UC San Diego UC San Diego Electronic Theses and Dissertations Title Cell type specific gene expression : profiling and targeting Permalink https://escholarship.org/uc/item/36m3m5n3 Author Nathanson, Jason Lawrence Publication Date 2008 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Cell type specific gene expression: profiling and targeting A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Jason Lawrence Nathanson Committee in charge: Professor Lanping Amy Sung, Chair Professor Edward M Callaway, Co-Chair Professor Fred H Gage Professor Vivian Hook Professor Xiaohua Huang Professor Gabriel A Silva 2008 Copyright Jason Nathanson, 2008 All rights reserved. The Dissertation of Jason Lawrence Nathanson is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-chair Chair University of California, San Diego 2008 iii DEDICATION I dedicate this dissertation in recognition of the untiring support and encouragement from family and friends. I am especially grateful to my parents for their support and love from the beginning of my days, to my mom’s parents for their unwavering financial and inspirational support of my undergraduate and graduate school endeavors, to my dad’s parents for their love and hugs, to my brothers and sisters for their laughter and encouragement, and to Noah for his tolerance of my late hours and grouchy moods. My appreciation also goes to all my teachers and co-workers for their guidance, patience and goodwill. iv TABLE OF CONTENTS Signature Page…………………………………………………………….... iii Dedication…………………………………………………………………... iv Table of Contents…………………………………………………………… v List of Figures………………………………………………………………. viii List of Tables……………………………………………………………….. x Acknowledgements…………………………………………………………. xi Vita………………………………………………………………………….. xii Abstract of the Dissertation…………………………………………………. xiii Chapter 1 Cell type profiling of hippocampal neurogenesis using laser capture microdissection, DNA microarrays and anatomical-based clustering……… 1 Abstract……………………………………………………………… 2 Introduction…………………………………………………………. 3 Materials and Methods……………………………………………… 8 Results………………………………………………………………. 13 1.1 DNA microarrays successfully identified genes enriched in the inner portion of the GZ and a number of these genes corresponded to known cell types…………………………….... 13 1.2 Anatomical-based clustering grouped genes into basic cell types……………………………………………………….. 14 1.3 Over-expressed gene ontology classifications identified functional roles for groups of clustered genes…………………. 19 1.4 Some SGZ enriched genes also showed running-induced increases in gene expression…………………………………… 22 Conclusions……………………………………………………….… 23 Figures………………………………………………………………. 28 v Tables……………………………………………………………….. 49 Chapter 2 Selective transduction of inhibitory and excitatory cortical neurons by endogenous tropism of AAV and lentiviral vectors………………………... 52 Abstract……………………………………………………………... 53 Introduction…………………………………………………………. 54 Materials and Methods……………………………………………… 56 Results………………………………………………………………. 61 2.1 AAV biased expression to inhibitory neurons…………………. 62 2.2 AAV tropism showed titer dependency………………………... 63 2.3 LV predominately labeled excitatory neurons………………... 64 Conclusions…………………………………………………………. 67 Figures………………………………………………………………. 72 Tables………………………………………………………………... 77 Chapter 3 Short promoters drive inhibitory neuron class specific, but not subtype specific expression in the mammalian brain………………………………... 79 Abstract……………………………………………………………... 80 Introduction…………………………………………………………. 82 Materials and Methods……………………………………………… 87 Results………………………………………………………………. 97 3.1 Fugu promoters…………………………………………………. 97 3.1.1 Fugu promoters in AAV drove expression in mouse cortex………………………………………… 97 3.1.2 Fugu promoters in AAV drove expression in vi rat and monkey cortex………………………………. 99 3.1.3 Fugu promoters in AAV drove expression in dentate gyrus and thalamus…………………………. 99 3.1.4 AAV-fSST expression strongly favored cortical inhibitory neurons…………………………………… 100 3.1.5 Fugu promoters failed to generate inhibitory cell subtype specific expression…………………………. 101 3.1.6 Fugu promoter in lentivirus drove expression in excitatory neurons…………………………………... 102 3.2 Mammalian promoters…………………………………………. 103 3.3 Transcription factor binding site assembled promoters……….. 105 Conclusions………………………………………………………… 107 Figures……………………………………………………………… 113 Tables………………………………………………………………. 129 References………………………………………………………………….. 135 vii LIST OF FIGURES Chapter 1 Figure 1 The SGZ of the dentate gyrus consists of many different cell types…………………………………………………………. 28 Figure 2 Laser capture microdissection (LCM) isolated the inner and outer regions of the dentate gyrus………………………..