--- _id: '12875' abstract: - lang: eng text: The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny. acknowledged_ssus: - _id: Bio - _id: M-Shop - _id: LifeSc - _id: PreCl acknowledgement: "We thank Liqun Luo for his continued support, for providing essential resources for generating Fzd10-CreER mice which were generated in his laboratory, and for comments on the manuscript; W. Zhong for providing Nestin-Cre transgenic mouse line for this study; A. Heger for mouse colony management; R. Beattie and T. Asenov for designing and producing components of acute slice recovery chamber for MADM-CloneSeq experiments; and K. Leopold, J. Rodarte and N. Amberg for initial experiments, technical support and/or assistance. This study was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Imaging & Optics Facility (IOF), Laboratory Support Facility (LSF), Miba Machine Shop, and Pre-clinical Facility (PCF). G.C. received funding from European Commission (IST plus postdoctoral fellowship). This work was supported by ISTA institutional\r\nfunds; the Austrian Science Fund Special Research Programmes (FWF SFB F78 Neuro Stem Modulation) to S.H. " article_processing_charge: Yes (via OA deal) article_type: original author: - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung orcid: 0000-0001-8457-2572 - first_name: Florian full_name: Pauler, Florian id: 48EA0138-F248-11E8-B48F-1D18A9856A87 last_name: Pauler orcid: 0000-0002-7462-0048 - first_name: Peter full_name: Koppensteiner, Peter id: 3B8B25A8-F248-11E8-B48F-1D18A9856A87 last_name: Koppensteiner orcid: 0000-0002-3509-1948 - first_name: Thomas full_name: Krausgruber, Thomas last_name: Krausgruber - first_name: Carmen full_name: Streicher, Carmen id: 36BCB99C-F248-11E8-B48F-1D18A9856A87 last_name: Streicher - first_name: Martin full_name: Schrammel, Martin id: f13e7cae-e8bd-11ed-841a-96dedf69f46d last_name: Schrammel - first_name: Natalie Y full_name: Özgen, Natalie Y id: e68ece33-f6e0-11ea-865d-ae1031dcc090 last_name: Özgen - first_name: Alexis full_name: Ivec, Alexis id: 1d144691-e8be-11ed-9b33-bdd3077fad4c last_name: Ivec - first_name: Christoph full_name: Bock, Christoph last_name: Bock - first_name: Ryuichi full_name: Shigemoto, Ryuichi id: 499F3ABC-F248-11E8-B48F-1D18A9856A87 last_name: Shigemoto orcid: 0000-0001-8761-9444 - first_name: Simon full_name: Hippenmeyer, Simon id: 37B36620-F248-11E8-B48F-1D18A9856A87 last_name: Hippenmeyer orcid: 0000-0003-2279-1061 citation: ama: Cheung GT, Pauler F, Koppensteiner P, et al. Multipotent progenitors instruct ontogeny of the superior colliculus. Neuron. 2024;112(2):230-246.e11. doi:10.1016/j.neuron.2023.11.009 apa: Cheung, G. T., Pauler, F., Koppensteiner, P., Krausgruber, T., Streicher, C., Schrammel, M., … Hippenmeyer, S. (2024). Multipotent progenitors instruct ontogeny of the superior colliculus. Neuron. Elsevier. https://doi.org/10.1016/j.neuron.2023.11.009 chicago: Cheung, Giselle T, Florian Pauler, Peter Koppensteiner, Thomas Krausgruber, Carmen Streicher, Martin Schrammel, Natalie Y Özgen, et al. “Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus.” Neuron. Elsevier, 2024. https://doi.org/10.1016/j.neuron.2023.11.009. ieee: G. T. Cheung et al., “Multipotent progenitors instruct ontogeny of the superior colliculus,” Neuron, vol. 112, no. 2. Elsevier, p. 230–246.e11, 2024. ista: Cheung GT, Pauler F, Koppensteiner P, Krausgruber T, Streicher C, Schrammel M, Özgen NY, Ivec A, Bock C, Shigemoto R, Hippenmeyer S. 2024. Multipotent progenitors instruct ontogeny of the superior colliculus. Neuron. 112(2), 230–246.e11. mla: Cheung, Giselle T., et al. “Multipotent Progenitors Instruct Ontogeny of the Superior Colliculus.” Neuron, vol. 112, no. 2, Elsevier, 2024, p. 230–246.e11, doi:10.1016/j.neuron.2023.11.009. short: G.T. Cheung, F. Pauler, P. Koppensteiner, T. Krausgruber, C. Streicher, M. Schrammel, N.Y. Özgen, A. Ivec, C. Bock, R. Shigemoto, S. Hippenmeyer, Neuron 112 (2024) 230–246.e11. date_created: 2023-04-27T09:41:48Z date_published: 2024-01-17T00:00:00Z date_updated: 2024-03-05T09:43:02Z day: '17' ddc: - '570' department: - _id: SiHi - _id: RySh doi: 10.1016/j.neuron.2023.11.009 external_id: pmid: - '38096816' file: - access_level: open_access checksum: 