DEVELOPMENT AND ORGANIZATION OF

THE DROSOPHILA OLFACTORY CIRCUIT

A DISSERTATION

SUBMITTED TO THE DEPARTMENT OF BIOLOGY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Bing Wu

August 2017
 © 2017 by Bing Wu. All Rights Reserved.
Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-

Noncommercial 3.0 United States License.
http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/vq704cj6821

ii
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Liqun Luo, Primary Adviser

I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Thomas Clandinin

I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Kang Shen

I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.

Thomas Sudhof

Approved for the Stanford University Committee on Graduate Studies.

Patricia J. Gumport, Vice Provost for Graduate Education

This signature page was generated electronically upon submission of this dissertation in
electronic format. An original signed hard copy of the signature page is on file in
University Archives.

iii
Abstract

The formation of complex but highly organized neural circuits requires precise

recognition and interactions between cells that constitute the nervous system. A growing

body of research has discovered numerous neuro-neuronal interactions underlying

neural development, while the important roles played by glia are appreciated only more

recently. In this thesis, I utilized the olfactory circuit of Drosophila melanogaster as a

model to study the roles of both neurons and glia which together establish the unique

structure of the olfactory system.

During the assembly of the Drosophila olfactory circuit, ~50 olfactory receptor

neuron (ORN) classes and ~50 projection neuron (PN) classes form one-to-one synaptic

connections in ~50 glomerular compartments in the antennal lobe, each of which

represents a discrete olfactory information processing channel. Several cell surface

molecules have been reported to mediate neuro-neuronal interactions and determine the

specific connectivity of olfactory neurons. However, we are still far from a complete

understanding of this process. In a genetic screen to look for additional wiring molecules

in this process, I identified Fish-lips (Fili), a leucine-rich repeat transmembrane protein,

to be expressed in a subset of olfactory neurons. Loss- and gain-of-function experiments

indicate that Fili can instruct PN dendrites to project to proper glomerular targets.

Besides olfactory neurons, the antennal lobe is also permeated by several types of glia.

Specifically, each glomerular compartment is separated from the adjacent

compartments by membranous processes from the ensheathing glia. In a genetic screen

iv
designed to reveal molecular mechanisms underlying glia morphogenesis, I identified

that Thisbe, a fibroblast growth factor released from olfactory neurons particularly local

interneurons (LNs), controls ensheathing glia to wrap each glomerulus. The FGF

receptor, Heartless, acts cell-autonomously in ensheathing glia to regulate process

elaboration so as to insulate each neuropil compartment. Overexpressing Thisbe in

ORNs or PNs causes over-wrapping of glomeruli to which their axons or dendrites

target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon

targeting and discrete glomerular formation.

In summary, I have identified two molecular mechanisms underpinning the assembly

of the olfactory circuit. Fili, combined with previously identified cell surface cues, can

define dendrites from different PN classes and instruct them to target correct glomeruli.

After the initial innervation of PN dendrites and ORN axons, ensheathing glia respond

to a neuronal cue, Thisbe, to pattern the boundaries of the nascent glomerular

compartments; neural compartments in turn require such glial barriers to separate

themselves from neighboring compartments, so as to ensure the correct organization of

the olfactory circuit. These findings highlight the synergism of neurons and glia in

shaping the intricate network of the nervous system.

v
Preface

Collaborations

Section 2.3.6 in chapter 2 would not have been possible without my collaborator Dr.

Ya-Hui Chou, who generously shared the unpublished information about local

interneurons and related reagents with me. Jiefu Li assisted in establishing some of the

Drosophila stocks for this study. Chapter 2 is modified from the publication, Fibroblast

growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory

glomeruli (Wu et al., PNAS, 2017).

The genetic screen in section 3.3.1 of Chapter 3 was accomplished by the joint effect

from Dr. Xin Wang and myself. Dr. Wang contributed to establishing the stock for the

RNAi screen, and was dedicated to testing 50% of the candidate RNAi lines.

All embryonic injections for generating transgenic Drosophila lines were conducted

by David Luginbuhl.

vi
Acknowledgement

My advisor, Prof. Liqun Luo, is the foremost firm support for my research in graduate

school. Being deeply inquisitive, creative, knowledgeable, and open-minded, Liqun

inspired me to pursue my interests, develop my projects, and learn my lessons. Besides

providing all the freedom I could ever ask for, Liqun is also a rich and constant source

for ideas, wisdom, and criticism, as well as a role model exemplifying efficient

communication, hard-working, and self-discipline. I cannot thank Liqun enough for

taking me under his wing and extending his support to my career beyond graduate

school.

I am very grateful to all the members of the Luo lab for their suggestions and

discussions around my research projects. In particular, Alex Ward and Weizhe Hong

supervised me during my rotation, and continuously mentored me during my early years

in the lab. Xiaojing Gao and Jan Lui consecutively shared the bay with me in the lab,

and supplied me with numerous scientific thoughts and mental energies. People who

have directly contributed to chapter 2 and 3 are mentioned in the Collaborations section.

In addition, I have worked with Xiaojing Gao, Hongjie Li, and Felix Horns on projects

not described here, and have benefited from their superb research skills and their spirit

of sharing. The Luo lab would have been an absolutely different place without all the

colleagues and friends, who have shared their life stories, opinions, and emotions with

me. Thanks to Alex Ward, Xiaojing Gao, Jan Lui, Casey Guenthner, Xin Wang,

Dominic Berns, Brady Weissbourd, Weizhe Hong, Liang Liang, Lindsay Schwarz,

Xiang Wang, Jing Ren, Jiefu Li, Hongjie Li, Andrew Shuster, Kevin Beier, Chen Ran,

vii
Laura DeNardo, Wei-Hsiang Huang, Tongchao Li, Mark Wagner, David Luginbuhl,

Tim Mosca, William Joo, Drew Friedmann, Vincenzo Favaloro, Cindy Liu, Katherine

DeLoach, Kazunari Miyamichi, William Allen, Ethan Richman, Simon Hippenmeyer,

Xiaomeng Yu, Yanyang Ge, Carlota Manalac, and Stephanie Wheaton, who have made

my graduate school easier and more lively.

I am indebted to my committee members, Prof. Thomas Clandinin, Prof. Kang Shen,

Prof. Thomas Südhof, and Prof. Ben Barres, who went out of their way to help my

research, pushed me to my full potential, and provided complete support for my career.

Prof. Südhof generously trained me for electrophysiology in my first year of graduate

school as a rotation student, and kept delivering thought provoking and sometimes

philosophical questions, teaching me to think more critically. Prof. Shen and Prof.

Südhof, together with other members in Stanford neuroscience community composed

the Synapse Club, a casual and nurturing platform where I have presented and learned.

As friendly neighbors in our research building, Prof. Shen and his lab members

(Pengpeng Li, Xintong Dong, and Callista Yee to name a few) have been endless

sources of encouragement and refreshing insights. Prof. Clandinin not only brought his

excellent expertise in Drosophila genetics to our meetings, but also gave great patience

and guidance in the long process of my development. Prof. Barres has served as a steady

defense for my glia project from the very beginning of that work, and presented intense

enthusiasm for my studies, as well as tremendous responsiveness to my request even

when in confrontation with his life challenges. It has been a privilege to have these

esteemed and relatable figures looking after me throughout graduate school.

viii
 I am appreciative of my parents, who always give me their unconditional love and

blessings, even though my life and career trajectories may not have aligned well with

their expectations. My life has been more exciting and adventurous with my partner

and my closest friend, Yangye Zhu, who was my partner in crime during our PhDs, and

evolves with me, hikes with me, as well as looks outward in the same direction with me.

Last but not least, I also need to thank Su Shi, Pyotr Ilyich Tchaikovsky, Murasaki

Shikibu, and a long list of artists, who have unwittingly kept me accompanied in the

long journey.

ix
Table of Contents
Abstract ..................................................................................................................................... iv

Preface ....................................................................................................................................... vi

Acknowledgement .................................................................................................................... vii

Table of Contents ....................................................................................................................... x

Table of Figures ....................................................................................................................... xii

1 Chapter 1 Introduction........................................................................................................ 1

1.1 Overview of Neural Development Leading to Circuit Assembly .............................. 1

1.2 Cell Surface Proteins Mediate Cell-cell Interactions in Neuronal Wiring ................. 3

1.3 Wiring Specificity in Drosophila Olfactory System .................................................. 7

2 Chapter 2 Fibroblast Growth Factor Signaling Controls Ensheathing Glia Wrapping of

Drosophila Olfactory Glomeruli .............................................................................................. 12

2.1 Abstract .................................................................................................................... 12

2.2 Introduction .............................................................................................................. 13

2.3 Results ...................................................................................................................... 15

2.3.1 Pupal Development of Antennal Lobe Ensheathing Glia. ................................ 15

2.3.2 Heartless Knockdown in Ensheathing Glia Reduces Processes and Disrupts the
Ensheathment Pattern ....................................................................................................... 22

2.3.3 Heartless Promotes Ensheathing Glia Survival and Cell-autonomously Regulates

Process Elaboration .......................................................................................................... 26

2.3.4 Thisbe, an FGF Ligand, Is Required for the Wrapping of Glomeruli by

Ensheathing glia ............................................................................................................... 33

2.3.5 Thisbe Is Produced by ORNs, PNs, and Antennal Lobe Local Interneurons ... 36

2.3.6 LN-derived Thisbe Is Necessary for Ensheathing Glia Wrapping and Antennal
Lobe Compartmentalization ............................................................................................. 38

