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Temporal and Spatial Patterning of Esrrb Expression During Cerebellar Development Tsai, Casper; Ramirez, Miguel; Robert, Remi; Yeung, Joanna; FANTOM5 Consortium; Goldowitz, Dan 2018-03-17

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Casper Tsai1,2, Miguel Ramirez1,2,3, Remi Robert5, Joanna Yeung1,2, FANTOM5 Consortium6, Dan Goldowitz1,2,3,4To characterize the spatial (eg. Cell types) and temporal expression of Esrrb during cerebellar developmentTemporal and Spatial Patterning of Esrrb Expression During Cerebellar Development1Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada; 2BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, Canada; 3Genome Science and Technology; 4Neurosciencves, University of British Columbia, Vancouver, Canada; 5Université de Rennes 1, Rennes, France; 6RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, JapanB A C KG R O U N D• Cerebellum controls motor coordination• Defective cerebellum causes impairment of fine motor functions such as balance and coordination• General morphological characteristics of cerebellar development is understood but the genetic regulation that drive these morphological changes are unknown• Recently, in collaboration with the FANTOM5 consortium, transcriptomic data was collected from the developing mouse cerebellum at 12 time points through Cap Analysis Gene Expression (CAGE) sequencing• A gene transcription network was constructed using the transcriptomic data to identify important genes in cerebellar development• Set of criteria used to select a gene of interest:• No previous research related to cerebellum development• Differentially expressed in time course• Is a transcription factor• Identified in gene transcription network• Expression in cerebellum identified by in situ hybridization in the Allen Brain Atlas• Neural or motor phenotype identified in KO mice• Estrogen related receptor beta (Esrrb) satisfied all criteriaFigure  1. A. Cerebellar development time points included in the FANTOM5 CAGE-sequencing dataset B. Gene network from FANTOM5 CAGE-sequencing dataset.O B J E C T I V ER E S U LT SC O N C L U S I O N S  &  F U T U R E  D I R E C T I O N Swww.cmmt.ubc.caABFigure 3. In situ hybridization of Esrrb in cerebellum at A. E13.5 B. E15.5 and C. E18.5. Presence of staining of Esrrb in the ventricular zone confirms the expression of Esrrb in the developing cerebellum. CP, choroid plexus; EGL, external granule layer; PCP, Purkinje cell progenitors; VZ, ventricular zone.A B CFigure 5. Immunofluorescent co-staining of Esrrbwith Ptf1a in cerebellum at E12.5. A. shows Esrrbin green B. shows Ptf1a in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Ptf1a is a marker for a spatial zone where progenitor cells of the GABAergic lineage (includes Purkinje cell progenitors and interneuron progenitors) arise and migrate from. Positive co-staining of Ptf1a with Esrrb indicates that Esrrb is expressed in GABAergic progenitor cells. RL, rhombic lip; VZ, ventricular zone.Figure 6. Immunofluorescent co-staining of Esrrb with Pax2 in cerebellum at E15.5. A. shows Esrrb in green B. shows Pax2 in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Pax2 is a marker for interneuron progenitors. The lack of co-staining between Esrrb and Pax2 indicates that Esrrb is not expressed in interneuron progenitor at this time. EGL, external granular layer; RL, rhombic lip; VZ, ventricular zone.Figure 7. Immunofluorescent co-staining of Esrrbwith Pax2 in cerebellum at E18.5. A. shows Esrrbin green B. shows Pax2 in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Pax2 is a marker for interneuron progenitors. The lack of co-staining between Esrrb and Pax2 indicates that Esrrb is not expressed in interneuron progenitors at this time as well. This, along with Figure 6 likely suggests Esrrb is not expressed in interneuron progenitors in embryonic cerebellum. EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.Figure 8. Immunofluorescent co-staining of Esrrbwith Calb in cerebellum at E18.5. A. shows Esrrb in green B. shows Calb in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Calb is a marker for Purkinje cell progenitors. Positive co-staining between Esrrb and Calb indicates that Esrrb is expressed in Purkinje cell progenitors at E18.5. In conjunction with Figure 5, 6, and 7, this shows that Esrrb is selectively expressed in Purkinje cell progenitors. CP, choroid plexus; EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.