The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis.
Neural stem cells (NSCs) acting as a source of various cell types are a subpopulation of cells that can self-renewal and proliferate identical cells. They are multipotent to generate diversity neural lineages, encompassing neurons, astrocytes, and oligodendrocytes [
The elemental discrimination between embryonic and adult neural stem cells is that the process of adult NSC is not orchestrated and massively paralleled progression as that in the embryonic developmental stages because such stages can occur at any time point [
NSCs, a headspring of progenitor cells in the central nervous system (CNS), are born with proliferation capacity of self-renewal and generation of both neurons and glia through a multistep process [
With the various technologies development, a quiet number of molecular biomarkers have been emerging like mushrooms after rain, which will favor the further research in the neuroscience field. However, there is not a systematic framework to illustrate the specific markers’ detailed characters and functions. And our summary is tempting to provide such a commentary on these particular cell types for the best use of these powerful cells.
During the embryogenesis, there are two crucial proliferative zones: ventricular zone (VZ) and subventricular zone (SVZ), which are the springheads of cortical neurons and glia cells [
The schematic of embryogenesis and the specific markers expressed in specific time-line. (a) The process of embryogenesis. With the beginning of neuroepithelial cells, a series of cell types are produced, including radial glial cells, neurogenic intermediate progenitor cells, oligogenic intermediate progenitor cells, neurons, and astrocytes. (b) The specific markers indicate the specific cell types generated during the process of neurogenesis [
NSCs in the VZ divide symmetrically and asymmetrically to preserve the stem cell pool and generate progenitor cells, which subsequently migrate to SVZ and then perform the capability of proliferation or differentiation [
The embryogenesis originates from the neural plate which is composed of neuroepithelial cells (NECs). Initially, the NECs divide symmetrically to amplify their own cohorts which are identified as the earliest form of embryonic NSCs [
Given the general frame of embryogenesis, we provide a subsection of this process to get a detailed understanding. The mouse cerebral neocortex can be factitiously partitioned into 6 layers horizontally, each of which contains a specific subpopulation of cells distinguished by singular or multiple markers identifying the characteristics functionally and molecularly [
And at E11.5, the cortical projection neurons present firstly in PP layer and migrate to establish the seminal cortical plate (CP), which whereafter progress to form L2–L6 layers. Before the program of embryonic neurogenesis being launched, the neural progenitor cells (NPCs) in VZ divide symmetrically to amplify the neural progenitor pool. At around E11.5, NPCs get down to divide asymmetrically for self-renewal and to produce neurons which will subsequently migrate to mantle layers (MZ) along the scaffold acted by radial-glia cells (RGCs).
The projection neurons formed at the initial stage locate at the PP and build the nascent CP, which will thenceforth convert into the neocortex L2–6. The increasing CP neurons subsequently crack the PP into SP and MZ. With the neurogenesis progressing, many projection neurons are created in sequence through the continuous asymmetric divisions of NPCs. Gathered together, neurons residing in SP are formed firstly, then those locating at deep layers, and at last those locating at the upper layers (L4, 3, and 2).
The formation of neocortex composed by neurons starts with deep layers; then the newborn neurons will migrate across the older ones to build upper layers. A part of daughter cells of NPCs transform into the IPCs, which will migrate away from VZ and go through symmetric divisions in SVZ, contributing to upper-layer neurons. At around E17.5, closing to the end of neurogenesis, the NPCs convert into gliogenesis, which produce cortical and subependymal zone (SEZ) astrocytes and form the layer of ependymal cells (EL) layer.
During the procedure of embryogenesis, different cell lineages generate different cell types in the different time-courses. And the specific cell types show exclusive cell surface proteins which can be applied to discriminate the particular cell type in specific stage from the NSCs pool. According to the characters above, various cell surface proteins can function as cell markers. And abundant biomarkers have been reported to identify the different cell lineages and different time-courses. Here, we listed the major ones for an illustration.