2.3.7 Thisbe Can Instruct Glomerular Wrapping with a High Spatial Specificity .... 41

2.3.8 FGF Signaling Ensures Accurate Neuronal Targeting ..................................... 43

x
 2.4 Discussion ................................................................................................................ 46

2.5 Experiment Methods ................................................................................................ 49

3 Chapter 3 Fish-lips Directs Drosophila Olfactory Neuron Wiring .................................. 51

3.1 Abstract .................................................................................................................... 51

3.2 Introduction .............................................................................................................. 52

3.3 Results ...................................................................................................................... 54

3.3.1 Develop New Genetic Tools to Extend Wiring Specificity Studies to Additional
Regions of the Antennal Lobe .......................................................................................... 54

3.3.2 Identification of LRR Protein Fish-lips (Fili) as a Wiring Specificity Molecule

58

3.3.3 Fili Expression Pattern in the Developing Antennal Lobe ............................... 63

3.3.4 Fili Expressed in ORNs Can Control PN Targeting ......................................... 72

3.3.5 Overexpressing Fili Alters PN Dendrite and ORN Axon Projection Patterns . 74

3.4 Discussion ................................................................................................................ 77

3.5 Experiment Methods ................................................................................................ 78

Bibliography ............................................................................................................................. 80

xi
Table of Figures
Figure 1-1 Schematic of the fly olfactory system. ................................................................... 11
Figure 2-1 Ensheathing glia morphogenesis during antennal lobe development. .................... 18
Figure 2-2 Two FlyLight-GAL4 lines and SPARC-GAL4 label antennal lobe ensheathing glia.
.................................................................................................................................................. 20
Figure 2-3 Heartless knockdown causes an altered ensheathing glia wrapping pattern and
disrupts antennal lobe compartmentalization. .......................................................................... 24
Figure 2-4 Heartless controls ensheathing glia number and cell-autonomously control their
morphology. ............................................................................................................................. 28
Figure 2-5 P35 suppresses the reduction of ensheathing glia number caused by htl knockdown.
.................................................................................................................................................. 30
Figure 2-6 Additional MARCM clone examples and analysis. ............................................... 32
Figure 2-7 Thisbe is expressed in olfactory neurons and required for ensheathing glia to wrap
glomeruli. ................................................................................................................................. 34
Figure 2-8 Orb0449-GAL4 labels local interneurons in the antennal lobe. .................................... 39
Figure 2-9 Loss of Thisbe from ORNs and PNs does not cause ensheathing glia or glomerulus
defects....................................................................................................................................... 40
Figure 2-10 Changes in ensheathing glia processes and number in response to local
overexpression of ths. ............................................................................................................... 42
Figure 2-11 Loss of ths results in olfactory receptor neuron targeting defects. ....................... 44
Figure 3-1 Identify enhancer-GAL4 lines specifically label certain classes of PNs in adult
antennal lobe. ........................................................................................................................... 56
Figure 3-2 Developmental analysis showing expression patterns for 4 of the enhancer-GAL4
lines during the pupae stage. .................................................................................................... 56
Figure 3-3 Enhancer-LexA lines recapitulate GAL4 patterns. ................................................. 57
Figure 3-4 Identify Fili as a candidate molecule for olfactory wiring...................................... 59
Figure 3-5 Generation of a Fili loss-of-function allele. ........................................................... 61
Figure 3-6 VM5v and/or VM5d PN dendrites target ectopically in Fili-/-................................ 62
Figure 3-7 Fili-GAL4 labels a subset of glomeruli in the developing antennal lobe. ............... 65
Figure 3-8 Fili-GAL4 labels a subset of ORNs during antennal lobe development. ................ 66
Figure 3-9 Fili-GAL4 labels a subset of PNs during antennal lobe development. ................... 67
Figure 3-10 A Fili-GAL4+ MARCM clone reveals a potential LN. ........................................ 68

xii
Figure 3-11 Fili is enriched in a subset of glomeruli, including VM5v and VM5d. ................ 71
Figure 3-12 Fili from ORNs controls PN dendrite targeting. ................................................... 73
Figure 3-13 Fili overexpression causes DA1 PNs to mistarget. ............................................... 75
Figure 3-14 Fili overexpression causes ORN mistargeting. ..................................................... 76

xiii
1 Chapter 1 Introduction

1.1 Overview of Neural Development Leading to Circuit Assembly

The nervous system functions by receiving, transmitting, and processing information in

a network of interconnected neuronal cells. A series of developmental events largely

governed by genetic programs control the assembly of the neural network. The initial

induction of neural tissues is regulated by signaling of secreted morphogens such as

bone morphogenetic proteins, fibroblast growth factors, Wingless and Hedgehog

families of proteins (Munoz-Sanjuan and Brivanlou, 2002; Stern, 2005), followed by

further arealization, differentiation and amplification of the cells giving rise to an

enormous diversity of neurons and glia — the two most abundant cell types in the

nervous system (Hebert and Fishell, 2008; Kriegstein and Alvarez-Buylla, 2009;

O'Leary and Sahara, 2008; Rowitch and Kriegstein, 2010). Afterwards, cooperation

between external signals and internal transcriptional profile changes drives cell

migration from their birth places to appropriate positions to integrate into the circuit

(Klambt, 2009; Kriegstein and Noctor, 2004; Marin et al., 2010). These neurons and

glia also undergo substantial morphogenesis to acquire special shapes and protrusion

patterns. For example, in response to a series of extracellular guidance cues, neurons

utilize the corresponding cell surface receptors and their cytoskeleton-associated

effectors to steer axons and dendrites towards specific targets. A variety of intracellular

events including vasicular transport, changes in transcription factor profile, and Golgi

outpost dynamics all contribute to the development of neuronal processes (Dong et al.,

1
2015; O'Donnell et al., 2009). Finally, the contact of axons with their dendritic partners

is followed by the generation of synapses, which display dramatic plasticity in response

to life experiences (Chung et al., 2015a; McAllister, 2007). A growing body of evidence

has revealed essential roles played by glia in synaptic formation and elimination

(Stogsdill and Eroglu, 2017), as well as other events including neurological diseases

(Stork et al., 2012; Zuchero and Barres, 2015). However, we know surprisingly little

about the molecular mechanisms that mediate such close interactions between glia and

neurons.

This thesis will focus on the genetic basis that underlies the formation of an accurate

and specific projection pattern of neuronal axons and dendrites, particularly the cellular

and molecular interactions among axons, dendrites, and the glial processes, which

establish the physical framework on which functional neural circuits can be built.

2
1.2 Cell Surface Proteins Mediate Cell-cell Interactions in Neuronal

Wiring

In order to organize a network of neurons and glia, the involved cells respond to

extrinsic cues, and extend their membranous processes (axonal and dendritic projections

from neurons, as well as extended membrane structures from glia) to reach their targets.

A wealth of instructive cues presented in either the extracellular environment or on the

surface of other cells encountered by the ones making target choice have been identified,

as well as their cell surface receptors. These cell surface proteins, either secreted or

transmembrane, mediate the intercellular communication that enable cells in the

nervous system to find their destination. A canonical example is the molecular

interaction between Ephrins and Eph receptors that mediate the communication between

retinal ganglion cells (RGCs) and their target tissues, and therefore direct the formation

of topographic map in the visual system (Feldheim and O'Leary, 2010). RGCs in the

mouse project their axons to the superior colliculus (SC) among multiple targets in the

brain. EphA3 receptor is expressed at a high level in the temporal retina and decreases

along the temporal-nasal axis. Its repellent ligand, ephrin-A5, displays an increasing

gradient along the anterior-posterior axis of SC. As a result for each RGC, the more

temporal it locates, the higher level of EphA3 receptor it expresses, and the more

sensitive its axons are to the repellent cue, ephrin-A5, which results in more anterior

targeting (Feldheim et al., 2000; Flanagan, 2006; Frisen et al., 1998; Pfeiffenberger et

al., 2006). Such target specificity ensures the preservation of neighbor-neighbor

relationship in the projection field, and therefore transmit the spatial information of

vision.

3
 A list of other cell surface molecules, including Semaphorins (Tran et al., 2007),

Cadherins (Takeichi, 2007), leucine-rich repeat proteins, and immunoglobulin

superfamily members (de Wit and Ghosh, 2016), have all been involved in the cell-cell

interactions that enable axons and dendrites to navigate the brain, recognize, and

communicate with intermediate and final targets during the assembly of the neural

network.

It is noteworthy that some of the cell surface cues can be produced by glia to guide

the pathfinding of neuronal processes. For example, the ipsilateral projecting axons in

Drosophila embryonic ventral nerve cord use Roundabout (Robo), a receptor for Slit,

the midline glia derived cue, to avoid midline crossing. In addition, Slit also signals to

the commissural axons to prevent them from re-crossing after they have crossed the

midline (Kidd et al., 1999; Kidd et al., 1998). Furthermore, to establish the longitudinal

pathways through which these axons can extend laterally, the longitudinal glia also

transiently express Robo which keeps them away from the midline (Kinrade et al., 2001).

The close contact and interplay between neurons and glia make it imperative to consider

both cell types in the assembly of the neural circuit.