• Esrrb is expressed in Purkinje cell progenitors and not interneuron progenitors between E12.5 and E18.5, suggesting it could be important in cerebellar development• These results could help us validate our gene network which aims to identify important genes in cerebellar development• Future studies could include look into functional significance of Esrrb in cerebellar developmentFigure 4. Immunofluorescent staining of Esrrb in cerebellum at A. E12.5 B. E15.5, and C. E18.5. The leftmost column shows the DAPI stain in blue, which stains all nuclei. The middle column shows Esrrb staining in green. The rightmost column shows the overlaid DAPI (blue) and Esrrb (green) staining. A. shows the Esrrb-positive cells staining the ventricular zone. B. shows the Esrrb-positive cells migrating and spreading towards the cortex. C. shows the Esrrb-positive cells arranging itself in a layer directly beneath the cortex. EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.ABCCPVZCPVZEGLCPPCPEGLRLVZEGLRLVZEGLRLVZEGLRLVZEGLPCLRLVZEGLPCLRLVZEGLPCLFigure 2. Expression of Esrrb in transcripts per million (TPM) in embryonic and postnatal cerebellum at 12 different time points as determined by CAGE sequencing.Esrrb peaks in expression during embryonic time points, suggesting its importance during that timeExpression (TPM)EsrrbRLVZRLVZRLVZE13.5 E13.5 E13.5E15.5 E15.5 E15.5E18.5 E18.5 E18.5RLVZRLVZRLVZ VZA BC DRL VZEGLRL VZEGLRL VZEGLVZBAC DRLVZEGL PCLRLVZEGL PCLRLVZEGL PCLA BC DA C K N O W L E D G E M E N T S• Funding from NSERC Discovery Award and NeuroDev Net/KidsBrainHealth Network• Special thanks to all current and former members of the Goldowitz labA BC DRLVZEGLPCLCP RLVZEGLPCLCPRLVZEGLPCLCPPCLEGLStudent Author: Casper TsaiSupervisor: Dan GoldowitzDate of presentation: March 17, 2018MURC Casper Tsai1,2, Miguel Ramirez1,2,3, Remi Robert5, Joanna Yeung1,2, FANTOM5 Consortium6, Dan Goldowitz1,2,3,4To characterize the spatial (eg. Cell types) and temporal expression of Esrrb during cerebellar developmentTemporal and Spatial Patterning of Esrrb Expression During Cerebellar Development1Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada; 2BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, Canada; 3Genome Science and Technology; 4Neurosciencves, University of British Columbia, Vancouver, Canada; 5Université de Rennes 1, Rennes, France; 6RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, JapanB A C KG R O U N D• Cerebellum controls motor coordination• Defective cerebellum causes impairment of fine motor functions such as balance and coordination• General morphological characteristics of cerebellar development is understood but the genetic regulation that drive these morphological changes are unknown• Recently, in collaboration with the FANTOM5 consortium, transcriptomic data was collected from the developing mouse cerebellum at 12 time points through Cap Analysis Gene Expression (CAGE) sequencing• A gene transcription network was constructed using the transcriptomic data to identify important genes in cerebellar development• Set of criteria used to select a gene of interest:• No previous research related to cerebellum development• Differentially expressed in time course• Is a transcription factor• Identified in gene transcription network• Expression in cerebellum identified by in situ hybridization in the Allen Brain Atlas• Neural or motor phenotype identified in KO mice• Estrogen related receptor beta (Esrrb) satisfied all criteriaFigure  1. A. Cerebellar development time points included in the FANTOM5 CAGE-sequencing dataset B. Gene network from FANTOM5 CAGE-sequencing dataset.O B J E C T I V ER E S U LT SC O N C L U S I O N S  &  F U T U R E  D I R E C T I O N Swww.cmmt.ubc.caABFigure 3. In situ hybridization of Esrrb in cerebellum at A. E13.5 B. E15.5 and C. E18.5. Presence of staining of Esrrb in the ventricular zone confirms the expression of Esrrb in the developing cerebellum. CP, choroid plexus; EGL, external granule layer; PCP, Purkinje cell progenitors; VZ, ventricular zone.A B CFigure 5. Immunofluorescent co-staining of Esrrbwith Ptf1a in cerebellum at E12.5. A. shows Esrrbin green B. shows Ptf1a in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Ptf1a is a marker for a spatial zone where progenitor cells of the GABAergic lineage (includes Purkinje cell progenitors and interneuron progenitors) arise and migrate from. Positive co-staining of Ptf1a with Esrrb indicates that Esrrb is expressed in GABAergic progenitor cells. RL, rhombic lip; VZ, ventricular zone.Figure 6. Immunofluorescent co-staining of Esrrb with Pax2 in cerebellum at E15.5. A. shows Esrrb in green B. shows Pax2 in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Pax2 is a marker for interneuron progenitors. The lack of co-staining between Esrrb and Pax2 indicates that Esrrb is not expressed in interneuron progenitor at this time. EGL, external granular layer; RL, rhombic lip; VZ, ventricular zone.Figure 7. Immunofluorescent co-staining of Esrrbwith Pax2 in cerebellum at E18.5. A. shows Esrrbin green B. shows