The Cerebellum https://doi.org/10.1007/s12311-018-0935-4
SHORT REPORT
Centrosome Inheritance Does Not Regulate Cell Fate in Granule Neuron Progenitors of the Developing Cerebellum Anindo Chatterjee 1 & Kaviya Chinnappa 1 & Narendrakumar Ramanan 1 & Shyamala Mani 1,2
# Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited. Keywords Mother centrosome . Pericentriolar material (PCM) . Cell fate . Cerebellar granule neuron progenitors (CGNPs)
Introduction Centrosomes are cell organelles that are composed of two parts—the centrioles and the PCM. The centriolar structure is polarized, comprising a proximal and a distal end. The distal end of the centriole houses the distal and sub-distal appendages [1]. Centrioles are duplicated in the S-phase. Each pre-existing centriole (referred to as mother centriole) nucleates a nascent procentriole at its base, which elongates as the S-phase proceeds. Thus, at the end of the S-phase, the cells have two centrosomes, having two centrioles each. The two centrosomes disengage as the cell proceeds to mitosis, and they move to opposite poles of the cell for the formation and correct positioning of the mitotic spindles. Following mitosis, each cell possesses one centrosome that has one mother * Anindo Chatterjee
[email protected] * Shyamala Mani
[email protected] 1
Centre for Neuroscience, Indian Institute of Science, Bengaluru 560012, India
2
Present address: Curadev Pharma, Pvt. Ltd., B-87, Sector 83, Noida, Uttar Pradesh 201305, India
centriole from the previous cycle and the newly duplicated daughter centriole (Fig. 1). During the S-phase, the centrioles duplicate, and at the end of this duplication, there will exist three generations of centrioles, a mother centriole-procentriole pair (referred to as the mother centrosome), and a daughter centriole-procentriole pair (referred to as the daughter centrosome). As centrioles mature, they acquire additional structural characteristics. The mother centriole has two extra appendages at its distal end—the distal and sub-distal appendages. The distal appendage is important for basal body and cilia formation while in addition to the PCM, the sub-distal appendage nucleates microtubules. Given the nature of centrosome duplication, there is an inherent asymmetry in the structural maturity of the centrosomes in a dividing cell [2]. Inherent asymmetry in centrosomes seems to have been used during evolution as a mechanism for determination of cell fate. In mollusk post-fertilization divisions, mRNAs localize preferentially to one of the centrosomes, and this centrosome is consistently inherited by a daughter cell with a subsequent specific cell fate [3]. In human embryonic stem cells, proteins marked for degradation localize to one of the two centrosomes. This mechanism may allow for degradation and thus regulation of proteins important for cell fate [4]. Consistent patterns of mother/daughter centrosome inheritance and resultant cell fate of the daughter cell have been reported during asymmetric cell divisions. In embryonic stem
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Fig. 1 The centrosome cycle. Post, Mitosis [1], one of the daughter cell inherits the mother centriole (light brown with yellow crown) with its procentriole (green), that is, the mother centrosome. The other daughter cell inherits the daughter centriole (dark brown with black pattern) with its procentriole (green), that is, the daughter centrosome. In the ensuing cell cycle, the daughter centriole acquires all the required appendages (yellow
crown) and matures to become a mother centriole. In the S-phase, each centriole gives rise to a procentriole (blue). The two centrosomes migrate to opposite ends of the cell prior to cell division. In Post, Mitosis [2], the two daughter cells inherit two different sets of centrosomes. The type of centrosome inherited might determine their subsequent cell fate
cell divisions, the mother centrosome is inherited by the daughter cell which remains a stem cell post division [5]. Drosophila male germline stem cells undergo divisions to give rise to one stem cell and one gonialblast, which differentiates, and the mother centrosome is always inherited by the daughter cell which retains its stemness [6]. In the developing mammalian cortex, the mother centrosome is inherited by the daughter cell which remains a radial glial progenitor, while the daughter cell inheriting the daughter centrosome migrates dorsally and differentiates in to a neuron [7]. However, in the case of human neuroblastoma cell lines, such as NB69 and TBW, the mother centrosome is inherited by the daughter cell which presumably exits the cell cycle [8]. Similarly, in Drosophila neuroblast divisions, the mother centrosome is inherited by the ganglion mother cell which subsequently exits the cell cycle [9]. Although the consequence of the asymmetric inheritance of the mother or daughter centrosome is context dependent, it is nevertheless the case that there is a correlation between the fate of a daughter cell (in terms of remaining in or exiting the cell cycle) and the age of the centrosome it inherited during cell division. The goal of the current study was to determine whether asymmetric inheritance of the centrosome by cerebellar granule neuron progenitors (CGNPs) is important for cell fate.
