Prominin‐1 controls stem cell activation by orchestrating ciliary dynamics
2018; Springer Nature; Volume: 38; Issue: 2 Linguagem: Inglês
10.15252/embj.201899845
ISSN1460-2075
AutoresDonald A. Singer, Kristina Thamm, Heng Zhuang, Jana Karbanová, Yan Gao, Jemma Victoria Walker, Heng Jin, Xiangnan Wu, Clarissa Coveney, Pauline Marangoni, Dongmei Lu, Portia Rebecca Clare Grayson, Tülay Gülşen, Karen Liu, Stefano Ardu, A.K. Wann, Shouqing Luo, Alexander C. Zambon, Anton M. Jetten, Christopher Tredwin, Ophir D. Klein, Massimo Attanasio, Peter Carmeliet, Wieland Β. Huttner, Denis Corbeil, Bing Hu,
Tópico(s)Cancer Cells and Metastasis
ResumoArticle6 December 2018Open Access Transparent process Prominin-1 controls stem cell activation by orchestrating ciliary dynamics Donald Singer Donald Singer Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Kristina Thamm Kristina Thamm Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Heng Zhuang Heng Zhuang Peninsula Dental School, University of Plymouth, Plymouth, UK Department of Cariology, Endodontology and Operative Dentistry, Peking University School and Hospital of Stomatology, Beijing, China Search for more papers by this author Jana Karbanová Jana Karbanová Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Yan Gao Yan Gao Peninsula Dental School, University of Plymouth, Plymouth, UK Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China Search for more papers by this author Jemma Victoria Walker Jemma Victoria Walker Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Heng Jin Heng Jin Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Department of Emergency Medicine, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Xiangnan Wu Xiangnan Wu Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Search for more papers by this author Clarissa R Coveney Clarissa R Coveney Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute, Nuffield Department for Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, UK Search for more papers by this author Pauline Marangoni Pauline Marangoni Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Search for more papers by this author Dongmei Lu Dongmei Lu Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Portia Rebecca Clare Grayson Portia Rebecca Clare Grayson Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Tulay Gulsen Tulay Gulsen Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Karen J Liu Karen J Liu Centre for Craniofacial and Regenerative Biology, King's College London, London, UK Search for more papers by this author Stefano Ardu Stefano Ardu Division of Cariology & Endodontology, Dental School, University of Geneva, Geneva, Switzerland Search for more papers by this author Angus KT Wann Angus KT Wann Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute, Nuffield Department for Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, UK Search for more papers by this author Shouqing Luo Shouqing Luo orcid.org/0000-0002-7998-3059 Peninsula Medical School, University of Plymouth, Plymouth, UK Search for more papers by this author Alexander C Zambon Alexander C Zambon Biopharmaceutical Sciences, Keck Graduate Institute, Claremont, CA, USA Search for more papers by this author Anton M Jetten Anton M Jetten orcid.org/0000-0003-0954-4445 Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Christopher Tredwin Christopher Tredwin Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Ophir D Klein Ophir D Klein orcid.org/0000-0002-6254-7082 Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA Search for more papers by this author Massimo Attanasio Massimo Attanasio Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Peter Carmeliet Peter Carmeliet orcid.org/0000-0001-7961-1821 Department of Oncology, Laboratory of Angiogenesis and Vascular Metabolism, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium Search for more papers by this author Wieland B Huttner Wieland B Huttner orcid.org/0000-0003-4143-7201 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Denis Corbeil Corresponding Author Denis Corbeil [email protected] orcid.org/0000-0003-1181-3659 Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Bing Hu Corresponding Author Bing Hu [email protected] orcid.org/0000-0001-5085-3114 Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Donald Singer Donald Singer Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Kristina Thamm Kristina Thamm Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Heng Zhuang Heng Zhuang Peninsula Dental School, University of Plymouth, Plymouth, UK Department of Cariology, Endodontology and Operative Dentistry, Peking University School and Hospital of Stomatology, Beijing, China Search for more papers by this author Jana Karbanová Jana Karbanová Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Yan