Pax2 in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Pax2 is a marker for interneuron progenitors. The lack of co-staining between Esrrb and Pax2 indicates that Esrrb is not expressed in interneuron progenitors at this time as well. This, along with Figure 6 likely suggests Esrrb is not expressed in interneuron progenitors in embryonic cerebellum. EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.Figure 8. Immunofluorescent co-staining of Esrrbwith Calb in cerebellum at E18.5. A. shows Esrrb in green B. shows Calb in red C. shows an overlay of the two stains as well as DAPI in blue D. shows a magnified view of the co-stained region. Calb is a marker for Purkinje cell progenitors. Positive co-staining between Esrrb and Calb indicates that Esrrb is expressed in Purkinje cell progenitors at E18.5. In conjunction with Figure 5, 6, and 7, this shows that Esrrb is selectively expressed in Purkinje cell progenitors. CP, choroid plexus; EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.• Esrrb is expressed in Purkinje cell progenitors and not interneuron progenitors between E12.5 and E18.5, suggesting it could be important in cerebellar development• These results could help us validate our gene network which aims to identify important genes in cerebellar development• Future studies could include look into functional significance of Esrrb in cerebellar developmentFigure 4. Immunofluorescent staining of Esrrb in cerebellum at A. E12.5 B. E15.5, and C. E18.5. The leftmost column shows the DAPI stain in blue, which stains all nuclei. The middle column shows Esrrb staining in green. The rightmost column shows the overlaid DAPI (blue) and Esrrb (green) staining. A. shows the Esrrb-positive cells staining the ventricular zone. B. shows the Esrrb-positive cells migrating and spreading towards the cortex. C. shows the Esrrb-positive cells arranging itself in a layer directly beneath the cortex. EGL, external granular layer; PCL, Purkinje cell layer; RL, rhombic lip; VZ, ventricular zone.ABCCPVZCPVZEGLCPPCPEGLRLVZEGLRLVZEGLRLVZEGLRLVZEGLPCLRLVZEGLPCLRLVZEGLPCLFigure 2. Expression of Esrrb in transcripts per million (TPM) in embryonic and postnatal cerebellum at 12 different time points as determined by CAGE sequencing.Esrrb peaks in expression during embryonic time points, suggesting its importance during that timeExpression (TPM)EsrrbRLVZRLVZRLVZE13.5 E13.5 E13.5E15.5 E15.5 E15.5E18.5 E18.5 E18.5RLVZRLVZRLVZ VZA BC DRL VZEGLRL VZEGLRL VZEGLVZBAC DRLVZEGL PCLRLVZEGL PCLRLVZEGL PCLA BC DA C K N O W L E D G E M E N T S• Funding from NSERC Discovery Award and NeuroDev Net/KidsBrainHealth Network• Special thanks to all current and former members of the Goldowitz labA BC DRLVZEGLPCLCP RLVZEGLPCLCPRLVZEGLPCLCPPCLEGLStudent Author: Casper TsaiSupervisor: Dan GoldowitzDate of presentation: March 17, 2018MURC Temporal and Spatial Patterning of Esrrb Expression During Cerebellar Development The cerebellum is a part of the brain responsible for fine motor skills such as balance and coordination. Defective cerebellar development can often cause impairment of motor functions. While we have a good understanding of the anatomical changes during cerebellar development, genetic regulation that drives these changes are largely unknown. We previously collected mRNA transcripts from developing mouse cerebellum and sequenced them to obtain gene expression levels at several time points. A gene expression network was then constructed using this data in hopes of identifying important genes during cerebellar development. Among the genes in the network, Estrogen-related receptor beta (Esrrb) was determined through a list of biological criteria to likely be involved in cerebellar development. To understand the role of Esrrb in cerebellar development, we sought to identify the spatial distribution of Esrrb and Essrb-expressing cell-type in the developing cerebellum. In-situ hybridization (ISH) and immunofluorescence staining of Esrrb were performed on embryonic cerebellum to determine the spatial distribution of Esrrb at different time points in cerebellar development. ISH confirmed Esrrb expression in the cerebellum while immunofluorescence localized Esrrb expression to the ventricular zone at early embryonic stages and to the Purkinje cell layer at late embryonic stages. In addition, Esrrb was co-stained with other cell-type specific markers to identify the cell-type of Esrrb-expressing cells. Positive immunofluorescence co-staining with Ptf1a suggested Esrrb expression in GABAergic cells. Furthermore, co-staining with other markers suggest that Esrrb is specific to Purkinje cell progenitors in the developing cerebellum, a cell-type important in normal cerebellar function.  

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