……… 29 Figure 3 In situ hybridization verified SGZ enrichment for genes found in the microarray screen………………………….............. 30 Figure 4 Canonical cell type expression patterns suggested classifiers for anatomical-based clustering ………………………….…….. 35 Figure 5 Clustering of SGZ enriched genes revealed cell type groupings ………………………………………………………. 38 Figure 6 Genes in each cluster group showed similar, but not identical expression patterns………………………………….... 39 Chapter 2 Figure 1 Virus injections in GAD67-GFP mice………………………. 72 Figure 2 Differences in promoter, titer and virus type affect neuron transduction efficiencies……………………………………….. 73 Figure 3 Dilution of virus shifts rAAV2/1 transduction away from excitatory neurons……………………………………………… 74 Figure 4 mCAMK promoter expresses in inhibitory neurons in rAAV2/1……………………………………………………….. 75 Supplemental Figure S1 NeuN labeling identifies neurons in rAAV2/1-CAG-RFP sections………………………………….. 76 Chapter 3 Figure 1 Vector design………………………………………………... 113 Figure 2 Fugu promoters in AAV expressed in mouse cortex………... 114 Figure 3 AAV-fSST, AAV-fNPY and AAV-fCAMK expressed in rat and monkey cortex…………………………………………. 116 Figure 4 Fugu promoters in AAV expressed in mouse dentate gyrus (DG) and thalamus………………………………………. 117 Figure 5 AAV-fSST predominately labeled inhibitory neurons in mouse cortex…………………………………………………… 118 Figure 6 AAV-fSST expression co-labeled with CR, PV, SST and VIP antibodies…………………………………………………. 119 Figure 7 Percentage of virus label in inhibitory neuron subtypes…….. 121 Figure 8 Fugu promoters in LV drove expression in large numbers of excitatory neurons…………………………………………... 122 Figure 9 Mammalian promoters drove expression in the rodent brain.. 123 Figure 10 Mammalian conserved TFBS promoters drove expression in mouse brain…………………………………………………. 124 Figure 11 AAV-DR1 drove expression in only a few cells…………… 126 viii Figure 12 AAV-E1 drove strong expression in a scattered population of cells ………………………………………………………… 127 ix LIST OF TABLES Chapter 1 Table 1 Enriched gene ontology terms………………………………… 49 Table 2 SGZ enriched genes up-regulated with running………………. 50 Table 3 SGZ enriched genes down-regulated with running…………… 51 Chapter 2 Table 1 Genomic and infectious titers………………………………….. 77 Supplemental Table S1 Virus expression counts in GAD67-GFP mice.. 78 Chapter 3 Table 1 AAV and LV vectors…………………………………………… 129 Supplemental Table 1 Co-expressed genes in the G30 (VIP), G42 (PV) and GIN (SST) cell type populations………………… 131 Supplemental Table 2 TFBS enriched in the G30 (VIP) population of co-expressed genes…………………………………………… 132 Supplemental Table 3 TFBS enriched in the G42 (PV) population of co-expressed genes……………………………………………… 133 Supplemental Figure 4 TFBS enriched in the GIN (SST) population of co-expressed genes…………………………………………… 134 x ACKNOWLEDGMENTS I would like to acknowledge all of my committee members, professors and co- workers for their support and encouragement. I especially acknowledge Professor Edward Callaway for his guidance, support and trust, and for the friendly atmosphere he creates in his lab; Lanping Amy Sung for her encouragement and giving me a start in research; and Edward Lein, who has been a such a wonderful mentor and to whom I owe so many of the skills I have learned in graduate school. And surely, without the friendly support of the Gage and Callaway labs my work would not have been possible. Thank you. The work in Chapter 1 was done in a joint effort with Edward Lein, a postdoc in the lab of Fred Gage and Director of Neuroscience at the Allen Brain Institute. The work in Chapter 2 was done in the lab of Edward Callaway. The dissertation author was the primary investigator of this work. The work in Chapter 3 was done in the lab of Edward Callaway. The dissertation author was the primary investigator of this work. Roberto Jappelli contributed to the work with fugu promoters. Eric Scheeff contributed to the bioinformatics analysis in the work with mammalian promoters from co-expresssed genes. Each of the contributors to this work has granted permission to include their work in this dissertation. xi VITA 2001 Bachelor of Science, University of California, San Diego 2002 Master of Science, University of California, San Diego 2003-2005 Teaching Assistant, Department of Bioengineering, University of California, San Diego 2001-2008 Research Assistant, Salk Institute for Biological Studies 2008 Doctor of Philosophy, University of California, San Diego PUBLICATIONS Yao W, Nathanson J, Lian I, Gage FH, Sung LA. “Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter” Gene Expr Patterns.