key genes differentially expressed between the efferent and
afferent nerve fibres at each developmental time point, gene network diagrams were created using the IPA database
(Fig.?6). We found that Gabra1, Gabrb2, and Slit2 were differentially expressed between efferent and afferent
nerve fibres as early as P0, and that multiple types of GABA receptors (GABARs) were consistently differentially
expressed at later developmental stages. For instance, Gabra1, Gabra2, and Gabrb2 were found to be differentially
expressed at 1 W; Gabra3 and Gabrb1 were still differentially expressed at 3 W; and Gabra2 and Gabra3 were
consistently differentially expressed between the efferent and afferent nerve fibres during development. Intriguingly,
the neurexin family members Nrxn1 and Nrxn2 were significantly differentially expressed between the efferent
and afferent nerve fibres at 1W, and Nrxn1 was differentially expressed even at later developmental stages. Nlgn1,
which encodes neuroligin 1, the post-synaptic binding partner of neurexin, was differentially expressed between
the efferent and afferent nerve fibres at 3W and 5 W. This suggests that the developmental signature of the
neuroligins was different from that of the neurexins during efferent and afferent nerve fibre development.
Dynamic Changes in Differentially Expressed Genes Associated with Motor and Sensory Nerve
Fibre Development. The regulatory networks for the genes differentially expressed during motor and
sensory nerve fibre development were further analysed using the IPA database. The gene network diagram suggests
that the differentially expressed genes were in highly correlated clusters in well-regulated networks during
development (Fig.?7). During the motor nerve fibre development, Gabrg2, Gabrb2, and Nrxn1 were dramatically
downregulated at 1 W when compared with P0. This downregulation was sustained thereafter. Notably, a larger number
of GABA receptors, such as Gabra1, Gabra4, and Gabra5, were further downregulated during motor nerve fibre
development from 3 W to 5 W. Nlgn1 was involved in motor nerve fibre development from 3 W to 5 W. In
comparison, the slit guidance ligand (SLIT) family members Slit2 and Slit3 were downregulated in the extracellular space
in sensory nerve fibres at 1 W when compared with P0. These mRNAs interacted with roundabout homologue
1 (ROBO1), which was downregulated in the plasma membrane. In addition, GABAR family members, such
as Gabra3, Gabrb2, and Gabrg3, were down-regulated in sensory nerve fibres at 1 W when compared with P0.
However, changes in the expression levels of these proteins in sensory nerve fibres were not as obvious as those
observed in motor nerve fibres during development. For instance, the Gabrb2 was dramatically downregulated
together with Nrxn3 at 3 W and 5 W, while Gabra3 was only upregulated at 5 W. Furthermore, Nrxn2 and Nrxn3,
but not Nrxn1, were downregulated together with Nlgn3, which is involved in sensory nerve fibre development.
RT-qPCR Validation of Key Genes Associated with Motor and Sensory Nerve Fibre Development.
Several genes were considered representative regulatory genes. These genes were selected from the microarray
analysis data for RT-qPCR validation. They included Mpz, Slit2, Unc13c, Ncam1, Nrxn1, Nlgn1, Gabrb2, Gabra1,
and Npy. The heat map in Fig.?8A shows the expression pattern for each selected gene in all 24 samples (8 groups
in triplicate). The RT-qPCR results are summarized in the histograms (Fig.?8B). Slit2 mRNA expression in afferent
nerve fibres was significantly up-regulated when compared with that in efferent nerve fibres at P0, although this
trend was reversed thereafter. Nrxn1 mRNA expression in afferent nerve fibres was significantly up-regulated
when compared with that in efferent nerve fibres at P0. Interestingly, this differential expression pattern was even
more obvious than that for Slit2 because Nrxn1 mRNA expression levels were decreased during development in
efferent nerve fibres. Coincidentally, the expression patterns of Nlgn1, Gabrb2, and Gabra1 mRNA were similar
to that of Nrxn1. These similarities indicated that the above genes may have essential roles in motor and sensory
nerve fibre development. The RT-qPCR results were consistent with the data from the microarray analysis.
Under normal physiological circumstances, neurons maintain proper functioning condition by transporting
subcellular components to growth cone terminals via axoplasmic flow. However, when the continuity of axons is
completely disrupted, the distal stump of the nerve trunk initiates a process known as Wallerian degeneration22,23.