Materials and Methods Cerebellar Cultures Cultures were prepared as described previously [10]. Additionally, the pellet was resuspended in serum containing media and plated on poly-D-lysine coated wells (SigmaAldrich, 250 μg/ml) for 10 min during which time the heavier cells settled down while the lighter and smaller cells like granule progenitors remained in media. This was done twice, and finally, the cells were plated and allowed to settle down on poly-Dlysine coated wells. Such a culture had around 70–75% CGNPs. Experiments in which sister pairs of daughter cells had to be analyzed, the final pellet was diluted ten times, of which 1– 2 μl was then seeded on to poly-D-lysine coated wells. After the cells settled down, each well was checked under the microscope to ensure that evenly placed single cells have attached. Any well with clumps of two or more cells was discarded. Twenty four hours after plating, the required staining was done and sister cells were sought for analysis.
Immunocytochemistry Twelve (Figs. 2 and 3) to twenty four (Fig. 4) hours post-plating, the serum was aspirated and a 5-min wash with 1 × PBS was
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Fig. 2 Centrosome asymmetry in CGNPs. a P5 CGNP cultures stained for Pax6 (green), which marks CGNPs. DAPI (blue) stains the nucleus. Scale bar = 10 μm. b P5 CGNP cultures stained for GFAP (green, shown using a yellow arrow), which marks glial cells. DAPI (blue) stains the nucleus. Scale bar = 10 μm. c Cells stained for a PCM marker (γ-tubulin -
red), and a mother centrosome marker (ODF2 -green) showed the presence of two centrosomes during interphase. The presence of ODF2 on only of the centrosomes shows that there is centrosome asymmetry in CGNPs. DAPI (blue) stains the nucleus. Scale bar = 1 μm
given. The cells were fixed using pre-chilled methanol at − 30 °C for 10 min and then washed three times using 1 × PBS for 5 min each. Non-specific binding was blocked using 5– 10% normal horse serum (NHS) for an hour and permeabilized using 0.02–0.1% Triton X-100 for 10 min. The cells were incubated overnight at 4 °C with the required antibody and then washed three times with 1 × PBS for 5 min each. Antibodies used were anti-PCNA (CST, 2586S, 1:500), anti γ-tubulin (Sigma, T5192, 1:300), and anti ODF2 (Abcam, ab43840, 1:200). Fluorophore-labeled secondary antibodies (Alexa Fluor 1:1000 in 1 × PBS containing 0.5% BSA) were used; incubated for an hour at room temperature. Three washes with 1× PBS (5 min each) were given after which the excess PBS was drained and a coverslip placed on the slide after putting DAPI containing fluorescent medium (Vectashield). Images were captured using the Imager. M2 (Zeiss Apotome) microscope and the Eclipse 80i (Nikon) microscope, at either × 20 or × 63 magnification. ImageJ software was used to quantitate PCM fluorescence and area.
normal horse serum and 4% bovine serum albumin) containing anti-ODF2 (Abcam, ab43840, 1:100) and anti- γ-tubulin (Sigma, T5192, 1:100) antibodies.
Statistics One-sample t test was used for comparison of PCM area and quantity between mother and daughter centrosome pairs, wherein a value of 1 was assigned to the values obtained from the mother centrosome. For in-vitro centrosome inheritance experiments, chi-square test was used assuming probability of mother centrosome inheritance as 50%. Paired Student’s t test was used to analyze ODF2+ mother centrosome distribution data in the EGL. GraphPad Prism software was used for analysis and generating the graphs.