32b3788f7085cf44a84108d8faaff3ce content_type: application/pdf creator: dernst date_created: 2024-02-06T13:56:15Z date_updated: 2024-02-06T13:56:15Z file_id: '14944' file_name: 2024_Neuron_Cheung.pdf file_size: 5942467 relation: main_file success: 1 file_date_updated: 2024-02-06T13:56:15Z has_accepted_license: '1' intvolume: ' 112' issue: '2' language: - iso: eng month: '01' oa: 1 oa_version: Published Version page: 230-246.e11 pmid: 1 project: - _id: 059F6AB4-7A3F-11EA-A408-12923DDC885E grant_number: F07805 name: Molecular Mechanisms of Neural Stem Cell Lineage Progression publication: Neuron publication_identifier: issn: - 0896-6273 publication_status: published publisher: Elsevier quality_controlled: '1' related_material: link: - description: News on ISTA Website relation: press_release url: https://ista.ac.at/en/news/the-pedigree-of-brain-cells/ scopus_import: '1' status: public title: Multipotent progenitors instruct ontogeny of the superior colliculus tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 112 year: '2024' ... --- _id: '14683' abstract: - lang: eng text: "Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice.\r\nFor complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1" acknowledged_ssus: - _id: Bio - _id: PreCl acknowledgement: This research was supported by the Scientific Service Units (SSU) at IST Austria through resources provided by the Imaging & Optics Facility (IOF) and Preclinical Facilities (PCF). N.A. received support from FWF Firnberg-Programme (T 1031). G.C. received support from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 754411 as an ISTplus postdoctoral fellow. This work was also supported by IST Austria institutional funds, FWF SFB F78 to S.H., and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 725780 LinPro) to S.H. article_number: '102771' article_processing_charge: No article_type: review author: - first_name: Nicole full_name: Amberg, Nicole id: 4CD6AAC6-F248-11E8-B48F-1D18A9856A87 last_name: Amberg orcid: 0000-0002-3183-8207 - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung orcid: 0000-0001-8457-2572 - first_name: Simon full_name: Hippenmeyer, Simon id: 37B36620-F248-11E8-B48F-1D18A9856A87 last_name: Hippenmeyer orcid: 0000-0003-2279-1061 citation: ama: Amberg N, Cheung GT, Hippenmeyer S. Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry. STAR Protocols. 2023;5(1). doi:10.1016/j.xpro.2023.102771 apa: Amberg, N., Cheung, G. T., & Hippenmeyer, S. (2023). Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry. STAR Protocols. Elsevier. https://doi.org/10.1016/j.xpro.2023.102771 chicago: Amberg, Nicole, Giselle T Cheung, and Simon Hippenmeyer. “Protocol for Sorting Cells from Mouse Brains Labeled with Mosaic Analysis with Double Markers by Flow Cytometry.” STAR Protocols. Elsevier, 2023. https://doi.org/10.1016/j.xpro.2023.102771. ieee: N. Amberg, G. T. Cheung, and S. Hippenmeyer, “Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry,” STAR Protocols, vol. 5, no. 1. Elsevier, 2023. ista: Amberg N, Cheung GT, Hippenmeyer S. 2023. Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry. STAR Protocols. 5(1), 102771. mla: Amberg, Nicole, et al. “Protocol for Sorting Cells from Mouse Brains Labeled with Mosaic Analysis with Double Markers by Flow Cytometry.” STAR Protocols, vol. 5, no. 1, 102771, Elsevier, 2023, doi:10.1016/j.xpro.2023.102771. short: N. Amberg, G.T. Cheung, S. Hippenmeyer, STAR Protocols 5 (2023). date_created: 2023-12-13T11:48:05Z date_published: 2023-12-08T00:00:00Z date_updated: 2023-12-18T08:06:14Z day: '08' ddc: - '570' department: - _id: SiHi doi: 10.1016/j.xpro.2023.102771 ec_funded: 1 external_id: pmid: - '38070137' intvolume: ' 5' issue: '1' keyword: - General Immunology and Microbiology - General Biochemistry - Genetics and Molecular Biology - General Neuroscience language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1016/j.xpro.2023.102771 month: '12' oa: 1 oa_version: Submitted Version pmid: 1 project: - _id: 268F8446-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: T0101031 name: Role of Eed in neural stem cell lineage progression - _id: 260C2330-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '754411' name: ISTplus - Postdoctoral Fellowships - _id: 059F6AB4-7A3F-11EA-A408-12923DDC885E grant_number: F07805 name: Molecular Mechanisms of Neural Stem Cell Lineage Progression - _id: 260018B0-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '725780' name: Principles of Neural Stem Cell Lineage Progression in Cerebral Cortex Development publication: STAR Protocols publication_identifier: issn: - 2666-1667 publication_status: epub_ahead publisher: Elsevier quality_controlled: '1' scopus_import: '1' status: public title: Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 5 year: '2023' ... --- _id: '14783' abstract: - lang: eng text: Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy. acknowledgement: 'This research was funded by grants from the European Research Council (Consolidator grant #683154) and European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie Innovative Training Networks, grant #722053, EU-GliaPhD) to N.R., as well as from FP7-PEOPLE Marie Curie Intra-European Fellowship for career development (grant #622289) to G.C. We thank Elena Dossi, Grégory Ghézali, and Jérémie Teillon for support with setting up the MEA system for the two-photon microscope. We would also like to thank Tayfun Palaz for their technical assistance with the EM preparations.' article_number: '1133' article_processing_charge: Yes article_type: original author: - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung orcid: 0000-0001-8457-2572 - first_name: Oana full_name: Chever, Oana last_name: Chever - first_name: Astrid full_name: Rollenhagen, Astrid last_name: Rollenhagen - first_name: Nicole full_name: Quenech’du, Nicole last_name: Quenech’du - first_name: Pascal full_name: Ezan, Pascal last_name: Ezan - first_name: Joachim H. R. full_name: Lübke, Joachim H. R. last_name: Lübke - first_name: Nathalie full_name: Rouach, Nathalie last_name: Rouach citation: ama: Cheung GT, Chever O, Rollenhagen A, et al. Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses. Cells. 2023;12(8). doi:10.3390/cells12081133 apa: Cheung, G. T., Chever, O., Rollenhagen, A., Quenech’du, N., Ezan, P., Lübke, J. H. R., & Rouach, N. (2023). Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses. Cells. MDPI. https://doi.org/10.3390/cells12081133 chicago: Cheung, Giselle T, Oana Chever, Astrid Rollenhagen, Nicole Quenech’du, Pascal Ezan, Joachim H. R. Lübke, and Nathalie Rouach. “Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.” Cells. MDPI, 2023. https://doi.org/10.3390/cells12081133. ieee: G. T. Cheung et al., “Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses,” Cells, vol. 12, no. 8. MDPI, 2023. ista: Cheung GT, Chever O, Rollenhagen A, Quenech’du N, Ezan P, Lübke JHR, Rouach N. 2023. Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses. Cells. 12(8), 1133. mla: Cheung, Giselle T., et al. “Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.” Cells, vol. 12, no. 8, 1133, MDPI, 2023, doi:10.3390/cells12081133. short: G.T. Cheung, O. Chever, A. Rollenhagen, N. Quenech’du, P. Ezan, J.H.R. Lübke, N. Rouach, Cells 12 (2023). date_created: 2024-01-10T09:46:35Z date_published: 2023-04-11T00:00:00Z date_updated: 2024-01-16T09:29:35Z day: '11' ddc: - '570' department: - _id: SiHi doi: 10.3390/cells12081133 external_id: isi: - '000977445700001' pmid: - '37190042' file: - access_level: open_access checksum: 6798cd75d8857976fbc58a43fd173d68 content_type: application/pdf creator: dernst date_created: 2024-01-16T09:26:52Z date_updated: 2024-01-16T09:26:52Z file_id: '14808' file_name: 2023_Cells_Cheung.pdf file_size: 7931643 relation: main_file success: 1 file_date_updated: 2024-01-16T09:26:52Z has_accepted_license: '1' intvolume: ' 12' isi: 1 issue: '8' keyword: - General Medicine language: - iso: eng month: '04' oa: 1 oa_version: Published Version pmid: 1 publication: Cells publication_identifier: issn: - 2073-4409 publication_status: published publisher: MDPI quality_controlled: '1' status: public title: Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 12 year: '2023' ... --- _id: '10764' abstract: - lang: eng text: Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes. acknowledgement: 'We thank D. Mazaud and. J. Cazères for technical assistance. This work was supported by grants from the European Research