Emx2, empty spiracles homolog 2, belongs to a homeobox-containing gene [
Sox5, sex determining region Y-box 5 (Sox5), is a transcription factor belonging to Sox family. It can be detected exclusively in postmitotic neurons of SP and in projection neurons of L6 at high level and in a cohort of L5 projection neurons. And the detectable phenotype persists from E14.5 to P7 (postnatal 7 days). Meanwhile, Sox5 can also be detected in a few of upper-layer neurons at low levels [
Otherwise, investigation reported that Sox5 is absent in the progenitor cells residing in VZ and SVZ [
Bcl11b (also called Ctip2), B cell leukemia/lymphoma 11B, is a zinc finger transcription factor, which takes part in the development of L5 subcortical axon and is exclusively expressed in L5 [
Tbr1, T-box brain factor 1, is a transcription factor, which cooperates with Sox5 to regulate early born neurons in multiple lines during the embryonic development. The deletion of Tbr1 inmice indicated that it is necessary for numerous processes in cortical development, such as laminar location, molecular differentiation, and axonal expeditions [
Fezf2 (also known as FEZL, ZFP312) belongs to FEZ family zinc finger 2, which also functions as a transcription factor. Fezf2 can be found in L5 cortical spinal (CS) neurons at a high level and plays a pivotal role in the CS tract development. Fezf2 is also found enriching in early progenitors of VZ and in their neuronal progenies which launch into the deep-layer of subcortex. Yet it disappeared in late progenitor cells and upper-layer neurons. Fezf2 is downregulated in L6 neurons during the late embryonic development [
Satb2, special AT-rich sequence-binding protein 2 (DNA-binding protein), is a matrix-attachment region interacting transcription factor, which can exclusively conjunct the nuclear matrix-attachment regions and plays a role in regulating the transcription and remodelling chromatin. It regulates the position of laminin and helps to identify the late-born neurons. It enriched the postmitotic neurons of corticocortical L2–L5, which begins to emerge in the E13.5 neurons when these neurons migrate into the IZ (intermediate zone) and will persist postnatally. Yet, in the subcortical projection neurons, it is not expressed [
Cux1/Cux2, cut-like homeobox 1/2, are upper layer-specific markers for neurons, which participate in the fundamental regulation of the late neuronal differentiation, spine formation, dendritic branching, and synaptogenesis in upper-layer (L2-3) neurons of the cortex [
Pou3f2 (POU class 3 homeobox 2, also called Brn-2) and Pou3f3 (POU class 3 homeobox 3, also called Brn-1) both are the members of the class III POU family transcription factors involved in neural differentiation [
Pax6, paired box 6, plays a pivotal role in the neuronal fate determination and NSCs proliferation. It participates in the neocortex positioning and upper-layer neurons generation via identifying the SVZ progenitor cells [
Nr2f1 (also known as Coup-tf1), nuclear receptor subfamily 2, group F, member 1, plays a crucial role in the neocortical regionalization. And the late-born neurons migration and the callosal projection neurons (CPNs) differentiation are modulated by Nr2f1 [
Sox1 is expressed exclusively in the CNS and probably functions as the earliest marker for neural fate decision of embryonic stem cells. Furthermore, it marks the proliferating progenitors residing in the neural tube [
Adult neural stem cells are peculiar cell subpopulations with the character of structural plasticity [
The schematic of adult neurogenesis and specific markers for specific cell types in different time-courses. (a) A model of the two major NSCs niches (labeled by the red panes) in the adult brain. (b) The process of adult neurogenesis originates from the active radial glial cells RGCs, (type-1 cells), generating intermediate progenitor cells (IPCs, type-2a, type-2ab, and type-2b cells), subsequently immature neurons, and finally mature neurons. The specific cell types emerging in the certain strategies are traced by various special markers [
Radial-glia-like neural stem/precursor cells existing in the SGZ are usually regarded as relatively quiescent but can be activated by internal and external stimulus. They compose a pool of neuroblasts including transit-amplifying and proliferative cells produced by symmetrically and asymmetrically dividing. Only a small bunch of cells in this pool can survive and differentiate into immature neurons. After the postdivision of 7–10 days, cells start to enter a neuronal fate [
Radial-glia cells act dual-status during CNS development. On the one hand, they play as neural progenitor cells for neuronal generation and a scaffolding facilitating neuronal migration. And on the other hand, they act as a source of most neurons life-long in the CNS [
Adult neurogenesis in the hippocampus germinates from progenitor cells and leads to the birth of granule cell neurons, which goes through approximately six distinct stages experiencing type-1 cells, type-2a cells, type-2b cells, type-3 cells and immature and mature neurons [
NSCs can be defined as a cohort that possesses both self-renewal and neurons/glia cells production from a unicell according to their potential capacities [
During the process of neurogenesis, a number of diverse biomarkers can be utilized by immunohistochemical means labeling the specific cell types in diverse states. Following statements will delineate the developmental procedures of neurogenesis with the key identifications, accompanied with generally utilized stage-specific markers.
