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| A flow cytometric approach for the isolation and characterization of primary and motile cilia. Flowchart of the experimental procedure for cilia identification and characterization according to size and marker expression. The * symbol represents a conjugated fluorophore. (A,B) FACS plots showing the distribution of reference beads and a sample of cilia according to forward (FSC) and side (SSC) scatter. (C) Representative FACS plot illustrating the distribution of 2 µm particles upon immunostaining with antibodies directed against adenylate cyclase type iii (AC3; AC3-FITC) and prominin-1 (Prom-APC). The gates indicate AC3 positive (AC3+), Prominin positive (Prom+) and double positive (DP) particles.
Source publication
In the adult mammalian brain, the apical surface of the subependymal zone (SEZ)
is covered by many motile ependymal cilia and a few primary cilia originating from
rare intermingled neural stem cells (NSCs). In NSCs the primary cilia are key for the
transduction of essential extracellular signals such as Sonic hedgehog (SHH) and
platelet-derived gro...
Contexts in source publication
Context 1
... schematically illustrated in Figure 1, we took advantage of flow cytometry to set up a novel approach to identify and isolate primary cilia from the murine SEZ. We performed this method with samples obtained from the SEZ of adult mice, in which motile ependymal cilia are much more abundant than primary cilia, and the corresponding germinal area dissected from embryos at embryonic day (E) 18, when primary cilia represent the vast majority of the cilia. ...
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... SEM images of the apical surface of the lateral ventricle wall of E18 ( Figure 2B) and adult mice (Figure 2C), after deciliation or no treatment (Control), showed that the treatment was effective in removing primary but not motile cilia. In untreated embryonic tissue, primary cilia were readily visible (Figures 2Ba,a , highlighted in green) whereas, after deciliation, they were largely absent (quantification in Supplementary Figure S1A). Sometimes a remaining stump could be detected (Figures 2Bb,b , highlighted in green). ...
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... suggests that deciliation also gives rise to fragments of different sizes, as previously reported (Mitchell et al., 2009). In contrast, many tufts of ependymal cilia were still present after deciliation, (Figure 2C) although their number was significantly reduced (quantification in Supplementary Figure S1B). In order to distinguish motile from primary cilia we have taken advantage of differential expression of prominin-1 (Prom) and AC3 in the two cilia types. ...
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... whereas in motile cilia prominin-1 was expressed at consistently high levels ( Figure 3B, white arrow), primary cilia displayed high variability in expression levels, ranging from very high (Figure 3B, red arrow) to undetectable (Figure 3B, green arrow). To address variability in cilia length, ranging between 1 µm and 9 µm ( Dummer et al., 2016), we have used beads of known size to set the forward (FSC) and side light scatter (SSC) parameters (Figures 1A,B) and monitor changes in particle size during sorting. Although for bigger particles, like for example cells, the first parameter reflects cell size whereas the second indicates complexity (granularity), for smaller particles the measurement of both parameters provides a better detection of changes in size. ...
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... for bigger particles, like for example cells, the first parameter reflects cell size whereas the second indicates complexity (granularity), for smaller particles the measurement of both parameters provides a better detection of changes in size. Independent of size, we detected four types of particles: primary cilia particles immunopositive for either AC3 only (AC3 + ) or double immunopositive (DP) for both AC3 and Prom, motile cilia particles immunopositive for Prom only (Prom + ) and double immunonegative fragments (DN; Figure 1C). To further investigate their nature, the four populations of particles were separately collected by FACS, concentrated by ultracentrifugation and then analyzed by Scanning electron microscope (SEM) images of the apical surface of the lateral ventricular wall in whole mount preparations from E18 mice before (Control) and after deciliation. ...
Citations
... Whole lateral walls were dissected and fixed as described before (Mirzadeh et al, 2008;Monaco et al, 2019). In short, the mice were killed by CO 2 inhalation and subsequent cervical dislocation (adult) or by decapitation (E18), and the brain was dissected in a buffered sucrose solution. ...