Council (Consolidator grant #683154) and European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie Innovative Training Networks, grant #722053, EU-GliaPhD) to N.R. and from FP7-PEOPLE Marie Curie Intra-European Fellowship for career development (grant #622289) to G.C.' article_number: '753' article_processing_charge: No article_type: original author: - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung - first_name: Danijela full_name: Bataveljic, Danijela last_name: Bataveljic - first_name: Josien full_name: Visser, Josien last_name: Visser - first_name: Naresh full_name: Kumar, Naresh last_name: Kumar - first_name: Julien full_name: Moulard, Julien last_name: Moulard - first_name: Glenn full_name: Dallérac, Glenn last_name: Dallérac - first_name: Daria full_name: Mozheiko, Daria last_name: Mozheiko - first_name: Astrid full_name: Rollenhagen, Astrid last_name: Rollenhagen - first_name: Pascal full_name: Ezan, Pascal last_name: Ezan - first_name: Cédric full_name: Mongin, Cédric last_name: Mongin - first_name: Oana full_name: Chever, Oana last_name: Chever - first_name: Alexis Pierre full_name: Bemelmans, Alexis Pierre last_name: Bemelmans - first_name: Joachim full_name: Lübke, Joachim last_name: Lübke - first_name: Isabelle full_name: Leray, Isabelle last_name: Leray - first_name: Nathalie full_name: Rouach, Nathalie last_name: Rouach citation: ama: Cheung GT, Bataveljic D, Visser J, et al. Physiological synaptic activity and recognition memory require astroglial glutamine. Nature Communications. 2022;13. doi:10.1038/s41467-022-28331-7 apa: Cheung, G. T., Bataveljic, D., Visser, J., Kumar, N., Moulard, J., Dallérac, G., … Rouach, N. (2022). Physiological synaptic activity and recognition memory require astroglial glutamine. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-022-28331-7 chicago: Cheung, Giselle T, Danijela Bataveljic, Josien Visser, Naresh Kumar, Julien Moulard, Glenn Dallérac, Daria Mozheiko, et al. “Physiological Synaptic Activity and Recognition Memory Require Astroglial Glutamine.” Nature Communications. Springer Nature, 2022. https://doi.org/10.1038/s41467-022-28331-7. ieee: G. T. Cheung et al., “Physiological synaptic activity and recognition memory require astroglial glutamine,” Nature Communications, vol. 13. Springer Nature, 2022. ista: Cheung GT, Bataveljic D, Visser J, Kumar N, Moulard J, Dallérac G, Mozheiko D, Rollenhagen A, Ezan P, Mongin C, Chever O, Bemelmans AP, Lübke J, Leray I, Rouach N. 2022. Physiological synaptic activity and recognition memory require astroglial glutamine. Nature Communications. 13, 753. mla: Cheung, Giselle T., et al. “Physiological Synaptic Activity and Recognition Memory Require Astroglial Glutamine.” Nature Communications, vol. 13, 753, Springer Nature, 2022, doi:10.1038/s41467-022-28331-7. short: G.T. Cheung, D. Bataveljic, J. Visser, N. Kumar, J. Moulard, G. Dallérac, D. Mozheiko, A. Rollenhagen, P. Ezan, C. Mongin, O. Chever, A.P. Bemelmans, J. Lübke, I. Leray, N. Rouach, Nature Communications 13 (2022). date_created: 2022-02-20T23:01:30Z date_published: 2022-02-08T00:00:00Z date_updated: 2023-08-02T14:25:01Z day: '08' ddc: - '570' department: - _id: SiHi doi: 10.1038/s41467-022-28331-7 external_id: isi: - '000757297200017' pmid: - '35136061' file: - access_level: open_access checksum: 51d580aff2327dd957946208a9749e1a content_type: application/pdf creator: dernst date_created: 2022-02-21T07:51:33Z date_updated: 2022-02-21T07:51:33Z file_id: '10777' file_name: 2022_NatureCommunications_Cheung.pdf file_size: 7910519 relation: main_file success: 1 file_date_updated: 2022-02-21T07:51:33Z has_accepted_license: '1' intvolume: ' 13' isi: 1 language: - iso: eng month: '02' oa: 1 oa_version: Published Version pmid: 1 publication: Nature Communications publication_identifier: eissn: - '20411723' publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Physiological synaptic activity and recognition memory require astroglial glutamine tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 13 year: '2022' ... --- _id: '7815' abstract: - lang: eng text: Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present. acknowledged_ssus: - _id: Bio - _id: LifeSc - _id: PreCl article_number: e61147 article_processing_charge: No article_type: original author: - first_name: Robert J full_name: Beattie, Robert J id: 2E26DF60-F248-11E8-B48F-1D18A9856A87 last_name: Beattie orcid: 0000-0002-8483-8753 - first_name: Carmen full_name: Streicher, Carmen id: 36BCB99C-F248-11E8-B48F-1D18A9856A87 last_name: Streicher - first_name: Nicole full_name: Amberg, Nicole id: 4CD6AAC6-F248-11E8-B48F-1D18A9856A87 last_name: Amberg orcid: 0000-0002-3183-8207 - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung orcid: 0000-0001-8457-2572 - first_name: Ximena full_name: Contreras, Ximena id: 475990FE-F248-11E8-B48F-1D18A9856A87 last_name: Contreras - first_name: Andi H full_name: Hansen, Andi H id: 38853E16-F248-11E8-B48F-1D18A9856A87 last_name: Hansen - first_name: Simon full_name: Hippenmeyer, Simon id: 37B36620-F248-11E8-B48F-1D18A9856A87 last_name: Hippenmeyer orcid: 0000-0003-2279-1061 citation: ama: Beattie RJ, Streicher C, Amberg N, et al. Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM). Journal of Visual Experiments. 2020;(159). doi:10.3791/61147 apa: Beattie, R. J., Streicher, C., Amberg, N., Cheung, G. T., Contreras, X., Hansen, A. H., & Hippenmeyer, S. (2020). Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM). Journal of Visual Experiments. MyJove Corporation. https://doi.org/10.3791/61147 chicago: Beattie, Robert J, Carmen Streicher, Nicole Amberg, Giselle T Cheung, Ximena Contreras, Andi H Hansen, and Simon Hippenmeyer. “Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM).” Journal of Visual Experiments. MyJove Corporation, 2020. https://doi.org/10.3791/61147. ieee: R. J. Beattie et al., “Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM),” Journal of Visual Experiments, no. 159. MyJove Corporation, 2020. ista: Beattie RJ, Streicher C, Amberg N, Cheung GT, Contreras X, Hansen AH, Hippenmeyer S. 2020. Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM). Journal of Visual Experiments. (159), e61147. mla: Beattie, Robert J., et al. “Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM).” Journal of Visual Experiments, no. 159, e61147, MyJove Corporation, 2020, doi:10.3791/61147. short: R.J. Beattie, C. Streicher, N. Amberg, G.T. Cheung, X. Contreras, A.H. Hansen, S. Hippenmeyer, Journal of Visual Experiments (2020). date_created: 2020-05-11T08:31:20Z date_published: 2020-05-08T00:00:00Z date_updated: 2024-03-27T23:30:41Z day: '08' ddc: - '570' department: - _id: SiHi doi: 10.3791/61147 ec_funded: 1 external_id: isi: - '000546406600043' file: - access_level: open_access checksum: 3154ea7f90b9fb45e084cd1c2770597d content_type: application/pdf creator: rbeattie date_created: 2020-05-11T08:28:38Z date_updated: 2020-07-14T12:48:03Z file_id: '7816' file_name: jove-protocol-61147-lineage-tracing-clonal-analysis-developing-cerebral-cortex-using.pdf file_size: 1352186 relation: main_file file_date_updated: 2020-07-14T12:48:03Z has_accepted_license: '1' isi: 1 issue: '159' language: - iso: eng month: '05' oa: 1 oa_version: Published Version project: - _id: 264E56E2-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: M02416 name: Molecular Mechanisms Regulating Gliogenesis in the Cerebral Cortex - _id: 268F8446-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: T0101031 name: Role of Eed in neural stem cell lineage progression - _id: 260C2330-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '754411' name: ISTplus - Postdoctoral Fellowships - _id: 2625A13E-B435-11E9-9278-68D0E5697425 grant_number: '24812' name: Molecular Mechanisms of Radial Neuronal Migration - _id: 260018B0-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '725780' name: Principles of Neural Stem Cell Lineage Progression in Cerebral Cortex Development publication: Journal of Visual Experiments publication_identifier: issn: - 1940-087X publication_status: published publisher: MyJove Corporation quality_controlled: '1' related_material: record: - id: '7902' relation: part_of_dissertation status: public scopus_import: '1' status: public title: Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM) tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 year: '2020' ... --- _id: '7005' abstract: - lang: eng text: Activity-dependent bulk endocytosis generates synaptic