In forming specific projection patterns and selecting precise targets, many neurons

need to develop extensive dendritic morphology such that branches emanating from the

same neuron spread out over a large receptive field without overlapping, a phenomenon

known as self-avoidance (Bodmer and Jan, 1987; Grueber et al., 2002; Nicholls and

Baylor, 1968; Stacy and Wong, 2003). A growing number of molecules have been

discovered to underlie such a striking behavior (Grueber and Sagasti, 2010; Jan and Jan,

4
2010; Lefebvre et al., 2012; Smith et al., 2012; Zipursky and Sanes, 2010). Take the

Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) as an example. By

alternative splicing, Dscam1 can give rise to over 38,000 possible isoforms (Schmucker

et al., 2000) that show isoform specific homophilic binding (Wojtowicz et al., 2004).

This molecular interaction on the cell membrane will lead to repulsion of neuronal

processes that express the same set of isoforms (Hughes et al., 2007; Matthews et al.,

2007; Soba et al., 2007; Zhan et al., 2004). Since each individual cell expresses a broad

yet distinctive repertoire of Dscam1 splice forms (Neves et al., 2004), a tremendous

number of distinct Dscam1 isoform combinations can be generated, thus providing each

neuron with a unique identification code to allow self-recognition and therefore self-

avoidance. Consistently, mammalian Dscam proteins also promote homophilic

repulsion and contributes to self-avoidance (Fuerst et al., 2008). However, Dscam genes

in mammals are not alternatively spliced. It is the clustered protocadherins (Pcdhs) that

can produce a large number of cadherin-like transmembrane proteins through variable

splicing (Esumi et al., 2005; Kaneko et al., 2006; Kohmura et al., 1998; Wu and

Maniatis, 1999) and mirror the function of Dscam1 in flies. Distinct combinations of

cis-heteromeric protocadherin oligomers expressed in each cell confer trans-homophilic

interactions between sibling neurites, resulting in a repulsive signal that leads to self-

avoidance (Lefebvre et al., 2012; Schreiner and Weiner, 2010). While diverse isoforms

of Dscam1 and Pcdhs can endow each single neuron with a specific identity, the

Drosophila Dscam2 which is not so highly alternatively spliced (Millard et al., 2007),

and a single Pcdh isoform, Pcdhac2 (Chen et al., 2017), can mark a population of like-

5
type neurons with an unique cell surface code, so that these neurons can tile the entire

target field without redundancy.

After axons and dendrites arrive at the correct position, synaptic cell adhesion

molecules such as neurexins and neuroligins (Craig and Kang, 2007; Sudhof, 2008) can

mediate trans-synaptic interaction and contribute to establishing functional synapses. In

addition, both astrocytes and microglia have been shown to control synapse formation

and function through secreted signals and mechanisms that are contact dependent

(Chung et al., 2015a; Chung et al., 2015b; Eroglu and Barres, 2010; Hama et al., 2004).

In summary, the transmembrane and secreted proteins play a pivotal role in

identifying cells and the environment, and responding to these signals accordingly, to

establish specific connections, develop functional synapses, and ultimately build up the

nervous system.

6
1.3 Wiring Specificity in Drosophila Olfactory System

Many of the canonical circuit wiring principles aforementioned are utilized in

specifying the target pattern of the olfactory system. For instance, Dscam1 is again used

by olfactory neurons to ensure that dendritic branches from the same neuron avoid each

other as they elaborate appropriate receptive fields (Zhu et al., 2006a). Besides, the

implementation of continuous distribution of cell surface cues in gradients is repeatedly

observed in the olfactory circuit wiring. In Drosophila, the gradient of secreted Sema-

2a/2b along the dorsolateral-ventromedial axis repels olfactory projection neuron (PN)

dendrites dorsolaterally through the transmembrane Sema-1a in the dendrites opposing

the Sema-2a/2b gradient (Komiyama et al., 2007; Sweeney et al., 2011). In mice,

neuropilin-1 is implicated to work with its repulsive ligand Sema3A in olfactory

receptor neuron guidance (Imai et al., 2006; Sakano, 2010).

Although the olfactory system shares some wiring strategies with the other circuits,

it is notable that chemical information carried in odorants does not exhibit a discernible

continuous feature, as opposed to visual information for example which is represented

by continuous retinotopic maps in the brain. Hence, olfactory information is represented

in a discrete map symbolized by the physical separation between different olfactory

glomeruli in the brain; therefore besides a coarse map that can be set up by molecular

gradients, the olfactory system will likely exploit additional mechanisms in order to

establish the discreteness of the odorant map. Therefore, it is attractive to utilize the

olfactory system to find novel cell surface molecules that potentially confer new forms

and principles underpinning neural circuit assembly.

7
 The organization of the olfactory system in Drosophila shares striking resemblance

with its mammalian counterpart, but is numerically much simpler and experimentally

more amenable (Komiyama and Luo, 2006). In the Drosophila sensory organs, antenna

and maxillary pulp, olfactory receptor neurons (ORNs) express structurally distinct

olfactory receptors (ORs) which exhibit variable affinity to an array of odorant

chemicals. Each ORN expresses only one or two of the ORs (Clyne et al., 1999; Gao

and Chess, 1999; Goldman et al., 2005; Vosshall et al., 1999), and all of the ORNs that

express identical OR(s) converge their axons toward one specific structural unit called

the glomerulus that can be identified by its stereotypic shape and location (Couto et al.,

2005; Fishilevich and Vosshall, 2005; Gao et al., 2000). In each of the glomeruli, ORN

axons make synaptic connections with a predestined class of projection neurons (PNs)

so as to relay the olfactory information to higher brain centers. Similar to ORNs, the

majority of PNs project their dendrites to one of the glomeruli, based on their identities

(Jefferis et al., 2001). In total, ~50 such synaptic clusters composed of ORN axonal and

PN dendritic terminals, i.e. glomeruli, constitute the primary olfactory processing center

called the antennal lobe in Drosophila (Figure 1-1). In human, a similar structure called

the olfactory bulb houses on average 5568 glomeruli (Maresh et al., 2008), and in mice

this number is still as large as ~1800 (Royet et al., 1988).

The highly specific wiring pattern of neurons in only ~50 olfactory glomeruli in

Drosophila has served as a platform for us to study the molecular and cellular basis of

olfactory circuit wiring. In particular, two transmembrane proteins, Capricious (Caps)

and Tartan (Trn), whose extracellular domains contain leucine-rich repeat (LRR)

sequences which are protein recognition modules (Kobe and Kajava, 2001), are

8
expressed in a subset of glomeruli in a mosaic pattern to specify discrete target regions

for PNs in a partially redundant manner (Hong et al., 2009). The role of Caps in neuronal

target selection has also been reported elsewhere in the nervous system (Shinza-Kameda

et al., 2006; Shishido et al., 1998); however, unlike in these contexts, Caps in the

antennal lobe requires a heterophilic ligand, which is yet to be identified (Hong et al.,

2009). Besides revealing new molecular mechanisms for known wiring molecules, the

fly olfactory system has also enabled us to uncover unconventional proteins for neuronal

wiring, such as Toll-6 and Toll-7 which are members of the Toll receptor family best

known for roles in immunity and pattern formation in embryos (Ward et al., 2015). Glia

are also suggested to control antennal lobe wiring. Specifically, the transient

interhemispheric fibrous ring (TIFR) glia express a receptor tyrosine kinase, Derailed

(Drl), to modulate Wnt5 signal and hence contribute to the accurate pattern of ORN

axon projection (Sakurai et al., 2009; Yao et al., 2007).

Despite these advances, we are still far from a complete understanding of the wiring

mechanism for the fly antennal lobe and for the nervous system in general. One of the

open questions remains whether additional cell types, including local interneurons

(Chou et al., 2010) and a wide diversity of glia types (Doherty et al., 2009) around the

antennal lobe can play critical roles in circuit assembly. In Chapter 2 of this dissertation,

I examine the developmental course of antennal lobe ensheathing glia, and reveal its

importance for the discreteness of the olfactory glomeruli.

Besides new cell types, additional molecules will also be identified to enrich the

toolbox for establishing wiring specificity. It is intriguing to consider whether and how

9
these diverse and partially redundant cues make up a combinatorial coding system to

achieve high coding capacity and robustness in specifying neuronal targets. In Chapter

3 of the dissertation I look for new molecular players in olfactory wiring, and identify

Fish-lips (Fili), which is potentially part of the combinatorial LRR code that directs

PN dendritic targeting.

10
Figure 1-1 Schematic of the fly olfactory system.

Olfactory receptor neurons (ORNs) expressing the same receptor (same color) target

their axons to the same glomerulus in the antennal lobe (AL). Projection neuron (PN)

dendrites also target to single glomeruli, and their axons project to the mushroom body

(MB) and lateral horn (LH). AT: 3rd antennal segment; MP: maxillary palp. Adapted

from Komiyama & Luo, 2006.

11
2 Chapter 2 Fibroblast Growth Factor Signaling Controls

Ensheathing Glia Wrapping of Drosophila Olfactory

Glomeruli

2.1 Abstract

The formation of complex but highly organized neural circuits requires interactions

between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50

olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form

synaptic connections in 50 glomerular compartments in the antennal lobe, each of which

represents a discrete olfactory information processing channel. Each compartment is

separated from the adjacent compartments by membranous processes from ensheathing

glia. Here we show that Thisbe, a fibroblast growth factor (FGF) released from olfactory

neurons particularly local interneurons, controls ensheathing glia to wrap each

glomerulus. The FGF receptor, Heartless, acts cell-autonomously in ensheathing glia to

regulate process extension so as to insulate each neuropil compartment. Overexpressing

Thisbe in ORNs or PNs causes over-wrapping of glomeruli to which their axons or

dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise

ORN axon targeting and discrete glomerular formation.