... Immunostaining of brain sections and whole mounts was performed as previously described (Luque-Molina et al, 2019;Monaco et al, 2019). For antibodies used, please refer to Table S1. ...
We recently reported that Growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous Sonic Hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, GFD15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.
... Furthermore, mass spectrometric analyses of primary cilia are usually performed using cilia derived from cultured cells and by purifying them by ultracentrifugation in sucrose density gradients (Ishikawa, Thompson, Yates 3rd, & Marshall, 2012;Mitchell, Szabo, & Otero Ade, 2009;Narita et al., 2012), a lengthy and complicated procedure. Therefore, analysis of the protein content of the cilium has been slow, especially with cilia from specific animal tissues or subtypes of cilia, e.g., separating motile from primary cilia in the same sample (Monaco et al., 2019). ...
... Here we describe a method to isolate cilia from tissue, specifically from neural stem cells in the ventricular-subventricular zone (SVZ) from murine brain (Monaco et al., 2019). This method uses immunofluorescently labeled antibodies to identify ciliary particles shed from the tissue, which are then sorted by a flow cytometer. ...
... However, since the particle size is based on round objects and the process of flow cytometry damages the ciliary particles, the size is not an accurate representation of the actual length or thickness of individual cilia; the method can however be used to determine difference in size across populations, e.g. ependymal cilia (>10 μm) have a higher percentage of 8 μm particles than primary cilia (Monaco et al., 2019). Although the here described protocol was designed for cilia isolation and subsequent proteomic analysis such as Western Blot (Monaco et al., 2019), it may also be used to detect the amount of certain proteins present in the cilia, as well as changes in overall number. ...
Primary cilia provide a specialized subcellular environment favoring ordered and timely interaction and modification of signaling molecules, necessary for the sensing and transduction of extracellular signals and environmental conditions. Crucial to the understanding of ciliary function is the knowledge of the signaling molecules composing the ciliary compartment. While proteomes of primary cilia have been published recently, the selective isolation of primary cilia from specific cell types and whole tissue still proves difficult, and many laboratories instead resort to the analysis of cultured cells, which may introduce experimental artifacts. Here we present a flow cytometry-based method to isolate and characterize primary cilia from the murine ventricular-subventricular zone. After deciliation, primary cilia are immunolabeled with antibodies against ciliary markers. As an example, we here use a double-staining with acetylated tubulin, which stains the ciliary axoneme, and ciliary membrane protein ADP-ribosylation-like factor 13b (Arl13b); additionally, we triple-labeled primary cilia using the ciliary marker adenylate cyclase 3 (AC3). Besides analysis at the single particle level, fluorescence activated cell sorting (FACS) allows collection of pure preparations of primary cilia suited for subsequent proteomic analyses like mass spectrometry or western blot. As an example of analytical application, we performed triple immunostaining and FACS analysis to reveal cilia heterogeneity. Thus, our cilia isolation method, which can readily be applied to other tissues or cell culture, will facilitate the study of this key cellular organelle and shed light on its role in normal conditions and disease.
... A subset of quiescent NSCs in the adult V-SVZ also displays primary cilia (Khatri et al, 2014), and cilia signalling is upregulated in NSCs of the V-SVZ closely linked to cell cycle progression and signal transduction (Goto et al, 2013). Therefore, we next used adenylate cyclase 3 (AC3) as a primary cilia marker (Bishop et al, 2007;Monaco et al, 2019) in combination with Nestin and H2B-GFP to investigate the presence of primary cilia in apical and basal progenitors (Fig 2C-E). Ciliated progenitors represented a small subset of G + -tagged progenitors in the V-SVZ and they were more abundant at the apical side of the niche (Fig 2D). ...