vesicles (SVs) during intense neuronal activity via a two-step process. First, bulk endosomes are formed direct from the plasma membrane from which SVs are then generated. SV generation from bulk endosomes requires the efflux of previously accumulated calcium and activation of the protein phosphatase calcineurin. However, it is still unknown how calcineurin mediates SV generation. We addressed this question using a series of acute interventions that decoupled the generation of SVs from bulk endosomes in rat primary neuronal culture. This was achieved by either disruption of protein–protein interactions via delivery of competitive peptides, or inhibition of enzyme activity by known inhibitors. SV generation was monitored using either a morphological horseradish peroxidase assay or an optical assay that monitors the replenishment of the reserve SV pool. We found that SV generation was inhibited by, (i) peptides that disrupt calcineurin interactions, (ii) an inhibitor of dynamin I GTPase activity and (iii) peptides that disrupt the phosphorylation-dependent dynamin I–syndapin I interaction. Peptides that disrupted syndapin I interactions with eps15 homology domain-containing proteins had no effect. This revealed that (i) calcineurin must be localized at bulk endosomes to mediate its effect, (ii) dynamin I GTPase activity is essential for SV fission and (iii) the calcineurin-dependent interaction between dynamin I and syndapin I is essential for SV generation. We therefore propose that a calcineurin-dependent dephosphorylation cascade that requires both dynamin I GTPase and syndapin I lipid-deforming activity is essential for SV generation from bulk endosomes. article_processing_charge: No article_type: original author: - first_name: Giselle T full_name: Cheung, Giselle T id: 471195F6-F248-11E8-B48F-1D18A9856A87 last_name: Cheung orcid: 0000-0001-8457-2572 - first_name: Michael A. full_name: Cousin, Michael A. last_name: Cousin citation: ama: Cheung GT, Cousin MA. Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction. Journal of Neurochemistry. 2019;151(5):570-583. doi:10.1111/jnc.14862 apa: Cheung, G. T., & Cousin, M. A. (2019). Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction. Journal of Neurochemistry. Wiley. https://doi.org/10.1111/jnc.14862 chicago: Cheung, Giselle T, and Michael A. Cousin. “Synaptic Vesicle Generation from Activity‐dependent Bulk Endosomes Requires a Dephosphorylation‐dependent Dynamin–Syndapin Interaction.” Journal of Neurochemistry. Wiley, 2019. https://doi.org/10.1111/jnc.14862. ieee: G. T. Cheung and M. A. Cousin, “Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction,” Journal of Neurochemistry, vol. 151, no. 5. Wiley, pp. 570–583, 2019. ista: Cheung GT, Cousin MA. 2019. Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction. Journal of Neurochemistry. 151(5), 570–583. mla: Cheung, Giselle T., and Michael A. Cousin. “Synaptic Vesicle Generation from Activity‐dependent Bulk Endosomes Requires a Dephosphorylation‐dependent Dynamin–Syndapin Interaction.” Journal of Neurochemistry, vol. 151, no. 5, Wiley, 2019, pp. 570–83, doi:10.1111/jnc.14862. short: G.T. Cheung, M.A. Cousin, Journal of Neurochemistry 151 (2019) 570–583. date_created: 2019-11-12T14:37:08Z date_published: 2019-12-01T00:00:00Z date_updated: 2023-08-30T07:21:50Z day: '01' ddc: - '570' department: - _id: SiHi doi: 10.1111/jnc.14862 external_id: isi: - '000490703100001' pmid: - '31479508' file: - access_level: open_access checksum: ec1fb2aebb874009bc309adaada6e1d7 content_type: application/pdf creator: dernst date_created: 2020-02-05T10:30:02Z date_updated: 2020-07-14T12:47:47Z file_id: '7452' file_name: 2019_JournNeurochemistry_Cheung.pdf file_size: 4334962 relation: main_file file_date_updated: 2020-07-14T12:47:47Z has_accepted_license: '1' intvolume: ' 151' isi: 1 issue: '5' language: - iso: eng month: '12' oa: 1 oa_version: Published Version page: 570-583 pmid: 1 publication: Journal of Neurochemistry publication_identifier: eissn: - 1471-4159 issn: - 0022-3042 publication_status: published publisher: Wiley quality_controlled: '1' scopus_import: '1' status: public title: Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 151 year: '2019' ...