12
2.2 Introduction

Glia and neurons interact dynamically to coordinate the development and function of

neural circuits. For example, Drosophila midline glia serve as guideposts that provide

spatial cues to direct axonal pathfinding in the embryonic ventral nerve cord (Kidd et

al., 1999; Mitchell et al., 1996). Neurons in turn provide signals that weave glia into the

fabric of the nervous system. For instance, axonal Neuregulin-1 levels dictate the extent

of Schwann cell myelination (Michailov et al., 2004). A myriad of other biological

processes in the developing and adult brain, including synapse formation

(Christopherson et al., 2005; Mauch et al., 2001; Pfrieger and Barres, 1997; Ullian et

al., 2001), elimination (Paolicelli et al., 2011; Schafer et al., 2012), and regulation of

synaptic activity (Araque et al., 2014; Araque et al., 1998a; Araque et al., 1998b; Bezzi

et al., 1998), all require extensive communication and cooperation between neurons and

glia.

The Drosophila nervous system contains a diverse array of glial cell types (Stork et

al., 2012), offering opportunities to discover new mechanisms for glia-neuron

interactions. Ensheathing glia are a group of cells that insulate neighboring neuropil

structures by extending their membranous processes along the outer surface of synaptic

neuropil or their sub-compartments, without invading the inner part of the neuropil

(Awasaki et al., 2008). It remains unknown how ensheathing glia establish such a

barrier-like structure, what molecular signals orchestrate this process, and whether the

glial barrier is essential for the integrity of the encircled neuropil.

13
 The Drosophila antennal lobe provides an excellent experimental system for tackling

these questions with high resolution. The antennal lobe is organized into 50 discrete

neuropil compartments, the glomeruli, wherein specific types of olfactory receptor

neuron (ORN) axons and projection neuron (PN) dendrites form synapses. Each

glomerulus is surrounded by ensheathing glia processes (Awasaki et al., 2008; Jefferis

et al., 2004; Jhaveri et al., 2000). In addition, the specific and stereotypic projection

pattern of neurons in the antennal lobe (Gao et al., 2000; Vosshall et al., 2000) renders

the system convenient for exploring the potential neuronal disorganization caused by

malformation of glial structures. Previous studies have utilized this system to find that

in adults, ensheathing glia increase their process extension to injured axons (Doherty et

al., 2009), help clear degenerating axons (Doherty et al., 2009), and strengthen

excitatory interactions between surviving neurons (Kazama et al., 2011). Here we study

ensheathing glia in the antennal lobe formation and organization during development.

14
2.3 Results

2.3.1 Pupal Development of Antennal Lobe Ensheathing Glia.

To study the development and function of antennal lobe ensheathing glia, I searched for

genetic tools that can specifically label these cells during the pupal stage when the

olfactory circuit in the antennal lobe is being assembled. I preselected a number of

enhancer-GAL4 lines from the FlyLight GAL4 collection based on the published

expression pattern in adult animals (Jenett et al., 2012; Pfeiffer et al., 2008), and a few

MiMIC-GAL4 lines (Venken et al., 2011). I tested whether these GAL4 lines were active

at different pupal stages. I found that at 96 hours after puparium formation (h APF), a

late stage in antennal lobe morphogenesis, SPARC-GAL4 (Figure 2-1D), GMR56F03-

GAL4 (Figure 2-2) (Kremer et al., 2017), and GMR10E12-GAL4 (Figure 2-2) flies

expressed GAL4 predominantly in ensheathing glia. As indicated by the UAS-

mCD8GFP reporter that labeled the cell membranes and processes in the presence of

GAL4, these GAL4+ cells were located on the periphery of the antennal lobe and

extended their processes to wrap around the antennal lobe as well as individual

glomeruli within it, but with minimal invasion into each glomerulus. These are

characteristic morphological features of ensheathing glia. I also used UAS-nuclear-LacZ

to mark the nuclei of these GAL4+ cells (Figure 2-1E), and found that every LacZ+

nucleus around the antennal lobe (within 10-µm distance to the surface of the antennal

lobe) was also positive for Repo, a glia marker in Drosophila (Halter et al., 1995),

confirming that these GAL4+ cells are indeed glia. On average ~100 Repo+ cells were

detected around the antennal lobe, and ~56 were LacZ+ (Figure 2-1F). Repo+/LacZ–

cells are likely astrocytes and cortex glia present in the vicinity of ensheathing glia. To

15
further distinguish these GAL4+ glia from astrocytes, I used the GAT antibody (Stork

et al., 2014) to mark astrocytes, and found that the majority (~95%) of the GAL4+ glia

are negative for GAT (Figure 2-2). In summary, SPARC-GAL4, GRM56F03-GAL4, and

GMR10E12-GAL4 mark ensheathing glia around the antennal lobe in the late pupal

stage.

SPARC-GAL4 also drove reporter expression at 24 h APF, which enabled us to

investigate the development of antennal lobe ensheathing glia. At 24 h APF, prior to

glomerular formation, ensheathing glia processes were restricted to the periphery of the

antennal lobe (Figure 2-1A). From 24 h to 48 h APF, axon terminals of ORNs invade

the antennal lobe and target specific sub-regions to connect with their synaptic partners

such as dendrites from PNs (Jefferis et al., 2004). I found that around 48 h APF when

proto-glomeruli first emerged, ensheathing glia processes started to invade the antennal

lobe from the lobe surface (Figure 2-1B). Even at this initial stage of infiltration,

ensheathing glia processes displayed a preference to grow along the borders between

adjacent proto-glomeruli, instead of growing into them (Figure 2-1B3). At 72 h APF, as

glomerular structures became more clearly separable, ensheathing glia processes had

encircled most glomeruli (Figure 2-1C). These processes remained on glomerular

boundaries, with minimal extension into the glomeruli. Ensheathing glia wrapping of

olfactory glomeruli was complete by the end of the pupal stage (Figure 2-1D).

I also observed an increase in the number of SPARC-GAL4+ ensheathing glia around

the antennal lobe from 24 h to 72 h APF (Figure 2-1F). This increase is at least in part

due to glial cell proliferation, since I could generate glia clones via the MARCM

16
technique (Lee and Luo, 1999) with heat shock induced mitotic recombination at any

point from 24 to 72 h APF (see Figure 2-4 and Figure 2-6 below).

17
Figure 2-1 Ensheathing glia morphogenesis during antennal lobe development.

18
(A–D) Confocal sections for antennal lobe at 24 h (A), 48 h (B), 72 h (C), and 96 h (D)

APF. Ensheathing glia processes are labeled by SPARC-GAL4 driven (>) UAS-

mCD8GFP. Ensheathing glia nuclei are marked by UAS-nuclear-LacZ. Magenta shows

neuropil staining by the N-cadherin (Ncad) antibody. Dashed rectangles are enlarged on

the right column. D, dorsal; L, lateral. Scale bars are 10 µm. (E) Confocal sections of

96 h antennal lobe. Green shows glia nuclei stained by the Repo antibody, overlaid on

the nuclear-LacZ channel in E2. (F) Quantification of antennal lobe ensheathing glia

cell number at different developmental stages, and the number of total glia around the

antennal lobe at the end of pupae stage. Error bars represent standard deviation (SD).

N=5 for each time point.

19
Figure 2-2 Two FlyLight-GAL4 lines and SPARC-GAL4 label antennal lobe

ensheathing glia.

20
Confocal sections of adult antennal lobe. (A1, B1, C1, D1) Ensheathing glia processes are

labeled by GMR56F03-GAL4 (at 25℃) (A1, C1), GMR10E12-GAL4 (at 18℃) (B1), or

SPARC-GAL4 (D1) driven (>) UAS-mCD8GFP. Ensheathing glia nuclei are marked by

UAS-nuclear-LacZ. (A2, B2) Green shows glia nuclei stained by the Repo antibody. (A3,

B3) The channel for Repo staining is overlaid on the nuclear-LacZ channel. (C–D) Co-

labeling of ensheathing glia (mCD8GFP and nuclear-lacZ) and astrocyte marker GAT,

visualized by antibody staining (white). GAT staining is overlaid on the nuclear-LacZ

channel (C2, D2) to show that GAT does not label GAL4+ cells, with few exceptions

(arrows). (E) Co-labeling of ensheathing glia (SPARC-QF>QUAS-mtdT and QUAS-

nuclear-lacZ, green and magenta) and astrocytes (Alrm-GAL4>mCD8GFP, white). The

GFP channel is overlaid on the nuclear-LacZ channel (E2) to show that the group of glia

marked by SPARC-QF are not astrocytes. Scale bars are 10 µm.

21
2.3.2 Heartless Knockdown in Ensheathing Glia Reduces Processes and Disrupts

the Ensheathment Pattern

To identify the molecular signals that control the extension of ensheathing glia processes,

I tested ~50 candidate adhesion molecules, receptor kinases, and receptor phosphatases

by RNA interference (RNAi) and screened for potential defects in ensheathing glia

development and antennal lobe organization. I found that expression of two independent

RNAi targeting non-overlapping regions of heartless (htl), a fibroblast growth factor

receptor (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1993), caused a

70% reduction of ensheathing glia processes within the antennal lobe (Figure 2-3A-D).