... Since we here found that the majority of ciliated apical NSCs extend primary cilia, it is possible that cilia-dependent signals promote Nestin expression. In the V-SVZ, primary cilia do not only regulate quiescence in radial glia and adult NSCs (Beckervordersandforth et al, 2010;Khatri et al, 2014) but they are also key to sensing signals present in the cerebrospinal fluid filling the ventricular cavity (Silva-Vargas et al, 2016;Monaco et al, 2019). Consistent with this view, recent observations have highlighted the possibility that in the V-SVZ, acquisition of quiescence occurs over a prolonged time period (Borrett et al, 2020) and involves different stages as well as the integration of multiple regulatory signals. ...
... Whole-mount dissection and immunostaining were performed as described previously (Mirzadeh et al, 2008;Monaco et al, 2019). For DAPT treatment, freshly dissected E18 whole mounts were incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and with either a solvent control (dimethyl sulfoxide, DMSO) or 20 lM DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; Sigma-Aldrich, D5942) in DMSO, for 24 h at 37°C, 5% CO 2 . ...
According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone V-SVZ. Both processes will ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult-born OB interneurons largely derive from an additional NSC type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differs between the two NSC groups and Notch-activation is greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreased Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis was paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ.
... The cells separated using the above two methods were centrifuged at 1000 r/min for 3 min, then the supernatant was removed, and a DMEF/F12 medium (containing 2% B27, 20 μg/L of epidermal growth factor and 20 μg/L of basic fibroblast growth factors) was added. Thereafter, the cells were cultivated in a 37°C, 5% CO 2 incubator, then examined for the HNSC markers PE-CD133 (ab252128, ABCAM, UK), APC-Nestin (ab187-846, ABCAM, UK), HNCs marker PE-CD24 (ab25494, ABCAM, UK, 0.2 µg for 106 cells), and FITC-Tyrosine hydroxylase (TH, 10009396-1, Cayman, USA) using flow cytometry [19]. ...
Objective:
To explore the molecular mechanism by which hippocampal neural stem cell (HNSC) exosome (exo)-derived MIAT improves cognitive disorders in rats with vascular dementia (VD).
Methods:
Rat hippocampal tissues were collected, and HNSCs and hippocampal neuronal cells (HNCs) were isolated, purified, and identified. Then the exosomes (exo) of the HNSCs were extracted and identified. A VD rat model was constructed. HE staining was used to evaluate the hippocampal pathology in each group. The expressions of the RNAs in the HNSCs were intervened, and the cells were then grouped. ELISA was used to measure the of TNF-α, IL-1, and Aβ1-42 expression levels. The kits were used to determine the oxidative stress factor levels. The targeting relationships among MIAT, miR-34b-5p, and CALB1 were measured using dual-luciferase assays. The MIAT expressions in exo were measured using qRT-PCR. The proliferation and apoptosis of the HNCs were determined using CCK-8 and Annexin V-FITC/PI staining, respectively. The CALB1, TH, and Bcl-2 protein expressions were determined using Western blot. The Morris water maze test was used for the spatial learning and memory testing.
Results:
The hippocampal tissues in the model group were clearly damaged, but the pathological characteristics were significantly improved in the exo group. The exo group also showed an increased SOD level, decreased MDA and ROS levels, and down-regulated TNF-α, IL-1, and Aβ1-42 expressions (all P<0.05). MiR-34b-5p had a targeting relationship with both MIAT and CALB1, and MIAT was found to be expressed in exo. The oe-MIAT-exo group and the miR-34b-5p inhibitor group showed significantly up-regulated CALB1, TH, and Bcl-2 protein expressions in the HNCs, increased cell viability, as well as reduced apoptosis, but the si-MIAT-exo group showed the opposite results (all P<0.05). The MiR-34b-5p inhibitor partially reversed the effect on the si-MIAT-exo group. The miR-34b-5p mimic group showed significantly down-regulated CALB1, TH, and Bcl-2 protein expressions in the HNCs, inhibited cell viability, as well as increased apoptosis, but the oe-CALB1 group showed the opposite results (all P<0.05). Oe-CALB1 partially reversed the effect on the miR-34b-5p mimic group. The memory and learning abilities of the rats in the oe-MIAT-exo group and the model + exo group were significantly improved but not as much as they were in the normal rats.