With the progress of adult neurogenesis, a series of peculiar cell lineages emerge in turn. Here, we particularize the major biomarkers according to the following capabilities: proliferation, neurogenesis, and gliogenesis.
PCNA, proliferating cell nuclear antigen, is important in both the repair and the replication of DNA. The expression of PCNA is increased during the G1 and S phases and decreased upon the cell converting into G2 and M phases. Nevertheless, this marker can also be detected in the early G0 phase, which is caused by the long half-life period of eight to twenty hours [
Ki67, also known as MKI67, is a nuclear protein, which can be used as a marker for dividing cells. It can be found in all time-courses of cell cycle but G0 and early G1 phases and the same to quiescent cells [
PH3, phosphohistone H3, is expressed at the late stage of G2 phase and the entire course of M phase during cell division [
BrdU (5′-bromo-2′-deoxyuridine), thymidine analog, is usually used to label cells being in the cell cycle of S phase in both embryonic and adult dividing cells. However, BrdU uniquely labeling, without additional markers, is only a prevalent symbolization for neurogenesis. BrdU-positive cells may indicate a subtype of progenitors for new neurons but not progenitors for neuronal cells [
MCM2, minichromosome maintenance protein 2, is associated with the DNA replication. It is expressed at the early stage of G1 phase and is sustained throughout cell cycle. Besides, it can be detected in proliferating cells without DNA synthesis too. Its expression level is thus higher than Ki67 which is the short-lived cell proliferation marker [
GFAP, glial fibrillary acidic protein, is an intermediate filament (IF) protein which works as a holder of astrocyte mechanical strength. It is a well-known marker for astrocytes [
BLBP, brain lipid binding protein (BLBP), also called B-FABP or FABP7, is subjected to a member of fatty acid-binding proteins (FABPs) family, which are cytoplasmic proteins undertaking fatty acid intake, transportation, and targeting [
Sox2, known as SRY (sex determining region Y)box 2, is a member of Sox family of transcription factors. It encodes a highly conserved DNA-binding motif identified as HMG (high-mobility group) box, playing vital roles in distinct stages of mammalian development. Sox2, presenting a high expression in embryonic stem cells and adult NSCs during development [
On the other hand, Sox1 is also expressed in adult neural progenitor cells and multipotent neural stem cells
PSA-NCAM, polysialylated neural cell adhesion molecule, is highly expressed in neural progenitor cells or mostly glial progenitor cells during brain development [
NeuroD (neurogenic differentiation) is a transcription factor belonging to the family of basic helix-loop-helix protein. It is presented in later strategies of neuronal progression and is classified as a differentiation indicator for neurogenesis, which may serve as a neuronal determination gene. It can also serve as a specific marker of adult cells in SGZ and inner granule layer [
PAX6, paired box 6, is a member of the paired box (Pax) family. As a conserved transcription factor with different DNA-binding domains (PD, a paired domain, and HD, a paired-type homeodomain), Pax6 is intensely expressed in the cells originating from the embryonic neural development and adult neurogenic niches [
As a transcription factor, Pax6 emerges in embryonic progenitor cells and participates in the cell proliferation and the determination of neuronal fate as a pivotal regulator [
FoxO3 (forkhead box O3) is a transcription factor belonging to the O subclass of the forkhead family which are identified by an evident fork head DNA-binding domain. FoxO factors can lure a series of cellular responses, involving the arrest of cell cycle, cell differentiation, apoptosis, and the opposition to oxidative stress [
Nestin, neuroepithelial stem cell protein, is a sort of intermediate filament protein involved in classes VI and IV, which is expressed transiently in adult NSCs, immature neural progenitor cells, and vanishes upon the cells converting into differentiation [
In the adult mouse brain, nestin-positive cells can be observed extensively in restricted regions, where they might serve as a niche of stem/progenitor cells with the capacity of proliferation and differentiation [
TLX, an orphan nuclear receptor (also called NR2E1), is encoded by the tailless (TLX) gene. It can be found in both neural stem/progenitor cells (quiescent NSCs) and transit-amplifying neural progenitors (active NSCs) in the SVZ (subventricular zone) and SGZ (subgranular zone) of adult mouse brain [
TLX is uniquely expressed by astrocyte-like B cells of SVZ. And the deletion of TLX gene may result in an absolute loss of SVZ neurogenesis and the deprivation of NSC property of GFAP-positive cells [
The bHLH is a basic helix-loop-helix protein transcription factor family, which regulates vertebrate neurogenesis, showing the capacity of transforming nonneuronal fate to neuronal fate when it is expressed ectopically [