Gao Yan Gao Peninsula Dental School, University of Plymouth, Plymouth, UK Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China Search for more papers by this author Jemma Victoria Walker Jemma Victoria Walker Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Heng Jin Heng Jin Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Department of Emergency Medicine, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Xiangnan Wu Xiangnan Wu Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Search for more papers by this author Clarissa R Coveney Clarissa R Coveney Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute, Nuffield Department for Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, UK Search for more papers by this author Pauline Marangoni Pauline Marangoni Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Search for more papers by this author Dongmei Lu Dongmei Lu Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Portia Rebecca Clare Grayson Portia Rebecca Clare Grayson Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Tulay Gulsen Tulay Gulsen Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Karen J Liu Karen J Liu Centre for Craniofacial and Regenerative Biology, King's College London, London, UK Search for more papers by this author Stefano Ardu Stefano Ardu Division of Cariology & Endodontology, Dental School, University of Geneva, Geneva, Switzerland Search for more papers by this author Angus KT Wann Angus KT Wann Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute, Nuffield Department for Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, UK Search for more papers by this author Shouqing Luo Shouqing Luo orcid.org/0000-0002-7998-3059 Peninsula Medical School, University of Plymouth, Plymouth, UK Search for more papers by this author Alexander C Zambon Alexander C Zambon Biopharmaceutical Sciences, Keck Graduate Institute, Claremont, CA, USA Search for more papers by this author Anton M Jetten Anton M Jetten orcid.org/0000-0003-0954-4445 Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Christopher Tredwin Christopher Tredwin Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Ophir D Klein Ophir D Klein orcid.org/0000-0002-6254-7082 Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA Search for more papers by this author Massimo Attanasio Massimo Attanasio Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Peter Carmeliet Peter Carmeliet orcid.org/0000-0001-7961-1821 Department of Oncology, Laboratory of Angiogenesis and Vascular Metabolism, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium Search for more papers by this author Wieland B Huttner Wieland B Huttner orcid.org/0000-0003-4143-7201 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Denis Corbeil Corresponding Author Denis Corbeil [email protected] orcid.org/0000-0003-1181-3659 Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Bing Hu Corresponding Author Bing Hu [email protected] orcid.org/0000-0001-5085-3114 Peninsula Dental School, University of Plymouth, Plymouth, UK Search for more papers by this author Author Information Donald Singer1,‡, Kristina Thamm2,‡, Heng Zhuang1,3,‡, Jana Karbanová2, Yan Gao1,4, Jemma Victoria Walker1, Heng Jin5,6, Xiangnan Wu7, Clarissa R Coveney8, Pauline Marangoni7, Dongmei Lu9, Portia Rebecca Clare Grayson1, Tulay Gulsen1,19, Karen J Liu10, Stefano Ardu11, Angus KT Wann8, Shouqing Luo12, Alexander C Zambon13, Anton M Jetten14, Christopher Tredwin1, Ophir D Klein7,15, Massimo Attanasio5, Peter Carmeliet16,17, Wieland B Huttner18, Denis Corbeil *,2 and Bing Hu *,1 1Peninsula Dental School, University of Plymouth, Plymouth, UK 2Tissue Engineering Laboratories, Biotechnology Center and Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany 3Department of Cariology, Endodontology and Operative Dentistry, Peking University School and Hospital of Stomatology, Beijing, China 4Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China 5Department of Internal Medicine, University of Iowa, Iowa City, IA, USA 6Department of Emergency Medicine, Tianjin Medical University General Hospital, Tianjin, China 7Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA 8Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute, Nuffield Department for Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford, UK 9Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 10Centre for Craniofacial and Regenerative Biology, King's College London, London, UK 11Division of Cariology & Endodontology, Dental School, University of Geneva, Geneva, Switzerland 12Peninsula Medical School, University of Plymouth, Plymouth, UK 13Biopharmaceutical Sciences, Keck Graduate Institute, Claremont, CA, USA 14Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA 15Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA 16Department of Oncology, Laboratory of Angiogenesis and Vascular Metabolism, KU Leuven, Leuven, Belgium 17VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium 18Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 19Present address: University College London Cancer Institute, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +49 351 463 40118; E-mail: [email protected] *Corresponding author. Tel: +44 1752 437 804; E-mail: [email protected] The EMBO Journal (2019)38:e99845https://doi.org/10.15252/embj.201899845 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly–disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases. Synopsis Using the continuously growing mouse incisor tooth as a model for stem cell activation, primary cilia are shown to function at the onset of differentiation. The stem cell surface marker prominin-1 (Prom1, CD133) is required for sequential recruitment of ciliary components and coordination with sonic hedgehog (SHH) signalling, shedding new light on how stem cell state and differentiation are balanced. Mouse incisor tooth epithelial stem cells and transit-amplifying cells display distinct, dynamic primary cilia biogenesis. Deletion of the ciliary component IFT88 disrupts stem cell activation in vivo. Prom1 is differentially co-expressed with ciliary components Arl13b and Hdac6 in stem cells and transit-amplifying cells, respectively. Prom1 knockout or Lys138 mutation in mice impairs primary cilia biogenesis, quiescent stem cell proliferation, and lineage differentiation. GLIS2 and PROM1 cooperate in nuclei to regulate SHH signaling and its downstream target STAT3. Introduction Tissue homeostasis depends on proper stem cell maintenance and activation to guarantee that balanced cell lineages can be produced upon necessity (Li & Clevers, 2010; Cheung & Rando, 2013). Stem cell hypoactivation can have severe consequence such as regeneration failure (Chen et al, 2012), while stem cell hyperactivation or aging can either exhaust the stem cell pool (Flach et al, 2014) or initiate oncogenesis (Reya et al, 2001; Plaks et al, 2015). At quiescence, stem cells produce long primary cilia that are responsible for sensing and transmitting signals into the cells (Izawa et al, 2015; Sanchez & Dynlacht, 2016). Stem cell activation is accompanied by dynamic primary cilium size, architecture, and assembly–disassembly modifications (Izawa et al, 2015; Sanchez & Dynlacht, 2016) and release of factors from primary cilium to cytoplasm to trigger downstream cascades that are responsible for cell lineage differentiation (Berbari et al, 2009; Goetz & Anderson, 2010). Most evidence for primary cilium participation in stem cell activation and renewal has been based on cultured cells. Historically it has been difficult to investigate stem cell ciliary dynamics in vivo since very few model systems possess enriched stem cells and transit amplifying cells at the same time and location. Therefore, the molecular mechanisms of orchestrating primary cilium assembly and its impact on stem cell fate determination have not been fully understood yet in tissue/organ level. Here, we use continuously growing mouse incisor as a model where epithelial stem cells represent a large proportion of cells at the distal end of the tooth epithelium named cervical loop (CL) (Jussila & Thesleff, 2012; Biehs et al, 2013) that provides a dynamic system in which we are able to profile the functional associations between primary cilia and stem cells. To gain insight into the precise molecular cues orchestrating primary cilium elements recruitment, we focus on one key stem cell marker called Prominin-1 (Prom1, also known as CD133), which is specifically associated with plasmalemmal protrusions (e.g., microvilli and primary cilium) (Corbeil et al, 2001). Prom1 is widely used in identifying stem cells in various somatic tissues and initiating cancer stem cells (Grosse-Gehling et al, 2013). In cancer, numerous lines of evidences suggest Prom1high cancer stem cells are resistant to radio- and chemo-therapies and Prom1 neutralizing antibodies or targeted toxin have anti-tumor activity through unclear mechanism (Damek-Poprawa et al, 2011; Waldron et al, 2011). PROM1 mutations cause various human retinal disorders by disrupting the cilium-derived photoreceptor outer segment (Fargeas et al, 2015), a phenomenon phenocopied in Prom1 null mice (Zacchigna et al, 2009). In photoreceptors, Prom1 interacts with protocadherin 21 (Yang et al, 2008), while other lipid and protein interactors were identified in various biological systems (Röper et al, 2000; Karbanová et al, 2008; Mak et al, 2012) making this pentaspan membrane glycoprotein, or other members of prominin family (Fargeas