This phenotype is consistent with a previous discovery that FGF signaling is required

for astrocytes to infiltrate the larval ventral nerve cord of Drosophila (Stork et al., 2014).

However, the antennal lobe provides a unique opportunity to observe the response of

neuronal compartmentalization to glia morphogenesis defects.

I found that many glomeruli became less clearly separable (Figure 2-3A2, B2, C2,

arrowheads) when htl was knocked down. To quantify this effect, I used relative

standard deviation (RSD) of the N-cadherin neuropil staining intensity as an index for

the degree of compartmentalization, as incomplete antennal lobe compartmentalization

would cause obscure glomerular borders and hence smaller variance in the neuropil

staining signal. I found a significant reduction of RSD in htl knockdown antennal lobes

compared with wild type (Figure 2-3E). The remaining glomerular borders sometimes

also lacked ensheathing glia processes that normally would divide the adjacent

glomeruli (Figure 2-3A-C). I also observed ensheathing glia processes that extended

within glomeruli, which is rarely observed in wild type antennal lobes (Figure 2-3B1,

22
arrowhead). This is likely due to poor establishment of glomerular borders. I quantified

the localization pattern of the ensheathing glia processes relative to the glomerular

compartments by plotting the intensity of the GFP signal derived from glial membranes

together with the intensity of N-cadherin signal marking the neuropil (Figure 2-3A3-C3),

and calculated their correlation coefficient (Figure 2-3F). In control animals, N-

cadherin and GFP signals exhibited strong anti-correlation (Figure 2-3F), since

ensheathing glia processes are preferentially located on the borders of neuropil

compartments instead of within the glomeruli. However, such anti-correlation was

greatly diminished when htl was knocked down (Figure 2-3F), suggesting that loss of

htl results in a disrupted ensheathment pattern.

23
Figure 2-3 Heartless knockdown causes an altered ensheathing glia wrapping pattern

and disrupts antennal lobe compartmentalization.

(A–C) Confocal sections for the antennal lobe at 96 h APF of wild type (A) and two

different UAS-Htl RNAi constructs (RNAi#1: VDRC6692, RNAi#2: VDRC27180)

driven by SPARC-GAL4 (B, C). Ensheathing glia processes are labeled by SPARC-

24
GAL4>UAS-mCD8GFP. Neuropil compartments are stained with Ncad antibody. GFP

and Ncad intensities along a line between the center of DM6 and DA1 glomeruli

(indicated by dashed circles in A2 and asterisks in A2–C2) are plotted in A3–C3, with the

X-axis indicating the position of each point on the line from DM6 to DA1 (left to right);

fluorescence intensity quantified in arbitrary unit. (D) Quantification of total GFP

intensities normalized by antennal lobe size for wild-type and RNAi expressing flies.

(E) Quantification of relative standard deviation of Ncad intensities for each antennal

lobe of wild-type and RNAi flies. (F) Correlation coefficient of GFP and Ncad

intensities as plotted in A3–C3. Scale bar is 10 µm. Error bars represent SD. **, p<0.01;

***, p<0.001, ****, p<0.0001.

25
2.3.3 Heartless Promotes Ensheathing Glia Survival and Cell-autonomously

Regulates Process Elaboration

Heartless is involved in cell differentiation, directional migration, and survival in a wide

variety of tissues (Beiman et al., 1996; Franzdottir et al., 2009; Mandal et al., 2004;

Michelson et al., 1998; Shishido et al., 1997; Stork et al., 2014). Consistent with this

notion, I observed that htl knockdown caused a 40% reduction of ensheathing glia

number near the antennal lobe as assayed by the nuclear-LacZ marker (Figure 2-4A-D).

This decrease is likely due to cell death, since the expression of an apoptosis suppresser,

P35 (Hay et al., 1994), in ensheathing glia largely rescued the reduction of cell number

(Figure 2-5); however, P35 expression did not rescue the reduction of ensheathing glia

processes or the defect in glomerular compartmentalization (Figure 2-5), suggesting that

FGF signaling may also regulate morphogenesis of antennal lobe ensheathing glia in

addition to its role in supporting glia survival.

To dissociate the role of htl in controlling glia survival and process extension, I used

the MARCM technique (Lee and Luo, 1999) to generate single-cell clones of

ensheathing glia homozygous for the htlAb42(null) (Gisselbrecht et al., 1996) allele in an

otherwise heterozygous background. In this experiment, GMR10E12-GAL4 was used to

label the homozygous mutant cells. I used UAS-mCD8GFP to visualize glial processes

(Figure 2-4E-F), and UAS-nuclear-LacZ (Figure 2-4E1, F2, arrows) to visualize cell

bodies of ensheathing glia in these MARCM clones. Compared to wild type, htlAb42

single-cell clones exhibited a 50% reduction of the volume of glial processes (Figure

2-4G) and a 50% reduction of total fluorescence intensity (Figure 2-6). These reductions

were accompanied by a decrease in the number of glomeruli each ensheathing glia could

26
access, as quantified by the number of glomerular borders each glia extended to (Figure

2-4H). Thus, this mosaic experiment demonstrated that htl is cell-autonomously

required for ensheathing glia process extension.

To determine the subcellular localization of the Htl protein, I used a fosmid transgenic

line that produces Htl-GFP from an insertion that contains the extended genomic region

covering htl (Sarov et al., 2016) to analyze GFP signal within and around the antennal

lobe during pupal development. At 24 h APF, no Htl-GFP signal was detected inside

the antennal lobe (Figure 2-4I), consistent with the location of ensheathing glia

processes (Figure 2-1A). At 48 h APF, I started to detect Htl-GFP signal in between

proto-glomeruli (Figure 2-4J). By 72 h APF, Htl-GFP signal was detected at most

glomerular borders, though a lower level of signal was also detected within glomeruli

(Figure 2-4K), which may originate from cell types other than ensheathing glia, such as

astrocytes and potentially neurons. The signal on glomerular borders coincided well

with the ensheathing glia wrapping pattern over this developmental course (Figure 2-1).

These data support the hypothesis that Htl mediates FGF signaling to regulate

ensheathing glia process invasion of the antennal lobe and wrapping of individual

glomeruli.

27
Figure 2-4 Heartless controls ensheathing glia number and cell-autonomously control

their morphology.

(A–C) Projections of confocal sections along the Z-axis. Red: ensheathing glia cell

bodies marked by SPARC-GAL4>UAS-nuclear-LacZ. Blue: Ncad antibody stains for

antennal lobe neuropil. (D) Quantification of ensheathing glia cell number in each

antennal lobe. ****, p<0.0001. (E, F) Wild-type (E) and htlAb42/Ab42 (F) ensheathing glia

single cell MARCM clones in adult antennal lobe. Green shows ensheathing glia

processes labeled by GMR10E12-GAL4>UAS-mCD8GFP. Blue shows ensheathing

glia nuclei marked by UAS-nuclear-LacZ. Magenta shows Ncad antibody staining for

antennal lobe neuropil. (G) Quantification of the volume of processes from each

28
ensheathing glia labeled by MARCM. (H) Quantification of the number of borders each

MARCM-labeled ensheathing glia contacts. Error bars represent SD. **, p<0.01; ***,

p<0.001. (I–K) Confocal sections of antennal lobe at 24 h (I), 48 h (J), and 72 h (K) APF

with Htl-GFP signal. Dashed rectangle in J1 is enlarged and shown as J3–J5. The yellow

stars mark the center of a proto-glomerulus around which ensheathing glia is wrapping.

Scale bars are 10 µm.

29
Figure 2-5 P35 suppresses the reduction of ensheathing glia number caused by htl

knockdown.

(A–B) Projections of confocal sections along the Z-axis for wild type (A) and UAS-Htl

RNAi (VDRC 27180) together with UAS-P35 driven by SPARC-GAL4. Red:

ensheathing glia cell bodies marked by SPARC-GAL4>UAS-nuclear-LacZ. Blue: Ncad

antibody stains for antennal lobe neuropil. (C) Quantification of ensheathing glia cell

number in each antennal lobe. (D–E) Confocal sections for the antennal lobe at 96 h

APF of wild type (D) and UAS-Htl RNAi (VDRC 27180) together with UAS-P35 driven

by SPARC-GAL4 (E). Ensheathing glia processes are labeled by SPARC-GAL4>UAS-

mCD8GFP. Neuropil compartments are stained with Ncad antibody. (F–H)

30
Quantification of the intensity of ensheathing process within the antennal lobe (F),

relative standard deviation of Ncad staining (G), and the correlation coefficient for the

intensities of ensheathing glia processes and neuropil staining (H). Scale bars are 10 µm.

Error bars represent SD. **, p<0.01; ****, p<0.0001; ns, p>0.05.

31
Figure 2-6 Additional MARCM clone examples and analysis.

(A–C) Green shows wild-type ensheathing glia processes labeled by GMR10E12-

GAL4>UAS-mCD8GFP. Blue shows ensheathing glia cell bodies marked by UAS-

nuclear-LacZ. Magenta shows neuropil staining by the Ncad antibody. Scale bar is 10

µm. D, dorsal; L, lateral. An ensheathing glia located on the dorsal surface of the

antennal lobe (A3) extends processes ventrally and semi-circles the DA3 glomerulus

(A2). An ensheathing glia located on the medial surface of the antennal lobe (B3) extends

processes laterally to access glomeruli in the center of the antennal lobe (B2, B3). An

ensheathing glia located on the most anterior surface of the antennal lobe (C2) extends

processes into posterior antennal lobe (C4). (D) Quantification of the total intensity of

processes from each ensheathing glia (wild-type and HtlAb42/Ab42) labeled by MARCM.