Hes5 is a member of Hes genes (mammalian homologues of Drosophila hairy and Enhancer of split genes) which can encode a series of basic helix-loop-helix (bHLH) transcriptional repressors. There are three conserved domains (bHLH, Orange, and WRPW domains) involved in Hes5 factor, through which Hes5 factor can set up transcriptional activities [
Mash1, mammalian achaete-scute homolog (also called Ascl1), is a bHLH transcription factor, which is essential for embryonic neural differentiation [
REST, RE1-silencing transcription factor (also known as NRSF), is the GLI-Kruppel class C2H2 zinc finger protein expressed in various neuronal genes [
DCX, doublecortin, a protein facilitating microtubule polymerization, is expressed in migrating neuroblasts and immature neurons, which can be classified as a marker for adult neurogenesis in SGZ. However, not all newly born neurons express DCX. It can be found in newly generated hippocampal, striatal, and olfactory neurons, but not in newly generated neurons in the neocortex [
Vimentin and GFAP are the two main intermediate filament proteins which imply the property of glial cells. And vimentin is mainly exhibited in the radial-glia and immature astrocytes of the early brain development and vanishes at the terminal of gestation. Simultaneously, GFAP presents in the astroglia cells instead of vimentin. However, Seri’s research also suggests that vimentin is expressed in both radial-glia and horizontal cells in SGZ [
S100
GLAST (also known as EAAT1) is astrocyte-specific glutamate transporter and GLT1 (also known as EAAT2) is glutamate transporter; both are defined as markers of glial group [
Tbr2, T-box brain gene 2, is a member of the mammalian brain-specific T-box gene family, which was expressed in a couple of regions in the developing brain. Yet, it is absent in most regions of adult brain but hippocampus and OB [
NeuroD, the basic helix-loop-helix protein, is a transcription factor, which has been parted into the group of markers for differentiated cells during neurogenesis in the SGZ [
Tuj1, neuron-specific class III
Prox1, prosperorelated homeobox gene 1, is necessary for the preservation of IPCs (intermediate progenitor cells) and is needed to promote granule cells to maturate during the procedure of adult neurogenesis [
CNPase, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, presents specifically in oligodendrocytes in SVZ and SGZ, whose morphology reflected by the immunostaining refers to the beginning of oligodendrocyte differentiation. Furthermore, it is considered to play a leading role in the formation of myelination [
Subventricular zone (SVZ) is the other NSCs pool of the two restricted regions. The SVZ mainly consists of four main cell types: SVZ astrocytes/NSCs (type B cells), intermediate progenitor cells (type C cells), neuroblasts (type A cells), and ependymal cells [
And the slowly dividing astrocytes (B cells) continue to produce precursor C cells (intermediate progenitor cells), and C cells can produce the specific lineage A cells (neuroblasts) which will migrate along the RMS (rostral migratory stream) to the OB (olfactory bulb) where they finish their destined mission and convert into granule neurons [
GFAP specifically marks the NSCs and astrocytes residing in the SVZ but does not mark the subgroup of TAPs (transient amplifying progenitor cells) [
Sox2 marks the active astrocytes (NSCs) and neural progenitors (TAPs) of SVZ [
Besides the markers mentioned above, a number of biomarkers reported in abundant investigations are remaining. Here, we enumerate some of them that were not illustrated in this review.
Given so abundant investigations and reviews by so many prominent scientists, we can now sketch the outline of various biomarker functional characteristics and latent application in multiple aspects. Gathering all of the formulations above, we catch a sight of many milestones established in the development of neurogenesis as well as obstacles being unfathomed.
Neural stem cells (NSCs) are the fundamental source of all cell types in the CNS. To grasp the nature of niche components in the mouse brain, we need to understand the major characteristics and functions of each element in the niche, to comprehend what role each cellular member plays in the procedure of NSCs maintenance, proliferation, and differentiation. However, it is difficult, using a single marker, to identify a single cell lineage exclusively. And thus it is necessary to apply multiple markers to the analysis of one peculiar cell type. Recently, numerous methods and technologies have been emerging to classify and identify the specific cell types during the neurogenesis of both embryonic and adult brains. Nevertheless, each approach possesses shining points and flaws. We need more concerns to consummate them.
The authors declare that there is no actual or potential conflict of interests to this work, including any financial, personal, or other relationships with relevant people or organizations.
This work was supported by the Ministry of Science and Technology of China (2014CB964903) and Strategic Priority Research Program (XDA01020301). The work submitted has not been published previously, and it is not under consideration for publication elsewhere.