Results Centrosome Asymmetry Exists in CGNPs
Cerebellar Slice Preparation and Staining The midbrain-cerebellum-brainstem was carefully removed and placed in ice cold HBSS buffer. Using the midbrain and brainstem as support points, the cerebellum was cut out, and 200 μm slices were made using a McIlwain Tissue Chopper. Slices were fixed in methanol for 15 min at − 20 °C and permeabilized with 1% Tx100 for 15 min at room temperature. Slices were incubated overnight at 4 °C in 10% blocking solution (10%
P5 CGNP cultures were stained with Pax6 (Fig. 2a), GFAP (Fig. 2b), and NeuN 12 h post-plating. We found that our cultures had around 72% ± 5.3 (Mean ± SD) Pax6+ cells and around 2.3% ± 1.4 (Mean ± SD) GFAP+ cells. We did not detect NeuN+ cells in the culture (the working of the NeuN antibody was confirmed using a 72-h CGNP culture as a positive control). To assess the presence of mother and daughter centrosomes in CGNPs, we used an established marker for
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Fig. 3 Asymmetry in PCM between mother and daughter centrosome arises as they migrate away from each other. a Image showing a pair of mother [ODF2 (green) and γ-tubulin (red) positive] and daughter centrosomes [only γ-tubulin (red) positive]. DAPI stains the nucleus. b There was no significant difference in PCM area (graph on the top) or the amount of PCM (graph at the bottom) between the two centrosomes (one sample t test; p > 0.05; 50 pairs of centrosomes pooled from five animals). c CGNPs were stained for ODF2 (green) and γ-tubulin (PCM, red). DAPI
stains the nucleus. Mother-daughter centrosome pairs chosen for analysis had separated considerably and/or were on opposite ends of the cell. d The daughter centrosome had significantly less PCM area (graph on the left) and quantity (graph on the right) than the mother centrosome as they migrated away from each other (One sample t test; p < 0.02 for PCM area, p < 0.05 for PCM quantity; 16 pairs of centrosomes analyzed on pooling data from four animals). Scale bar = 1 μm, for all the images. Graphs plot Mean ± SD
the mature mother centrosome called outer density fiber protein 2 (ODF2) which localizes to the distal and subdistal appendages of the mother centrosome [11]. In order to stain both the mother and the daughter centrosome, we used γ-tubulin which stains the PCM [12]. On staining P5-P7 CGNP cultures with the above
markers, we observed two types of centrosomes. One of them only stained for γ-tubulin while the other stained for both γ-tubulin and ODF2 (Fig. 2c), the latter being the mother centrosome. These results show that centrosome asymmetry does exist in CGNPs of the developing mouse cerebellum.
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Fig. 4 Centrosome inheritance does not correlate with cell fate in CGNPs: a P5 CGNP cultures stained with Pax6 (green) and pHH3 (red). Actively dividing Pax6+ cells have been shown using yellow arrows. DAPI (blue) stains the nucleus. Scale bar = 10 μm. b Images showing a pair of daughter cells in which the cell inheriting the mother centrosome (ODF2 - red; shown using a white arrow) has exited the cell cycle. Its sister cell remains in a state of proliferation (PCNA positive -
green). DAPI (blue) stains the nucleus. c There was no significant difference in the number of daughter cells which had inherited the mother centrosome and remained progenitors versus those that had inherited the mother centrosome and exited the cell cycle (chi-square test; p > 0.05; 39 asymmetric divisions pooled from six animals). Scale bar = 1 μm. Gray line depicts the cleavage plane
PCM Asymmetry Arises as the Mother and Daughter Centrosome Migrate Away from each Other
centrosome (Fig. 3d). This suggests that the two centrosomes might have unequal capacities to nucleate microtubules by mitosis. This could have implications in asymmetric protein segregation during cell division.
Having shown that CGNPs have asymmetric centrosomes, we next asked whether the mother and daughter centrosome in CGNPs differ in terms of the amount of PCM they possess and therefore their microtubule nucleation capacity. Previous studies have reported that mother and daughter centrosomes differ in the amount of PCM they have. This is specially important considering that the microtubule nucleation capacity of a centrosome is considered to be proportional to the amount of PCM it has [13] due to the fact that γ-tubulin is one of the main attachment points for microtubules and is one of the main components of PCM [12]. We used γ-tubulin as a marker for PCM and ODF2 as a marker for the mother centrosome (Fig. 3a) and analyzed the PCM area. We also analyzed the intensity of γ-tubulin staining as a proxy for the amount of γ-tubulin present. On examination of the area and amount of PCM in mother-daughter centrosome pairs, we noticed no discernible difference in the above parameters (Fig. 3b). We reasoned that an asymmetry in PCM might develop as the centrosomes migrate away from each other and prepare for cell division. We chose mother-daughter pairs that had separated considerably and/or were on opposite ends of the cell for analysis (Fig. 3c). Interestingly, daughter centrosomes possessed significantly lesser PCM, both in terms of area and amount, once they had migrated away from the mother
Centrosome Inheritance Does Not Correlate with Cell Fate in CGNPs Next, we set out to determine whether the inheritance of mother or daughter centrosome during CGNP divisions can be linked to cell fate, in terms of either the daughter cell remaining as a progenitor or exiting the cell cycle. We stained P5 CGNP cultures with Pax6 (CGNP marker) and pHH3 (marker for cells undergoing division) 24-h post-plating (Fig. 4a). We found all the pHH3+ cells to be Pax6+. Experiments in which sister pairs of daughter cells had to be analyzed had been seeded to have only one cell per well. After 24 h, some of these cells had divided and formed a pair of juxtaposed daughter cells. Such cells were analyzed poststaining (Fig. 4b). The daughter cell pairs were stained for PCNA (a proliferation marker) and ODF2 (mother centrosome marker). PCNA expression in daughter cell pairs with asymmetric ODF2 staining was analyzed. We observed no correlation between centrosome inheritance and PCNA expression (Fig. 4c). This suggests that in CGNPs, the asymmetric inheritance of the centrosome is not correlated with the cell fate of the daughter cell in vitro.
