Neuronal Diversification in the Postembryonic Drosophila Brain: A Dissertation

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 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.Neuroscienc

The making of the Drosophila mushroom body

The mushroom body (MB) is a computational center in the Drosophila brain. The intricate neural circuits of the mushroom body enable it to store associative memories and process sensory and internal state information. The mushroom body is composed of diverse types of neurons that are precisely assembled during development. Tremendous efforts have been made to unravel the molecular and cellular mechanisms that build the mushroom body. However, we are still at the beginning of this challenging quest, with many key aspects of mushroom body assembly remaining unexplored. In this review, I provide an in-depth overview of our current understanding of mushroom body development and pertinent knowledge gaps.</jats:p

Assembly of the<i>Drosophila</i>mushroom body circuit and its regulation by Semaphorin 1a

SummaryTheDrosophilamushroom body (MB) is a learning and memory center in the fly brain. It is the most extensively studied brain structure in insects, but we know little about the molecular and cellular mechanisms underlying assembly of its neural circuit. The MB is composed of around 2200 intrinsic Kenyon cells (KCs), whose axons are bundled to form multiple MB lobes. The MB lobes are innervated by a large number of extrinsic neurons. Twenty types of dopaminergic neurons (DANs) and 21 types of MB output neurons (MBONs) have been identified. Each type of these extrinsic neurons innervates specific compartments or zones in the MB lobes. Here, we characterize the assembly of the MB circuit and reveal several intriguing features of the process. The DANs and MBONs innervate zones in the MB vertical lobes in specific sequential orders. Innervation of DAN axons in some zones precedes that of MBON dendrites, andvice versain other zones. MBON and DAN innervations are largely independent of each other. Removing one type of extrinsic neuron during early development has a limited effect on the MB lobe innervations of the other type of extrinsic neurons. However, KC axons are essential for zonal elaboration of DAN axons and MBON dendrites. Competition also exists between MB zones for some MBONs, so when the cognate zones for these MBONs are missing, their dendrites are misdirected to other zones. Finally, we identify Semaphorin 1a (Sema1a) as a crucial guidance molecule for MBON dendrites to innervate specific MB lobe zones. Ectopic expression of Sema1a in some DANs is sufficient to re-direct their dendrites to those zones, demonstrating a potential to rewire the MB circuit. Taken together, our work provides an initial characterization of the cellular and molecular mechanisms underlying MB circuit assembly.</jats:p

Imaging CaLexA and TRIC Signals in the Dissected Fly Central Nervous System

Calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC) are transcription-based genetically encoded calcium indicators (transcriptional GECIs). When expressed in neurons, CaLexA and TRIC report neuronal activity by converting intracellular calcium levels into transcription activities and, subsequently, reporter gene expression. CaLexA and TRIC have been used successfully in many studies to label neurons associated with particular behaviors, regulated by internal states, or evoked by specific sensory inputs. This protocol details procedures for generating flies expressing genetic components of CaLexA and TRIC in specific neurons, performing immunostaining to label CaLexA and TRIC signals, and quantifying the signals using an open-source imaging analysis software.</jats:p

Generating neuronal diversity in the Drosophila central nervous system

Co-author Suewei Lin is a student in the Neuroscience program in the Morningside Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.Generating diverse neurons in the central nervous system involves three major steps. First, heterogeneous neural progenitors are specified by positional cues at early embryonic stages. Second, neural progenitors sequentially produce neurons or intermediate precursors that acquire different temporal identities based on their birth-order. Third, sister neurons produced during asymmetrical terminal mitoses are given distinct fates. Determining the molecular mechanisms underlying each of these three steps of cellular diversification will unravel brain development and evolution. Drosophila has a relatively simple and tractable CNS, and previous studies on Drosophila CNS development have greatly advanced our understanding of neuron fate specification. Here we review those studies and discuss how the lessons we have learned from fly teach us the process of neuronal diversification in general.Neuroscienc

Transcriptional Genetically Encoded Calcium Indicators in<i>Drosophila</i>

Knowing which neurons are active during behavior is a crucial step toward understanding how nervous systems work. Neuronal activation is generally accompanied by an increase in intracellular calcium levels. Therefore, intracellular calcium levels are widely used as a proxy for neuronal activity. Many types of synthetic components and bioluminescent or fluorescent proteins that report transient and long-term changes in intracellular calcium levels have been developed over the past 60 years. Calcium indicators that enable imaging of the dynamic activity of a large ensemble of neurons in behaving animals have revolutionized the field of neuroscience. Among these, transcription-based genetically encoded calcium indicators (transcriptional GECIs) have proven easy to use and do not depend on sophisticated imaging systems, offering unique advantages over other types of calcium indicators. Here, we describe the two currently available fly transcriptional GECIs—calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC)—and review studies that have used them. In the accompanying protocol, we present step-by-step details for generating CaLexA- and TRIC-ready flies and for imaging CaLexA and TRIC signals in dissected brains after experimental manipulations of intact free-moving flies.</jats:p