When injured neurons have achieved homoeostasis after trauma-induced disruptions, the intrinsic neuronal
regenerative capacity is activated to prompt the proximal stump of the nerve trunk to initiate the
regenerative programme. The key neuronal element responsible for axonal regeneration is the growth cone, which is a
specialized structure in both motor and sensory neurons24,25. Generally, growth cone navigation for achieving
synaptic rearrangement exhibits a molecular correlation with neuronal differentiation/maturation26, indicating
that the regenerative procedure may be fundamentally similar to the process of axon sprouting in the developing
Peripheral motor and sensory nerves have distinct functions that contribute to the maintenance of normal
physiological activities, especially their unique abilities to accurately and selectively reinnervate terminal nerve
pathways. Recent studies have demonstrated that Schwann cells obtained from different anatomical contexts can
express distinct motor and sensory phenotypes in cell culture systems27. However, other evidences have indicated
that it is the artificial culture condition that altered the gene expression patterns, and resulted in the distinct
phenotypic characteristics of differentially expressed genes28. In addition, isobaric tags for relative and
absolute quantitation-based analysis of total protein levels in different types of nerves in a quantitative proteomics
approach revealed that different motor and sensory nerves have distinct protein expression profiles in naive rats29.
In the in vivo regeneration model, the growth factor expression profiles between denervated motor and sensory
nerves had obvious differences, further indicating that Schwann cells express distinct motor and sensory
phenotypes that specifically benefit motor and sensory axonal regeneration, respectively11,30.
In order to decipher the cellular and molecular events during motor and sensory neuron development,
microarray analysis was conducted to identify differential gene expression patterns in both efferent and afferent
nerve fibres during different developmental processes. Considering the genders of patients who have peripheral
nerve injury in the clinic, both sexes of the C57BL/6 mice were used equally across the analyses. In this study, the
efferent nerve fibres mainly comprise axons of motor neurons whose cell bodies are located in the ventral horn
of the spinal cord, while, the afferent nerve fibres mainly comprise the central branches of axons extending from
DRG neurons that are biochemically and anatomically comparable to the efferent nerve fibres. In the peripheral
nerve fibres, myelinating Schwann cells inevitably remain in the microarray samples collected using conventional
dissecting methods31,32, which may also play important roles in peripheral neural development and regeneration.
We will further discuss these phenomena with respect to the involvement of Schwann cells as well.
Summarizing the GO analysis results, genes involved in axon guidance, cell differentiation, and GABAA
receptor activity subcategories are significantly differentially expressed at all the four time points examined,
indicating that the fundamental molecular and cellular mechanisms underlying the regulation of motor and sensory
nerve differentiation/maturation may be biologically distinct. Commissural axons grow along stereotyped
dorsoventral trajectories in the early developing spinal cord. The projection patterns are encoded by the
spatiotemporal distributions of axon guidance cues, specifically netrin-1/ DCC signalling for ventral attraction and SLIT/
ROBO signalling for dorsal repulsion33. The SLIT proteins are best known for their classic roles in mediating
chemorepulsion through the ROBO receptors during axon guidance34. However, accumulating evidence
indicates that the functional repertoire of SLIT proteins and ROBO also plays essential roles in neurogenesis, stem
cell regulation, and organ development35. It has been reported that SLIT2 contributes to branching/arborisation
of sensory trigeminal axons in the developing mammalian CNS36. Intriguingly, several groups have reported
that SLIT1 is only expressed in peripheral neurons, while other SLIT family members, such as SLIT2 and its
receptors ROBO1 and ROBO2 are also expressed in Schwann cells37, and SLIT2 functions as a repellent in
cultured Schwann cells via Ras homologue gene family member A-myosin signalling pathway38. In this study, Slit2
mRNA expression was found to be higher in sensory nerve fibres than in motor nerve fibres at the P0
developmental stage. This is consistent with previous reports describing Slit expression patterns in the embryonic spinal
cord. Intriguingly, this developmental characteristic changes after 1 W. The Slit2 mRNA expression level was
dramatically up-regulated in motor nerve fibres from 1 W to 5 W, so that its expression in motor nerve fibres was
significantly higher than that in sensory nerve fibres at the 3W and 5 W developmental stages. The differential
expression patterns of SLIT proteins in efferent and afferent nerve fibres led to our speculation regarding the
specific regulatory role of SLIT/ROBO signalling in motor and sensory neuronal differentiation/maturation across
different developmental stages in mice.