et al, 2003), a potential mechanoprotein in the organization of plasma membrane protrusion, and hence influencing the signaling pathways associated with them. Furthermore, extracellular membrane vesicles found in various body fluids were identified and immunoisolated based on the presence of Prom1 (Marzesco et al, 2005). They are released either by the membrane budding from plasmalemmal protrusions (Dubreuil et al, 2007) or upon the fusion of multivesicular bodies with plasma membrane and discharged as exosomes (Bauer et al, 2011). Prom1+ vesicles are upregulated in cerebrospinal fluid of patients with gliomas or degenerative neurological disease (Huttner et al, 2008; Bobinger et al, 2017). If and how Prom1 can be used as a molecular biological tool in regulating stem cells for regenerative medicine and anti-cancer therapies are still uncertain. We now show that Prom1 has essential roles in recruiting the primary cilium molecular compartments and in nuclear translocation that are critical for stem cell activation and homeostasis. Results Mouse incisor epithelial stem cells have a dynamic primary cilium biogenesis The mouse incisor cervical loop epithelium (CLE) has a significant transit amplifying cell pool that is adjacent, but molecularly distinct from the stem cells (Appendix Fig S1A). The two cell populations could be distinguished for instance, at postnatal day 7 (P7) lower incisor CLE by immunofluorescence (IF) using markers Sox2 and Bmi1 for stem cells, and Ki67 for transit amplifying cells (Fig 1A and Appendix Fig S1B) or upon laser capture microdissection followed by real-time RT–PCR profiling using markers such as Sox2, Bmi1, CDKn1c, CDKn2b, and p16 for stem cells, and CDK5r1, c-Myc, and Sonic Hedgehog (SHH) for transit amplifying cells (Fig 1B and C). Neighboring to the transit amplifying cells, we identified also a stem cell zone harboring scattered clusters of Ki67-positive cells that represented activated and self-renewing stem cells (Fig 1A). Hence, the CLE system enabled us to monitor temporal and spatial changes in ciliary dynamics during stem cell activation and self-renewal. To validate the primary cilium profiles in the CLE, we performed immunostaining on the primary cilium axoneme (or the core) using anti-acetylated α-tubulin (AcTub) antibodies followed by three-dimensional (3D) measurements to determine whether cilium size and integrity were linked to the stem cell status in vivo (Appendix Fig S1C). Consistent with the conventional cilium dynamic and cell cycle linkage concept, we confirmed that the CLE-associated stem cells (CLESCs) had longer and larger primary cilia and possessed a higher number of cells retaining them comparing to the transit amplifying cells (Fig 1D–G and Appendix Figs S1D and E). Figure 1. Incisor CLE has distinct ciliary dynamics in the stem cells and transit amplifying cells A. Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, anterior; Post, posterior. B, C. The mRNA expression profiling on specific markers of stem cells (B) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real-time RT–PCR (C). qRT–PCR results are in arbitrary values after normalization for GapDH. Statistics was performed on triplicates using one-way ANOVA followed by Bonferroni's test. *P < 0.05; **P < 0.01. Data are presented as mean and standard deviation. D. Representative IF double staining of E-cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue). E. 3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining. F, G. Quantification of cilia length (F) and volume (G) based on 3D reconstruction (E) and comparison between those in stem cell and transit amplifying cell regions. Numbers of quantified cilia were indicated (n). Statistics was performed using one-way ANOVA. Data are presented as mean and standard deviation. **P < 0.01. H. Tamoxifen-induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88. For Cre staining on the same set of samples, please see Appendix Fig S1F. I, J. 3D reconstruction of Sox2-positive regions based on its immunolabeling of the indicated mouse strains (I) and their volume comparison between mouse strains as indicated (J). Statistics was performed using Student's t-test: **P < 0.01. Data are presented as mean and standard deviation. K, L. Representative IF double staining of ADP-ribosylation factor-like protein 13B (Arl13b, red, K) or histone deacetylase 6 (Hdac6, red, L) with AcTub (green) as a marker of the primary cilium core following stem cell to transit amplifying cell transition. M. Summary of the immunolabeling of Arl13b or Hdac6 with AcTub (K, L) along stem cell to transit amplifying cell transition. Download figure Download PowerPoint Primary cilium axoneme assembly–disassembly and cilium membrane formation are closely coordinated