Conclusion:
MIAT-containing exo from HNSCs can improve cognitive disorders in VD rats via the miR-34b-5p/CALB1 axis.
... After 7 days, the purity of hippocampal neurons was measured. Neuronal cell markers CD24 and tyrosine hydroxylase (TH) were detected by flow cytometry, and cells with a positive rate of more than 95% were included in subsequent experiments [13]. This study was approved by the local hospital Ethics Committee. ...
Objective:
To investigate the regulatory mechanism of sevoflurane-induced neuronal apoptosis through analyzing the expression of glial cell-derived neurotrophic factor (GDNF) mediated by miR-133, sponged by long non-coding RNA (lncRNA) CDKN2B-AS1.
Methods:
An in vitro cell injury model was established by using different concentrations of sevoflurane and primary hippocampal neurons. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8); caspase-3 and caspase-9 activities were detected by colorimetry, and apoptotic cells were determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Fluorescence in situ hybridization (FISH) analysis was used to detect localized expression of CDKN2B antisense RNA 1 (CDKN2B-AS1), and dual-luciferase reporter assay was employed to verify the correlation of CDKN2B-AS1 and miR-133, and of miR-133 and GDNF. The expression of CDKN2B-AS1, miR-133, and GDNF mRNA in the cell injury model were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot was utilized to detect the expression of GDNF protein in the cell injury model.
Results:
In the cell injury model, CDKN2B-AS1 was highly expressed in the cytoplasm, and CDKN2B-AS1 and GDNF were downregulated and miR-133 was upregulated as detected by qRT-PCR (all P<0.05). The connections between CDKN2B-AS1 and miR-133, and between miR-133 and GDNF were confirmed. That is, CDKN2B-AS1 regulated the expression of GDNF by adsorbing miR-133 (all P<0.05). In cells treated with sevoflurane, overexpression of CDKN2B-AS1 could inhibit caspase-3 and caspase-9 activities and the degree of apoptosis. miR-133 could partially alleviate the effect of overexpressing CDKN2B-AS1 on cells, and si-GDNF the effect of miR-133 inhibitor (all P<0.05).
Conclusion:
lncRNA CDKN2B-AS1 can up-regulate the expression of GDNF, inhibit neuronal apoptosis, and ease the toxic effect of sevoflurane on neural cells by acting as a sponge to adsorb miR-133.
... Alternatively, the decrease in ciliary alpha-tubulin acetylation may be associated with glioma cell differentiation. Indeed, differentiated neurons in the brain typically lack aaTub in their cilia (data not shown), unlike their radial glial precursors [37], through mechanisms that are still unclear. Moreover, HDAC6 inhibitors have been recently reported to drive stem-like cells into senescence [7,8] or a differentiated neuronal state [38], which is consistent with the 738-induced increased expression of TUJ1, a marker of neuronal differentiation, that we observed in human and murine glioma cells. ...
Simple Summary
Glioblastoma is the most common and lethal brain tumor in adults because it becomes resistant to virtually every treatment. Histone deacetylase 6 (HDAC6), which is located primarily in the cytoplasm, has a unique role in promoting the disassembly of cells’ primary cilium, a non-motile “antenna” that must be broken down before cells can progress through the cell cycle. The role of HDAC6 and its function in gliomas have not been investigated with respect to tumor cell cilia. We have found that inhibitors of HDAC6 cause rapid and specific changes inside glioma cilia, reducing tumor cell proliferative capacity and promoting cell differentiation. Importantly, the HDAC6 inhibitors did not affect the proliferation or differentiation of glioma cells that we genetically modified unable to grow cilia. Our findings reveal a conserved and critical role for HDAC6 in glioma growth that is dependent on cilia.