Trans-synaptic interactions involving neuroligin-neurexin complexes are also implicated in the organization
of excitatory glutamatergic and inhibitory GABAergic synapses, and play fundamental roles in the regulation
of synaptic cell adhesion39. Recent studies have indicated that glutamate plays an essential role in sensory input
transduction, especially in the nociceptive afferent signalling pathways. Peripheral neuropathic pain and the
involvement of inflammatory processes could be attenuated by pharmacologic manipulation of the
glutamatergic system40. Neurexins constitute a family of polymorphic cell-surface proteins with hundreds of alternatively
spliced isoforms that are essential to the synapse architecture. Recent studies have indicated that distinct spliced
isoforms of neurexins are selectively expressed in particular neurons, implying that each type of neuron may have
a unique neurexin expression pattern41. Notably, ?-neurexins are not strictly limited in their function as synaptic
terminal-specific proteins but are also involved in axon-Schwann cell and perineurial fibroblast interactions42. In
addition, contactin-associated protein (CASPR)/paranodin, which is a vertebrate homolog of neurexin IV, has
been reported to localize at the paranodal region of the Ranvier nodes; CASPR/paranodin links neuronal
membrane components with the axonal cytoskeletal network and Schwann cells43. In this study, neurexin 1 was found
to be significantly differentially expressed between efferent and afferent nerve fibres in the postnatal
developmental stages from 1 W to 5 W, suggesting that neurexin 1 may serve as a central hub gene involved in modulating
postnatal sensory neuron development. It is noteworthy that neurexin 1 is predominantly expressed both in
motor and sensory axons at P0, implying that neurexin 1 plays fundamental roles in embryonic neural
development. Neuroligins are thought to be necessary for maintaining synapse integrity, and function through
recruitment of GABARs and ionotropic glutamate receptors. The microarray results indicated that neuroligin 1 mRNA
expression level gradually decreased as motor neuron development proceeded, and that the significant differential
expression between motor and sensory neurons was only detected at relatively mature stages (postnatal 3 W and
5 W). These data suggested that neuroligin 1 may follow a different modulating mechanism than its trans-synaptic
partner neurexin 1 in regulating motor and sensory neuron development.
The principal inhibitory neurotransmitter, GABA, is broadly distributed in both CNS and PNS. Accumulating
evidence indicates that the formation of GABAergic synapses is crucial for the formation and maintenance of
stereotypic neural networks during CNS development44. Moreover, recent evidence has confirmed that GABAergic
inputs occur in the mouse embryonic spinal cord as early as at the embryonic age of 12.5 days, implying that
GABAergic functions may be essential at very early developmental stages in mice45. The GABA-induced
activities are mediated via its receptors, chloride-permeable ionotropic GABAA and GABAC receptors, as well as
G-protein-coupled metabotropic GABAB receptors46. Interestingly, accumulating evidence demonstrates that
the myelin-producing Schwann cells also express GABAA and GABAB receptors during neural development
and regeneration, and that GABAA and GABAB receptors cross-interact with neuroactive steroids47,48 via
activation of Src and phospho-focal adhesion kinase signalling49. The GABAB receptors are involved in myelination
in Schwann cells but not in their proliferation50, and GABAB receptors are fundamental in regulating Schwann
cell maturation and small nociceptive C-fibres51,52. The microarray data in this study indicated that Gabra1 and
Gabrb2 levels were dramatically decreased in motor and sensory nerve fibres after 1W. This was especially
obvious in motor neuron development, consistent with previous reports that GABAergic activity contributes to the
early stages of CNS development. Moreover, Gabra1 and Gabrb2 were significantly differentially expressed only
at relatively early developmental time points (P0 and 1 W). To complicate matters more, other GABAR subtypes
have been shown to be implicated in neuronal differentiation/maturation events in the following developmental
windows (3 W and 5 W), further indicating that GABA and its various receptors have substantial roles in
determining motor and sensory neuronal cell fate specification across development in mice.
The current findings reveal fascinating aspects of differential gene expression signatures between
peripheral efferent and afferent nerve fibres at different developmental stages in mice. The precise and balanced
trans-synaptic cell adhesion system is crucial for the establishment of functioning neuronal circuits. Thus,
accurate reinnervation of motor and sensory original targets requires appropriate synaptic plasticity, which is needed
to fine-tune neural development and regeneration. Generally, the physiological and molecular mechanisms that
modulate peripheral nerve regeneration are most intensively studied in the nerve injury model. These data serve
as valuable resources, and provide a new perspective regarding the possible cellular and molecular mechanisms
underlying motor and sensory development. Further in-depth studies are needed to identify the hub genes and
key molecular events that regulate peripheral motor and sensory neuronal cell fate determination.
Separation of Efferent and Afferent Nerve Fibres. C57BL/6 mice at different developmental stages
(P0, 1 W, 3 W, and 5 W) were supplied by the Experimental Animal Center of Nantong University. All animal
protocols were approved by the Committee of Nantong University, and were performed in accordance with the
guidelines of the Administration Committee of Experimental Animals, Jiangsu Province, China.
The animals (n= 9 for each developmental time point) were sacrificed using an overdose of CO2 by inhalation.
The lumbar vertebral canal was opened, and the spinal cord was exposed. After the spinal arachnoid was carefully
peeled off under a dissecting microscope, the efferent and afferent nerve fibres arising from spinal cord
segments L4?L6 were dissected and separately collected into 8 tubes containing ice-cold D-Hank?s solution (Gibco,
Carlsbad, CA, USA) corresponding to the 8 groups (four time points and two types of nerve fibres). Tissues were
then cut into small pieces (no larger than approximately 1 mm3) and transferred into liquid nitrogen, where they
were stored until further processing.