cellular events (Sanchez & Dynlacht, 2016). Intraflagellar Transport 88 (IFT88) is one of the key intrinsic cilium components associating with centrosomes and moving along ciliary microtubules during cilium biogenesis to transport molecules such as the SHH pathway members (Pazour et al, 2002). Deletion of IFT88 in neural crest-derived cells or mesenchymal cells causes severe craniofacial deformities (Tian et al, 2017). To investigate how primary cilium functions in the CLESCs, we therefore temporally deleted IFT88 by crossing IFT88flox/flox mice (Haycraft et al, 2007) with tamoxifen-inducible Rosa26CreER transgenic mice (Badea et al, 2003). After the Cre recombinase was activated for 10 days starting from postnatal day 21 (P21) (Fig 1H), we first analyzed the regions where Cre was expressed. Interestingly in the CLE, the Cre expression was highly focused on the stem cells, while in the mesenchyme, the expression is more homogenous (Appendix Fig S1F). Next, we observed a significant reduction of the total stem cell zone volume using 3D reconstruction based on Sox2-positive immunolabeling (Fig 1H–J). The amount of proliferating Ki67+ cells was considerably reduced (Fig 1H). Hence, disrupting cilium dynamics in the stem cells impeded stem cell renewal and activation. Besides general intrinsic cilium molecules such as IFT88, stem cells need to recruit temporary specific molecular players regulating signaling pathway(s) associated with primary cilia to respond to external stimulation. The SHH pathway is one key stem cell-related signaling in the incisor CLESCs (Seidel et al, 2010; Zhao et al, 2014). In response to SHH signal, a cell needs to recruit or express different elements into the primary cilium. Among them, the ADP-ribosylation factor-like 13B ATPase (hereafter Arl13b) is an unique cilium-associated molecule specifically required for mediating SHH signaling (Mariani et al, 2016). In the CLE, we found that Arl13b was preferentially expressed in primary cilia associated with stem cells while its expression disappeared in the transit amplifying cells (Fig 1K and Appendix Fig S1G). On the contrary, when stem cells transitioned into transit amplifying cells, the primary cilia begin to be disassembled and absorbed through deacetylase-mediated tubulin and cortactin deacetylation (Pugacheva et al, 2007; Perdiz et al, 2011). We observed that histone deacetylase 6 (Hdac6) recruited into primary cilia was particularly evident in the transit amplifying cells (Fig 1L and Appendix Fig S1G). The successive recruitment of Arl13b and Hdac6 therefore represented a dynamic primary cilium biogenesis along the stem cell–transit amplifying cell axis (Fig 1M). Prom1 differentially co-expresses with Arl13b at primary cilium during stem cell activation One major defect of the IFT88 mutation is the failure of photoreceptor outer segment assembly and maintenance (Pazour et al, 2002). Interestingly, alterations of Prom1 gene cause similar photoreceptor defects (Zacchigna et al, 2009), raising the possibility that Prom1 participates in stem cell fate determination through affecting cilium dynamics. Using two distinct antibodies targeting either the extracellular domain or cytoplasmic C-terminal end of Prom1, we detected an increased Prom1 expression along the stem cell–transit amplifying cell axis in CLE (Fig 2A and Appendix Fig S2A, respectively). From stem cell toward transit amplifying cell transition, Prom1 had an increased cell surface expression on the transit amplifying cells by comparison with stem cells (Fig 2B). The association of Prom1 with primary cilia that are highlighted with AcTub and Arl13b is evident (Fig 2C and Appendix Fig S2B). In addition to primary cilia, Prom1 labels microvilli as we previously demonstrated in other epithelial cells (Weigmann et al, 1997; Fig 2B and Appendix Fig S2C). The specificity of Prom1 antibodies was validated using the Prom1 KO mice where the respective immunoreactivity was almost abolished (Fig 2D, see below). Likewise, we could again validate the Prom1 antibodies using established primary CLESCs (Appendix Fig S2E, see Materials and Methods) where Prom1 expression (transcript and protein) was silenced by short hairpin RNA (shRNA; Fig 2E and F). Figure 2. Prom1 has a dynamic expression in the incisor CLE primary cilia and nuclei A. Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts. B. 3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited to primary cilium but also to microvilli. C. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%. D. Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice. E, F. The mRNA (E) and protein (F) profiling on shRNA-mediated Prom1 knockdown (3 different shRNAs were used, mar
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