Error bars represent SD. **, p<0.01.

32
2.3.4 Thisbe, an FGF Ligand, Is Required for the Wrapping of Glomeruli by

Ensheathing glia

I next assessed the identity and cellular source of FGF ligands that regulate ensheathing

glia development. Htl responds to two FGF ligands, Thisbe (Ths) and Pyramus (Pyr)

(Gryzik and Muller, 2004; Stathopoulos et al., 2004). To test whether ths is required for

antennal lobe ensheathing glia to form the wrapping pattern, I analyzed ensheathing glia

and antennal lobe morphology in animals that carried different ths mutant alleles

(Klingseisen et al., 2009). The phenotypes observed with loss of htl were all

recapitulated in trans-heterozygous combinations of ths alleles (thse02026/ths759 and

Df(2R)ED2238/ths759). There was an overall reduction of ensheathing glia processes

and cell number in the antennal lobe of trans-heterozygous flies (Figure 2-7A-B, E-F).

Similar to loss of htl from ensheathing glia, the antennal lobe of ths mutants also showed

defective compartmentalization as measured by reduced RSD values of N-cadherin

staining (Figure 2-7G) and a markedly reduced anti-correlation between the intensity of

ensheathing glia processes and neuropil signal (Figure 2-7H). These findings indicate

that ths is required for ensheathing glia to wrap around glomeruli in the antennal lobe,

and that failure to form a correct wrapping pattern could disrupt compartmentalization

of antennal lobe neuropil.

33
Figure 2-7 Thisbe is expressed in olfactory neurons and required for ensheathing glia to

wrap glomeruli.

(A–D) Ensheathing glia wrapping pattern in wild type (A), ths mutant (B; thse02 denotes

thse02026 allele), panneural C155-GAL4 (C) and LN orb0449-GAL4 (D) driven RNAi against

ths. Green shows ensheathing glia processes labeled by SPARC-QF>QUAS-mtdT.

Magenta shows ensheathing glia nuclei marked with QUAS-nuclear-LacZ. (E–H)

Quantification of ensheathing glia number (E), process intensity (F), relative standard

deviation of Ncad staining (G), and the correlation coefficient for the intensities of

ensheathing glia process and neuropil staining (H). Error bars represent SD. *, p<0.05;

**, p<0.01; ***, p<0.001; ****, p<0.0001; ns, p>0.05. (I) ths expression pattern

revealed by ths-GAL4>UAS-mCD8GFP (green). Magenta in I1 shows Ncad

counterstaining. (J) ey-FLP intersects with ths-GAL4 together with UAS-FRT-stop-

FRT-mCD8GFP to show ths expression pattern in ORNs. (K) GH146-FLP intersection

shows ths expression pattern in PNs. (L) ths-GAL4+ LN cell bodies and LN and ORN

34
processes, after GH146-GAL80 suppressing ths-GAL4 in most PNs. (M) MARCM

labels a single LN that is positive for ths-GAL4. Green: GFP. Magenta: Ncad. All

images are confocal sections of adult antennal lobe except panel (M), which is a

projection of Z stacks. Scale bars are 10 µm.

35
2.3.5 Thisbe Is Produced by ORNs, PNs, and Antennal Lobe Local Interneurons

To identify which cell types express Thisbe, I took advantage of a MiMIC insertion

(Venken et al., 2011) located between two coding exons of the ths gene. I converted the

MiMIC cassette to an artificial exon that contains the coding sequence for 2A-GAL4

(Diao et al., 2015). Thus, GAL4 can be produced along with the endogenous N-terminus

of Ths (encoded by the first two exons), and can drive reporter gene expression in the

ths pattern. I then used this Ths-GAL4 to express UAS-mCD8GFP to visualize the cell

bodies and projections of the Ths-producing cells. In the antennal lobe, neuronal

processes from PNs, ORNs, and local interneurons (LNs) are all labeled, suggesting that

all three major neuronal types produce Ths (Figure 2-7I).

To determine the contributions of each of these cell types, I used an intersectional

strategy, in which Ths-GAL4 was combined with Flp recombinases that are specifically

expressed in ORNs (ey-FLP) (Newsome et al., 2000) or PNs (GH146-FLP) (Hong et

al., 2009). With a FLP-out reporter, UAS-FRT-stop-FRT-mCD8GFP (Hong et al., 2009),

I found a number of ORN and PN classes were Ths-GAL4+ based on glomerular labeling

(Figure 2-7J-K). At least 45 cell bodies over all sections of the antennal lobe were Ths-

GAL4+ without intersection (Figure 2-7I); however, only 19 of them were GH146-Flp+

PNs (which constitutes the majority of PNs innervating ~40 glomeruli), consistent with

the small number of glomeruli (~7) labeled by the intersection of GH146-Flp and Ths-

GAL4 (Figure 2-7K). This difference is likely due to the contribution from LNs, whose

cell bodies are also located around the antennal lobe (Chou et al., 2010; Stocker et al.,

1990), whereas ORN cell bodies are located in the peripheral sensory organs.

36
 I used two approaches to validate that LNs produce Thisbe. First, I suppressed the

expression from most PNs by combining GH146-GAL80 (Hong et al., 2009) together

with Ths-GAL4. After PN-derived signal was largely eliminated, around 27 cell bodies

around the antennal lobe remained. The projection pattern of these neurons covered the

entire antennal lobe (Figure 2-7L), which is characteristic of the majority of LNs (Chou

et al., 2010). Second, I used Ths-GAL4 to label single cells by the MARCM technique.

I was able to identify MARCM clones for ORNs and PNs, as well as LNs (Figure 2-7M).

In summary, Ths is produced by a subset of ORNs, PNs, and LNs.

37
2.3.6 LN-derived Thisbe Is Necessary for Ensheathing Glia Wrapping and

Antennal Lobe Compartmentalization

To test in which cell type(s) Thisbe functions to regulate ensheathing glia

morphogenesis and antennal lobe compartmentalization, I used RNAi to knock down

ths in ORNs (Pebbled-GAL4), PNs (GH146-GAL4), LNs, and all neurons (C155-GAL4),

respectively, while labeling the ensheathing glia by SPARC-QF, which was converted

from SPARC-GAL4 (Venken et al., 2011). Pan-neuronal knockdown of ths recapitulated

the phenotypes observed in ths mutants (Figure 2-7C, E-H). Knocking down ths

specifically in LNs by orb0449-GAL4 (Figure 2-8), a GAL4 line identified from the InSite

screen (Gohl et al., 2011), resulted in a mild but significant defect in ensheathing glia

wrapping and antennal lobe glomerulus integrity (Figure 2-7D, E-H). Knocking down

ths in PNs did not cause significant defects (Figure 2-9). I have also used MARCM

combined with a cell lethal strategy (Newsome et al., 2000) to create a near pan-ORN

mutant background for ths, and did not find defects in ensheathing glia wrapping (Figure

2-9). The milder phenotypes in LN knockdown compared to pan-neuronal knockdown

could be explained by: 1) pan-neuronal GAL4 may have stronger and/or earlier

expression than the LN-GAL4, and therefore causes more effective knockdown; 2) LN-

GAL4 does not include all LNs; 3) Ths from ORNs and PNs synergize with Ths from

LNs. Regardless, these data indicate that LN is an essential cellular source of Ths in the

antennal lobe for directing ensheathing glia wrapping.

38
Figure 2-8 Orb0449-GAL4 labels local interneurons in the antennal lobe.

(A) Green shows orb0449-GAL4>UAS-mCD8GFP. (A1) A projection of confocal sections

along the Z-axis at 24 h APF. Around 23 local interneurons are labeled. (A2) A confocal

section of antennal lobe at 96 h APF. A total of ~55 local interneurons are labeled over

all confocal sections of the antennal lobe. (B) ey-FLP intersects with orb0449-GAL4

together with UAS-FRT-stop-FRT-mCD8GFP to show that orb0449-GAL4 is inactive in the

majority of ORNs, except two classes that project to the medial side of the antennal lobe

(arrow). (C) GH146-FLP intersection shows a rare case in which an interneuron is

labeled (1 cell out of 10 antennal lobes), whereas around other antennal lobes 0 cell is

labeled. Magenta shows neuropil staining by the Ncad antibody. Scale bar is 10 µm.

39
Figure 2-9 Loss of Thisbe from ORNs and PNs does not cause ensheathing glia or

glomerulus defects.

(A) GH146-GAL4 drives RNAi against ths. Ensheathing glia processes are labeled by

SPARC- QF>QUAS-mtdT. Magenta shows neuropil staining by the Ncad antibody.

Scale bar is 10 µm. (B) ey-FLP MARCM combined with cell lethal strategy creates ths

mutant in nearly all ORNs. Ensheathing glia processes are labeled by GMR56F03-

GAL4>UAS-mCD8GFP. (C-F) Quantification of ensheathing glia process intensities

(C, E), and relative standard deviation of Ncad staining intensities (D, F). Error bars

represent SD. ns, p>0.05.

40
2.3.7 Thisbe Can Instruct Glomerular Wrapping with a High Spatial Specificity

I have shown that FGF signaling is necessary for ensheathing glia to extend processes

into the antennal lobe and demarcate individual glomeruli. To test whether FGF

signaling can instruct ensheathing glia to wrap around selected neuropil compartments,

I expressed ths in only one class of ORNs (VA1v) using a GAL4 under the control of

the promoter of the odorant receptor specifically expressed in this class (Or47b-GAL4).