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Mother Centrosome Distribution Does Not Differ Based on Cell Fate In Vivo
Cell fate in CGNP is likely to involve both intrinsic and extrinsic signals. For example, we as well as others [5, 10] have shown that an asymmetric source of an extrinsic ligand can
bring about asymmetric changes in intrinsic factors (including pluripotency markers) which might determine cell fate. The purpose of this study was to determine if biased centrosome inheritance could be an intrinsic factor in determining cell fate. In our experiments, ODF2 was used as a marker for identifying the mother centrosome. A number of studies have reported that ODF2 exclusively localizes to the mother centrosome [14–16]. As part of the distal appendage, ODF2 has been implicated in the formation of the cilium; and as part of the sub-distal appendage, it is involved in nucleating microtubules; ODF2 mutants lack both these structures [11, 17]. This, we thought, makes ODF2 a definitive marker to observe centrosome asymmetry. However, asymmetric ODF2 localization in mother centrosomes of CGNPs has not been reported, to the best of our knowledge. Our stainings with ODF2 and γtubulin in pre- and post-mitotic interphase cells clearly show that ODF2 is a reliable mother centrosome marker in CGNPs. The PCM consists of proteins organized around the centrioles. The PCM contains the γ-tubulin ring complex (γ-TuRC) which captures cytoplasmic tubulin to nucleate microtubules. Mother and daughter centrosomes can differ in the amount of PCM they possess and are therefore capable of nucleating different amounts of microtubules [6]. The centrosome nucleating more microtubules can lead to it being more strongly anchored to the actin cytoskeleton within the cell, with the
Fig. 5 ODF2+ mother centrosome distribution in the EGL. a Representative images of P5 cerebellar slices stained for ODF2 and γtubulin. The centrosomes appear as distinct puncta. The solid yellow line(s) delineates the outer edge of the EGL. The blue dotted lines mark the boundary between the oEGL and the iEGL. The image on the left is that of a cerebellar fold, thereby forming two adjacent EGLs. oEGL, outerEGL; iEGL, innerEGL; ML, molecular layer. Scale bar = 20 μm. b
Expanded image of the red inset in Fig. 5a. Centrosomes were stained for ODF2 (red) and γ-tubulin (green); marked by yellow arrows. Scale bar = 10 μm. c Quantification of the number of ODF2+ mother centrosomes present in the oEGL and the iEGL, normalized to total number of centrosomes stained in the respective layers (seven cerebellar slices from three animals). There was no significant difference in the number of mother centrosomes between the two layers. Graph plots Mean ± SD
CGNPs are found in the external granule layer (EGL) of the developing cerebellum, wherein the actively proliferating CGNPs are found in the outerEGL (towards the pia), while the progenitors which have exited the cell cycle accumulate ventrally at the innerEGL [10]. Cerebellar slices harvested from P5 mouse pups were stained with ODF2 and γ-tubulin (Fig. 5a, b). The number of ODF2+ γ-tubulin+ centrosomes were quantitated from the two uppermost layers of the outerEGL and the two lowermost layers of the innerEGL (the middleEGL is believed to have a mixed population of actively proliferating and post-mitotic CGNPs and was therefore excluded from the analysis). No significant difference in ODF2+ mother centrosome distribution was observed between the outerEGL and the innerEGL (Fig. 5c). This further suggests that centrosome inheritance in CGNPs might not regulate cell fate.