Abstract
Histone deacetylase 6 (HDAC6) is an emerging therapeutic target that is overexpressed in glioblastoma when compared to other HDACs. HDAC6 catalyzes the deacetylation of alpha-tubulin and mediates the disassembly of primary cilia, a process required for cell cycle progression. HDAC6 inhibition disrupts glioma proliferation, but whether this effect is dependent on tumor cell primary cilia is unknown. We found that HDAC6 inhibitors ACY-1215 (1215) and ACY-738 (738) inhibited the proliferation of multiple patient-derived and mouse glioma cells. While both inhibitors triggered rapid increases in acetylated alpha-tubulin (aaTub) in the cytosol and led to increased frequencies of primary cilia, they unexpectedly reduced the levels of aaTub in the cilia. To test whether the antiproliferative effects of HDAC6 inhibitors are dependent on tumor cell cilia, we generated patient-derived glioma lines devoid of cilia through depletion of ciliogenesis genes ARL13B or KIF3A. At low concentrations, 1215 or 738 did not decrease the proliferation of cilia-depleted cells. Moreover, the differentiation of glioma cells that was induced by HDAC6 inhibition did not occur after the inhibition of cilia formation. These data suggest HDAC6 signaling at primary cilia promotes the proliferation of glioma cells by restricting their ability to differentiate. Surprisingly, overexpressing HDAC6 did not reduce cilia length or the frequency of ciliated glioma cells, suggesting other factors are required to control HDAC6-mediated cilia disassembly in glioma cells. Collectively, our findings suggest that HDAC6 promotes the proliferation of glioma cells through primary cilia.
... A lack of a functional primary cilium led to defects in the development of several organs, such as the heart, cranial structures, the tongue, and the submandibular gland [36][37][38][39][40][41][42]. Interestingly, NCC development at the trunk level involves signaling pathways, such as Wnt, Sonic Hedgehog, TGFβ, or chemokine signaling, which are linked to the primary cilium or have components that have been shown to localize in it in other cell types [43][44][45][46][47]. Thus, a cell-autonomous role for primary cilia in early NCC development in the trunk is very likely. ...
The primary cilium plays a pivotal role during the embryonic development of vertebrates. It acts as a somatic signaling hub for specific pathways, such as Sonic Hedgehog signaling. In humans , mutations in genes that cause dysregulation of ciliogenesis or ciliary function lead to severe developmental disorders called ciliopathies. Beyond its role in early morphogenesis, growing evidence points towards an essential function of the primary cilium in neural circuit formation in the central nervous system. However, very little is known about a potential role in the formation of the peripheral nervous system. Here, we investigate the presence of the primary cilium in neural crest cells and their derivatives in the trunk of developing chicken embryos in vivo. We found that neural crest cells, sensory neurons, and boundary cap cells all bear a primary cilium during key stages of early peripheral nervous system formation. Moreover, we describe differences in the ciliation of neuronal cultures of different populations from the peripheral and central nervous systems. Our results offer a framework for further in vivo and in vitro investigations on specific roles that the primary cilium might play during peripheral nervous system formation.
... In line with this, studies using animal models of ciliopathies suggested that cranial NCC migration and development are impaired in a cell-autonomous manner when primary cilia are compromised. A lack of a functional primary cilium led to defects in the development of several organs, such as cranial bone structures, the tongue, and the submandibular gland [37][38][39][40][41]. Interestingly, NCC development at the trunk level involve signaling pathways, such as Wnt, Sonic hedgehog, TGFβ, or chemokine signaling, which are linked to the primary cilium or have components that were shown to localize in it in other cell types [42][43][44][45][46]. Thus, a cell-autonomous role for primary cilia in early NCC development in the trunk is very likely. ...