I observed that overexpression resulted in hyper-wrapped VA1v glomerulus by

ensheathing glia (Figure 2-10B, D). This effect was highly localized, since in addition

to VA1v, only the adjacent VA1d glomerulus was slightly hyper-wrapped (Figure

2-10D), likely due to intensified ensheathing glia processes on its border shared with

VA1v. Hyper-wrapping did not extend to the DA1 glomerulus (Figure 2-10D), which

is one glomerulus away from the VA1v glomerulus. There was also an excess of glia

cells around the hyper-wrapped VA1v glomerulus (Figure 2-10F).

Similarly, overexpressing Ths in Mz19-GAL4+ PNs, which send dendrites to DA1

and VA1d, also caused local hyper-wrapping of these glomeruli, as well as a local

increase of ensheathing glia cells (Figure 2-10C, E, G). It has been consistently noticed

that Mz19-GAL4 activity is stronger in DA1 PNs than in VA1d PNs (Hong et al., 2012).

Accordingly, glial hyper-wrapping was more pronounced around DA1 than around

VA1d. These results suggest that Thisbe can act locally as a spatial cue to instruct

ensheathing glia to infiltrate the antennal lobe.

41
Figure 2-10 Changes in ensheathing glia processes and number in response to local

overexpression of ths.

(A–C) Confocal sections of adult antennal lobe of wild type (A), overexpression of UAS-

ths by Or47b-GAL4 (B) or by Mz19-GAL4 (C). Green: SPARC-QF>QUAS-mtdT labels

ensheathing glia processes. Magenta: Ensheathing glia nuclei marked with QUAS-

nuclear-LacZ. Scale bar is 10 µm. (D, E) Quantification of signal intensities of

ensheathing glia processes around each glomerulus normalized by the perimeter of the

corresponding glomerulus. (F, G) Quantification of ensheathing glia numbers within 5-

µm distance to the surface of each glomerulus. Error bars represent SD. *, p<0.05; **,

p<0.01; ***, p<0.001; ****, p<0.0001; ns, p>0.05.

42
2.3.8 FGF Signaling Ensures Accurate Neuronal Targeting

Lastly, I examined whether glia wrapping defects could disrupt neuronal projections to

the glomerular compartments. I used membrane proteins under the control of specific

odorant receptor promoters (Or88a-mtdT and Or47b-rCD2) to label two ORN classes

that project their axons to two adjacent glomeruli, VA1v and VA1d (Figure 2-11A).

When ths was knocked down with a pan-neuronal driver, C155-GAL4, VA1v and VA1d

ORN axons still reached their target area in the ventrolateral antennal lobe. However,

they failed to establish exclusive territories for their axonal arborization, and instead

had partially overlapped axonal terminals (Figure 2-11B). Likewise, in ths mutant

animals, VA1v and VA1d axon terminals partially intermingled, rather than confining

to their respective glomerular compartments (Figure 2-11C; quantified in Figure 2-11D).

Thus, FGF signaling between neurons and ensheathing glia is essential for antennal lobe

compartmentalization and targeting accuracy for ORN axons.

43
Figure 2-11 Loss of ths results in olfactory receptor neuron targeting defects.

(A–D) Confocal sections of the adult antennal lobe, with Or88a-mtdT and Or47b-rCD2

labeling ORN axons that target VA1d (magenta) and VA1v (green) glomerulus,

respectively. Images within the dashed rectangles in the first column are enlarged in

columns 2–4. (A) Wild type. (B) C155-GAL4 driven ths RNAi. (C-D) ths mutant flies

showed severe intermingling of VA1d and VA1v ORN terminals (C) and a mild spillover

of VA1d ORN axons (D, arrow). ths238 denotes the Df(2R)ED2238 allele. Scale bars are

44
10 µm. (E) Quantification of the accuracy of ORN axon targeting. 1, normal; 2, mild

spillover; 3, severe intermingling. Each symbol represents 1 fly with two antennal lobes

scored separately and averaged. Error bars represent SD. *, p<0.05; **, p<0.01.

45
2.4 Discussion

The use of discrete neuropil compartments for organizing and signaling information is

widespread in invertebrate and vertebrate nervous systems. In both the fly antennal lobe

and vertebrate olfactory bulb, axons from different ORN classes are segregated into

distinct glomeruli (Komiyama and Luo, 2006). The rodent barrel cortex also uses

discrete compartments, the barrels, to represent individual whiskers (Woolsey and Van

der Loos, 1970). In this study, I show that FGF signaling between neurons and glia

mediate neural compartment formation in the Drosophila antennal lobe.

Members of the FGF family have diverse functions in a variety of tissues in both

vertebrates and invertebrates (Muha and Muller, 2013; Ornitz and Itoh, 2015).

Vertebrate FGFs regulate not only neural proliferation, differentiation, axon guidance,

and synaptogenesis, but also gliogenesis, glial migration, and morphogenesis (Furusho

et al., 2012; Guillemot and Zimmer, 2011; Perraud et al., 1988; Reilly et al., 1998; Smith

et al., 2006). Many of these roles are conserved in invertebrates. For example, Thisbe

and Pyramus induce glial wrapping of axonal tracts (Franzdottir et al., 2009; Shishido

et al., 1997) much like the role of other FGF members played in regulating myelin

sheaths in mammals (Furusho et al., 2012). Thisbe and Pyramus also control Drosophila

astrocyte migration and morphogenesis (Stork et al., 2014). Likewise, FGF signaling

also promotes morphogenesis of mammalian astrocytes (Kang et al., 2014). Therefore,

studying the signaling pathways in Drosophila will extend our understanding of the

principles of neural development.

46
 In ensheathing glia, whose developmental time course and mechanisms have not been

well documented prior to this study, I observed a glial response to FGF signaling

reminiscent of the paradigm shown before (Franzdottir et al., 2009; Gibson et al., 2012;

Stork et al., 2014); however, the exquisite compartmental structure of the Drosophila

antennal lobe and genetic access allowed us to further scrutinize changes of neuropil

structure and projection patterns that occurred alongside morphological phenotypes in

ensheathing glia. I demonstrated a necessity of Ths in local interneurons, although it is

possible that ORNs and PNs also contribute. I also tested the function of the other ligand,

Pyramus, in antennal lobe development. I did not detect any change in ensheathing glia

morphology with pyramus RNAi, and double RNAi against thisbe and pyramus did not

enhance the phenotype compared to thisbe knockdown alone.

FGF signaling in glomerular wrapping appears to be highly local. In our

overexpression experiments, the hyper-wrapping effect was restricted to the glomerulus

where the ligand is excessively produced, and did not spread to nearby non-adjacent

glomeruli. These experiments suggest that Thisbe communicates locally to instruct glial

ensheathment of the glomeruli, rather than diffusing across several microns to affect

nearby glomeruli. Since heparan sulfate proteoglycans are known to act as FGF co-

receptors by modulating the activity and spatial distribution of the ligands (Baeg and

Perrimon, 2000; Muha and Muller, 2013; Shimokawa et al., 2011), I speculate that

Thisbe in the antennal lobe may be subject to such regulation to limit its diffusion and

long-range effect.

47
 My data showed that deficient ensheathment of antennal lobe glomeruli is

accompanied by imprecise ORN axon targeting. However, I cannot determine whether

these targeting defects reflect initial axon targeting errors, or a failure to stabilize or

maintain the discrete targeting pattern. Previous models for the establishment of

antennal lobe wiring specificity suggested that the glomerular map is discernable by the

time glia processes start to infiltrate the antennal lobe (Jefferis et al., 2004). Due to a

lack of class-specific ORN markers for early developmental stages, the relative timing

between when neighboring ORN classes refine their axonal targeting to discrete

compartments and when ensheathing glia barriers are set up still remains unclear.

Regardless, our discovery that FGF signaling functions in the formation of discrete

neuronal compartments in the antennal lobe highlights an essential role for glia in the

precise assembly of neural circuits.

48
2.5 Experiment Methods

Immunostaining. Tissue dissection and immunostaining were performed according to

previously described methods (Sweeney et al., 2007). Primary antibodies used in this

study include rat anti-DNcad (DN-Ex #8; 1:40; DSHB), chicken anti-GFP (1:1000;

Aves Labs), rabbit anti-DsRed (1:500; Clontech), mouse anti-rCD2 (OX-34; 1:200;

AbD Serotec), rat anti-HA (1 µg/ml, Roche), mouse nc82 (1:35; Developmental Studies

Hybridoma Bank, [DSHB]), mouse anti-Repo (1:50, DSHB), rabbit anti-β-

Galactosidase (MP Biomedicals, 1:125), rabbit anti-GAT (1:3000, a gift from Marc

Freeman (Stork et al., 2014)) and mouse anti-β-Galactosidase (1:1000; Promega).

Secondary antibodies were raised in goat or donkey against rabbit, mouse, rat, and

chicken antisera (Jackson Immunoresearch), conjugated to Alexa 405, FITC, 568, or

647. Confocal images were collected with a Zeiss LSM 780 and processed with Zen

software, ImageJ, and Imaris.