RNA Isolation and Microarray Hybridization. Samples were electronically homogenized on ice and
then centrifuged at 13,200 rpm for 15 minutes at 4 ?C. The resulting supernatant was collected and used in the
following steps. Total RNA was isolated using mirVana RNA Isolation Kit (Ambion, Austin, TX, USA) according
to the product manual. After the total RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA;
product number [p/n]: 74104), its quality was determined based on RNA integrity number (RIN) obtained using
a bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Samples with RIN values ?7 were used
for further procedures.
The cDNA was labelled using Cyanine-3-CTP Quick Amp Labeling Kit (Agilent Technologies, p/n: 5190?2305).
Agilent SurePrint G3 Mouse GE V2.0 (8 ? 60 K, Design ID: 074809) microarrays (Agilent Technologies) were
hybridized using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, p/n: 5188?5242). The
arrays were then scanned using an Agilent Scanner G2505C (Agilent Technologies). Agilent feature-extraction
software (version 10.7.1.1) was used to analyse the microarray images and obtain raw data. Further, GeneSpring
GX software (version 12.5) was used to complete the basic analysis. The raw data were normalized using the
quantile algorithm. The R software (version 2.13.0) was used for deeper analysis of the microarray data. The limma
(linear regression model) package was used to statistically analyse differentially expressed genes.
The microarray analysis was performed by Shanghai OEBiotech Technology Co., Ltd. (Shanghai, China). For
each group, data from three independent experiments were assessed to ensure reproducibility. All data (raw and
normalized data) was Minimum Information About a Microarray Experiment-compliant, and has been
deposited to the National Center for Biotechnology Information database (accession number: GSE113820). Genes
and pathways with p value < 0.05 and a mean expression fold change greater than 2 were considered statistically
Bioinformatic Analyses. Principal Component Analysis, Z-scores, and Hierarchical Clustering. We
performed PCA using the ?Population PCA? tool on the Harvard Medical School webpage. The Z-scores (standard
scores) were calculated and hierarchical clustering was performed based on log2-transformed mean-centred
datasets, as described previously17. Hierarchical clustering analysis was performed using Multi Experiment
Viewer software (version 4.9). In addition, the STEM software (version 1.3.11) was used to visualize the
enrichment analysis of the temporal expression profiles of differentially expressed genes.
Gene Ontology Analysis. The differentially expressed genes were classified using the following GO categories:
Biological Processes, Cellular Components, and Molecular Functions. Database for Annotation, Visualization,
and Integrated Discovery (DAVID) bioinformatic resources were used for further analysis of GO category
enrichment53, and the expression profiles for each GO subcategory were calculated as previously described17,23.
Kyoto Encyclopedia of Genes and Genomes Pathway. The KEGG bioinformatics database was integrated with
DAVID tools, and used to systematically screen differentially expressed genes, essentially as previously described23.
Ingenuity Pathway Analysis. The IPA (Ingenuity Systems; www.ingenuity.com; Redwood City, CA, USA) is an
online software package used to identify canonical pathways and gene networks, and to further categorise specific
physiological processes. The Ingenuity Pathway Knowledge Base was used for deep analysis of the global
molecular network, and revealed interactions among the differentially expressed genes.
Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction. We performed
RT-qPCR analysis of the mRNA expression levels of the differentially expressed genes identified in the microarray
analysis, including Mpz, Slit2, Unc13c, Ncam1, Nrxn1, Nlgn1, Gabrb2, Gabra1, and Npy, to validate the microarray
results. A total amount of 0.5 ?g RNA from the peripheral nerve fibre samples was used as a template to perform
RT-qPCR. The reverse-transcribed cDNA was synthesized using the Prime-Script Reagent Kit (Takara, Dalian,
China); PCR was performed using SYBR Green Premix Ex Taq (Takara) on the StepOne Real-Time PCR System
(Applied Biosystems) in triplicate for each sample. The relative mRNA level was calculated using the
comparative 2???Ct method (normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA [Gapdh] levels). The
sequences of the primer pairs are listed in Table?S1.
Statistical Analysis. All quantitative data are presented as mean ? standard deviation. The data were
analysed using SPSS 11.5 software (Chicago, IL, USA). Statistical analysis was performed using one-way analysis of
variance, followed by Scheffe?s post hoc t-tests; p values <0.05 were considered statistically significant.
We appreciate the financial support from the National Key Research and Development Program of China (No.
31730031), National Natural Science Foundation of China (Nos 81771999 and 81401796), and Six Talent Peaks
Program of Jiangsu Province (WSW-038).
X.T. and X.G. designed the research; H.W., Y.Z., M.C., L.Z. and X.T. performed the experiments; H.W., Y.Z., X.G.
and X.T. analyzed data; X.T. wrote the paper.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-30463-0.
Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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