The primary cilium plays a pivotal role during embryonic development of vertebrates. It acts as a somatic signaling hub for specific pathways, such as sonic hedgehog signaling. In humans, mutations in genes that cause dysregulation of ciliogenesis or ciliary function lead to severe developmental disorders called ciliopathies. Beyond its obvious role in early morphogenesis, growing evidence points towards an essential function of the primary cilium in neural circuit formation in the central nervous system. However, very little is known about a potential role in the formation of the peripheral nervous system. Here, we investigated the presence of the primary cilium in neural crest cells and their derivatives in the trunk of the developing chicken embryo in vivo. We found that neural crest cells, sensory neurons, and boundary cap cells all bear a primary cilium during key stages of early peripheral nervous system formation. Moreover, we described differences in the ciliation of neuronal cultures of different populations from the peripheral and central nervous system. Our results offer a framework for further in vivo and in vitro investigations on specific roles that the primary cilium might play during peripheral nervous system formation.
... Whole mount dissection and immunostaining was performed as previously described (Mirzadeh et al. 2008;Monaco et al. 2019). For coronal sections, mice sedated with narcorene (400 mg/kg) were perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS, after which the brains were removed and postfixed in 3% PFA / 4% sucrose in PBS for 48h at 4°C. ...
... Since we here found that very few basal NSCs extend primary cilia and express Nestin, it is possible that the expression of Nestin is mediated by cilia-dependent signals. In the V-SVZ, primary cilia do not only regulate quiescence in radial glia and adult NSCs (Beckervordersandforth et al. 2010;Khatri et al. 2014), but they are also key to sensing signals present in the cerebrospinal fluid filling the ventricular cavity (Silva-Vargas et al. 2016;Monaco et al. 2019). Consistent with this view, recent observations have highlighted that in the V-SVZ, acquisition of quiescence concerns a prolonged time period (Borrett et al. 2020) and involves different stages and integration of multiple regulatory signals. ...
Neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ) generate adult-born olfactory bulb (OB) interneurons. Recent observations indicate that NSCs uncouple self-renewing from differentiating divisions to maintain life-long OB neurogenesis. According to this model, NSCs detach from the ventricle and relocate at the basal side of the V-SVZ to differentiate, thereby leading to NSC consumption. Using genetic and viral NSC tagging and apical membrane labelling, we here found on the contrary that basal neurogenesis originates from an additional novel NSC type resident in the basal V-SVZ and that aging decreases NSC activation, rather than promoting NSC consumption. Apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. However, both apical and basal NSCs similarly express GFAP, SOX9 and Lrig1 and they are capable of self-renewal and long-term quiescence. Within six weeks, apical NSCs generated few neuroblasts in the basal V-SVZ and in the OB, indicating that adult-born OB neurons essentially originate from basal NSCs. Supporting this, we found that pregnancy, a physiological modulator of OB neurogenesis, affects the number of basal but not apical NSCs. Lastly, apical NSCs displayed the highest levels of Notch activation in the neural lineage. Selective apical downregulation of the Notch-signalling effector Hes1 decreased Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Thus, OB neurogenesis is driven by basal NSCs, whereas notch signalling inhibits neurogenesis from apical NSCs.
The advent of single‐cell cytometric technologies, in conjunction with advances in single‐cell biology, has significantly propelled forward the field of geroscience, enhancing our comprehension of the mechanisms underlying age‐related diseases. Given that aging is a primary risk factor for numerous chronic health conditions, investigating the dynamic changes within the physiological landscape at the granularity of single cells is crucial for elucidating the molecular foundations of biological aging. Utilizing hallmarks of aging as a conceptual framework, we review current literature to delineate the progression of single‐cell cytometric techniques and their pivotal applications in the exploration of molecular alterations associated with aging. We next discuss recent advancements in single‐cell cytometry in terms of the development in instrument, software, and reagents, highlighting its promising and critical role in driving future breakthrough discoveries in aging research.