Discussion
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other centrosome being freer to move to the opposite pole and thereby defining the plane of cell division [13]. Further, movement of proteins on microtubules can mean that depending on the nucleating capacity of the centrosome, asymmetric segregation of proteins during cell division may happen. These proteins may in turn have a role as a cell fate determinant [18]. Interestingly, our results show that though there is no difference in the area or quantity of PCM between the mother and daughter centrosome initially, the daughter centrosome tends to have lesser PCM when it migrates away from the mother centrosome. Rebollo et al. [13] have also reported shedding-off of PCM by one of the centrosomes as it migrates away. ODF2 overexpression has been shown to maintain a population of dividing radial glia progenitors in the developing ventricular zone in Pax6 mutants, which normally exhibit depleted number of progenitors [19]. We checked for correlation between cell fate in CGNPs and the type of centrosome it has inherited in vitro and found no relation between the two. We also studied the distribution of mother centrosomes in the EGL of the developing cerebellum and found no correlation between the proliferation status of CGNPs and the type of centrosome they stained for. This possibly suggests that different areas of the brain might employ non-identical strategies to regulate cell fate during development.
References 1. 2.
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Conclusions In conclusion, we show that mother and daughter centrosomes are present in CGNPs and that they possess different amounts of PCM. However, the inheritance of centrosomes is not correlated with the subsequent cell fate of the progenitor daughter cells. Acknowledgements The authors thank Parthiv Haldipur and Ajit Ray (National Brain Research Centre, India) for their inputs. Funding Information SM received funding from the Indo-French Centre for the Promotion of Advanced Research (CEFIPRA, grant no. 4903-02). KC received financial support from the Department of Biotechnology, Government of India (DBT Neurobiology Task Force, grant no. 0376). NR was supported by SwarnaJayanti Fellowship from the Department of Science and Technology, Government of India, and the DBT-IISc partnership program. AC received financial support from the Council of Scientific and Industrial Research (CSIR), Government of India.
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Compliance with Ethical Standards 19. Conflict of Interest The authors declare that they have no conflict of interest.
Bornens M. The centrosome in cells and organisms. Science. 2012;335(6067):422–6. Yamashita YM, Fuller MT. Asymmetric centrosome behavior and the mechanisms of stem cell division. J Cell Biol. 2008;180(2): 261–6. Lambert JD, Nagy LM. Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages. Nature. 2002;420(6916):682–6. Fuentealba LC, Eivers E, Geissert D, Taelman V, De Robertis EM. Asymmetric mitosis: unequal segregation of proteins destined for degradation. Proc Natl Acad Sci. 2008;105(22):7732–7. Habib SJ, Chen B, Tsai F, Anastassiadis K, Meyer T, Betzig E, et al. A localized Wnt signal orients asymmetric stem cell division in vitro. Science. 2013;1424(6126):1445–8. Yamashita YM, Mahowald AP, Perlin JR, Fuller MT. Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science. 2007;315(5811):518–21. Wang X, Tsai J-W, Imai JH, Lian W-N, Vallee RB, Shi SH. Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature. 2009;461(7266):947–55. Izumi H, Kaneko Y. Evidence of asymmetric cell division and centrosome inheritance in human neuroblastoma cells. Proc Natl Acad Sci. 2012;109(44):18048–53. Januschke J, Llamazares S, Reina J, Gonzalez C. Drosophila neuroblasts retain the daughter centrosome. Nat Commun. 2011;2:243. Haldipur P, Sivaprakasm I, Periasamy P, Govindan S, Mani S. Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum. Biol Open. 2015;4: 865–72. Ishikawa H, Kubo A, Tsukita S, Tsukita S. Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia. Nat Cell Biol. 2005;7(5):517–24. Mennella V, Agard DA, Huang B, Pelletier L. Amorphous no more: Subdiffraction view of the pericentriolar material architecture. Trends Cell Biol. 2014;24(3):188–97. Rebollo E, Sampaio P, Januschke J, Llamazares S, Varmark H, González C. Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev Cell. 2007;12(3):467–74. Lange BMH, Gull K. A molecular marker for centriole maturation in the mammalian cell cycle. J Cell Biol. 1995;130(4):919–27. Nakagawa Y, Yamane Y, Okanoue T, Tsukita S, Tsukita S. Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. Mol Biol Cell. 2001;12(6):1687–97. Anderson CT, Stearns T. Centriole age underlies asynchronous primary cilium growth in mammalian cells. Curr Biol. 2009;19(17): 1498–502. Donkor FF, Mönnich M, Czirr E, Hollemann T, Hoyer-Fender S. Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules. J Cell Sci. 2004;117(20): 4643–51. Sugioka K, Mizumoto K, Sawa H. Wnt regulates spindle asymmetry to generate asymmetric nuclear β-catenin in C. elegans. Cell. 2011;146(6):942–54. Tylkowski MA, Yang K, Hoyer-Fender S, Stoykova A. Pax6 controls centriole maturation in cortical progenitors through Odf2. Cell Mol Life Sci. 2015;72(9):1795–809.
