University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2011-08-31 Neuronal Diversification in the ostembrP yonic Drosophila Brain: A Dissertation Suewei Lin University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Animal Experimentation and Research Commons, Cells Commons, Nervous System Commons, and the Neuroscience and Neurobiology Commons Repository Citation Lin S. (2011). Neuronal Diversification in the ostembrP yonic Drosophila Brain: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/eh7z-7w05. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/565 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. NEURONAL DIVERSIFICATION IN THE POSTEMBRYONIC DROSOPHILA BRAIN A Dissertation Presented By Suewei Lin Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 31, 2011 iii Dedications To my parents for their patience and support & To my wife, for her selfless love that carries me through all the ups and downs in the five years of my PhD life. iv Acknowledgements I would like to thank Tzumin Lee for his guidance and support, and all my TRAC committee members for valuable suggestions. Thanks to Sen-Lin Lai and Hung- Hsiang Yu for their contributions on the study of “Lineage-specific effects of Notch/Numb signaling in postembryonic development of the antennal lobe lineages (Chapter IV)”. Thanks to Yaling Huang for her dedication to molecular cloning for the entire lab, and other lab members, especially Takeshi Awasaki, Chih-Fei Kao, Shun-Jen Yang, Lei Shi, Hui-Min Chen, Mark Schroeder, and Haojian Luan, for their comments and suggestions on my research. Thanks to Tara Keegan and Colleen Baldelli in UMASS and Crystal Sullivan in JFRC for their help on ordering experimental materials and taking care of lab equipment. Thanks to Todd Laverty, Karen Hibbard, and Monti Mercer for maintaining fly stocks. Finally, I would like to thank everyone in the Department of Neurobiology in UMASS and JFRC for creating fun, creative, and cooperative research environments. v Abstract A functional central nervous system (CNS) is composed of numerous types of neurons. Neurons are derived from a limited number of multipotent neural stem cells. Previous studies have suggested three major strategies nature uses to diversify neurons: lineage identity specification that gives an individual neural stem cell distinct identity based on its position in the developing CNS; temporal identity specification that gives neurons derived from a neural stem cell distinct identities based on their birth-order within the lineage; and binary cell fate specification that gives different identities to the two sister postmitotic neurons derived from the terminal division of a common precursor. Through the combination of the three strategies, almost unlimited neuron types can be generated. To understand neuronal diversification, we have to understand the underlying molecular mechanisms of each of the three strategies. The fruit fly Drosophila melanogaster, has been an excellent model for studying neuronal diversity, mainly due to its easily traceable nervous system and an impressive collection of genetic tools. Studies in fly have provided us fundamental insights into lineage identity, temporal identity, and binary cell fate specifications. Nevertheless, previous studies mostly centered on the embryonic ventral nerve cord (VNC) because of its simpler organization. Our understanding of the generation of neuronal diversity in the fly brain is still rudimentary. In this vi thesis work, I focused on the mushroom body (MB) and three antennal lobe neuronal lineages, studying their neuronal diversification during postembryonic brain development. In Chapter I, I reviewed the previous studies that have built our current understanding of the neuronal diversification. In Chapter II, I showed that MB temporal identity changes are instructed by environmental cues. In Chapter III, to search for the potential factors that mediate the environmental control of the MB temporal identity changes, I silenced each of the 18 nuclear receptors (NRs) in the fly genome using RNA interference. Although I did not identify any NR important for the regulation of MB temporal identities, I found that unfulfilled is required for regulating axon guidance and for the MB neurons to acquire all major subtype-specific identities. In Chapter IV, I demonstrated that the Notch pathway and its antagonist Numb mediate binary cell fate determination in the three classical antennal lobe neuronal lineages— anterodorsal projection neuron (adPN), lateral antennal lobe (lAL), and ventral projection neuron (vPN)—in a context-dependent manner. Finally, in Chapter V, I did detailed lineage analysis for the lAL lineage, and identified four classes of local interneurons (LNs) with multiple subtypes innervating only the AL, and 44 types projection neurons (PNs) contributing to olfactory, gustatory, and auditory neural circuits. The PNs and LNs were generated simultaneously but with different tempos of temporal identity specification. I also showed that in the lAL lineage the Notch pathway not only specifies binary cell fates, but is also involved in the temporal identity specification. vii Table of Contents Chapter I: Introduction....................................................................................1 Specification of distinct Neuroblasts...........................................5 Specification of neuronal temporal identity..............................10 Specification of binary cell fate..................................................19 Interplay between lineage identity, temporal identity, and binary cell fate specifications.....................................................21 The neuronal diversification in the Drosophila brain...............25 Chapter II: Environmental control of mushroom body neuronal temporal identity...........................................................................................33 Introduction..................................................................................33 Result............................................................................................38 MB temporal identity changes are delayed when larval growth is prolonged.......................................................................................38 MB temporal identity changes are regulated by environmental cues................................................................................................40 Critical weight gates MB temporal identity transitions....................41 PTTH and ecdysone are not required for the MB temporal identity transitions.......................................................................................43 Discussion....................................................................................45 viii Materials and Methods................................................................50 Chapter III: Nuclear receptor Unfulfilled regulates axonal guidance and cell identity of mushroom body neurons..........................................65 Introduction..................................................................................65 Result............................................................................................71 Silencing individual NRs by miRNA unveils a role of nuclear receptor unf in MB development....................................................71 unf is mainly required for the formation of adult-specific MB lobes...............................................................................................73 unf acts in mature neurons to govern axon re-extension, while supporting initial axonal morphogenesis in later types of MB neurons..........................................................................................75 Direct involvement of unf in later MB morphogenetic processes.......................................................................................76 unf regulates axon pathfinding of MB neurons...............................78 unf is expressed in MB neurons continuously to regulate neuron subtype identity...............................................................................79 Discussion....................................................................................82 Materials and Methods................................................................86 Chapter IV: Lineage-specific effects of Notch/Numb signaling in postembryonic development of antennal lobe neurons.........108 Introduction................................................................................109 ix Result...........................................................................................113 Production of adult adPNs occurs one by one, rather than in pairs..............................................................................................113 Each adPN GMC makes an adPN and a cell eliminated by programmed cell death.................................................................114 Numb antagonizes Notch to specify PNs in the