Mosaic Analysis. The hsFlp MARCM analyses were performed as previously described

(Komiyama et al., 2004; Lee and Luo, 1999) with slight modifications. GMR10E12-

GAL4 was used for labeling ensheathing glia in adult stage. Flies were kept at 18℃ and

were heat shocked for 30 min at 37℃ between 0-24h APF.

Data Analysis. Confocal sections of antennal lobes were analyzed by ImageJ software

for measuring integrated intensity value, area size, mean, and standard deviation for the

region of interest (ROI) manually selected based on N-cadherin counterstaining. Process

intensity was calculated using the integrated intensity value of the antennal lobe section

crossing DA1 and DM6 glomeruli normalized by the area size of the antennal lobe of

49
that section. The Plot Profile function in ImageJ was used to measure signal intensities

for ensheathing glia processes and Ncad along the selected lines. Glia cell number was

manually counted based on LacZ staining within 10-µm distance to the surface of the

antennal lobe determined by Ncad counterstaining. To quantify the MARCM clones

(Fig. 2-4 and Fig. 2-6), confocal sections of marked ensheathing glia were processed by

Imaris software through manually thresholding the images by a set of consistent

parameters, followed by automatic measurement of the volume and total intensity of

glia processes. In the overexpression experiment (Fig. 2-10), ROI was manually

selected by including the inner region and the boundaries of the glomerulus of interest

based on Ncad counterstaining. Process intensity was defined as the integrated intensity

value divided by the perimeter of the ROI selection. Glia number was manually counted

based on LacZ signal within 5-µm distance to the surface of each glomerulus. ORN

targeting defects (Fig. 2-11) were scored by an experimenter blind to the genotype.

Graphs were generated using the GraphPad Prism software. Mean ± SD were shown on

the graphs. Statistical significance was calculated with Graphpad Prism using two-tailed

Student’s t test (Fig. 2-3 ~ Fig. 2-10) or Mann-Whitney test (nonparametric data in Fig.

2-11).

50
3 Chapter 3 Fish-lips Directs Drosophila Olfactory Neuron

Wiring

3.1 Abstract

Wiring specificity is defined by combinatorial molecular codes on the surface of the

pre- and post- synaptic neurons. A systematic screen searching for novel molecules

required for neuronal wiring in the antennal lobe identified a leucine-rich repeat (LRR)

transmembrane protein, Fish-lips (Fili), which is differentially expressed in a subset of

olfactory receptor neurons (ORNs) and projection neurons (PNs) that target specific

glomeruli. Loss of Fili caused targeting defects in a subset of olfactory neurons, whereas

Fili mis-expression induced ORNs and PNs to target ectopically. Fili is potentially a

member in the combinatorial LRR code system that instructs wiring specificity of

olfactory neurons.

51
3.2 Introduction

The large number and the diverse functions of the molecules involved in wiring the

neuronal network make wiring specificity an attractive field to study. The Drosophila

antennal lobe provides an ideal system, in which each of the ~50 distinct glomeruli

encapsulates synapses between a single class of ORNs and a single class of PNs. To

visualize the antennal lobe at a single glomerular resolution, a number of tools have

been developed by our lab and others in the field. To visualize ORNs we typically rely

on olfactory receptor promoters that drive membrane targeted fluorescent proteins. To

observe PN dendrites we use enhancer trap lines that are specific for certain PN classes.

Moreover, the MARCM method (Mosaic Analysis with a Repressible Cell Marker) (Lee

and Luo, 1999) allows us to label specific neurons and their processes by creating

mosaic clones. The GAL4/UAS binary system enables us to manipulate gene expression

in the desired set of cells. LexA/LexAop (Lai and Lee, 2006; Szuts and Bienz, 2000)

and QF/QUAS (Potter et al., 2010) systems provide additional binary expression

systems that can be used in combination with the GAL4/UAS system.

With the help of the powerful fly genetic toolbox, the Drosophila antennal lobe has

become one of the best-understood models for neuronal wiring at the cellular and

developmental levels (Hong and Luo, 2014). In development, PN dendrites innervate

the developing antennal lobe before interacting with ORN axons (Jefferis et al., 2004).

The targeting pattern of PN dendrites is predetermined by their lineage and birth order

(Jefferis et al., 2001) through distinct transcription factors (Komiyama et al., 2003; Zhu

et al., 2006b), ending up with distinct cell surface identities. For example, in response

to Sema-2a/2b secreted by the larval antennal lobe, graded expression of Sema-1a in

52
PNs instructs their dendrites to target along the dorsolateral-ventromedial axis in the

Drosophila antennal lobe (Komiyama et al., 2007; Sweeney et al., 2011). Later on when

ORN axons reach the antennal lobe, the same gradient of Sema-2a/2b also specifies

which ORN axons take the dorsolateral trajectory and which take the ventromedial by

attracting the ventromedial ORN classes which express Sema-2b. This trajectory choice

is ultimately important for axon target choice (Joo et al., 2013). Semaphorins also

contribute to other steps of ORN axon targeting. For instance, the early-arriving axons

from the antenna can constrain the target choice of late-arriving axons from maxillary

pulp, through Sema-1a/PlexinA mediated repulsion (Sweeney et al., 2007). When PN

dendrites and ORN axons get to their approximate position, synaptic matching

molecules, such as Ten-m and Ten-a, refine the glomerular map by matching the PN-

ORN pairs through homophilic interactions (Hong et al., 2012). Although the

aforementioned molecules (and others not discussed here) provide a foundation for

understanding wiring specificity in the fly olfactory system, they are not sufficient to

describe the mechanism by which different classes of ORN axons and PN dendrites

organize to form ~50 glomeruli. This chapter will focus on identifying new cell surface

proteins that are required for antennal lobe wiring, in the quest for a deeper

understanding of the molecular logic by which the olfactory system is organized, and in

doing so, to improve our general understanding of neuronal wiring principles.

53
3.3 Results

3.3.1 Develop New Genetic Tools to Extend Wiring Specificity Studies to

Additional Regions of the Antennal Lobe

Due in large part to the lack of good tools and reagents for labeling specific classes of

PNs outside of the lateral antennal lobe (mostly DA1 and VA1d glomeruli), much of

our understanding of olfactory system wiring is derived from a few glomeruli. While

certain molecules may have global functions (Zhu et al., 2006a; Zhu and Luo, 2004),

others have been found to be region- and even glomerulus-specific (Hong et al., 2012;

Hong et al., 2009; Joo et al., 2013; Ward et al., 2015). Therefore, in order to fully

understand how the entire antennal lobe is wired, it is essential that we identify new

tools that allow us access to other regions of the antennal lobe.

To this end, I screened through a collection of enhancer-GAL4 lines from the

FlyLight project (Jenett et al., 2012; Pfeiffer et al., 2008) and selected six lines that show

highly expressed, specific, and consistent PN labeling patterns. As shown in Figure 3-1,

the labeled glomeruli cover distinct regions including the dorsolateral, ventromedial and

middle regions of the antennal lobe. Furthermore, in developmental expression analysis,

as shown in Figure 3-2, many of these enhancer-GAL4 lines were active at early stages.

For two of the GAL4 lines, GMR71D09-GAL4 and GMR91G04-GAL4, the onset of

expression was as early as 24 hours after puparium formation (h APF) — the initial

stage when PNs are establishing their dendritic territories in the developing antennal

lobe. These lines have turned out valuable for a series of studies focusing on the course

of development (Li et al., 2017; Ward et al., 2015).

54
 In order to perform a GAL4-based RNAi screen with these PN classes labeled, I

converted these enhancer-GAL4 lines into another binary system, LexA/LexAop (Lai

and Lee, 2006), by fusing each of the specific enhancer fragments to the LexA sequence,

and creating transgenic flies by integrase-mediated site-specific transgenesis.

55
Figure 3-1 Identify enhancer-GAL4 lines specifically label certain classes of PNs in

adult antennal lobe.

Neuronal processes are labeled by UAS-mCD8GFP driven by 6 different enhancer-

GAL4 lines from the FlyLight collection. Magenta stains the synaptic marker, nc82,

for glomeruli visualization.

Figure 3-2 Developmental analysis showing expression patterns for 4 of the enhancer-

GAL4 lines during the pupae stage.

Neuronal processes are labeled by enhancer-GAL4 driven (>) UAS-mCD8GFP.

Magenta stains for N-cadherin (Ncad) for visualizing the developing antennal lobe.

56
 The resulting enhancer-LexA fly lines mostly recapitulated the labeling patterns of

the original enhancer-GAL4 in adults (Figure 3-3), with minor variance in labeling

intensity and specificity. For example, 91G04-LexA weakly labels an additional

glomerulus next to DC2 and DA2 on the medial side, which could be glomerulus 1

(Figure 3-3). I decided to base the new RNAi screen on GMR86C10-LexA that is

active in VM5v and VM5d PNs, because of its labeling stability and that the labeled

region is far away from the lateral side of the antennal lobe which has been

extensively scrutinized in previous studies.

Figure 3-3 Enhancer-LexA lines recapitulate GAL4 patterns.

Confocal sections for adult antennal lobe are shown. Neuronal processes are labeled

by enhancer-LexA > LexAop-mCD8GFP. Magenta shows neuropil staining by the

Ncad antibody.

57
3.3.2 Identification of LRR Protein Fish-lips (Fili) as a Wiring Specificity

Molecule

To increase the variety of neuronal wiring phenotypes that can be revealed by my

labeling strategy, such as PN mistargeting, ORN ectopic projection, and PN-ORN

mismatching, I combined the PN marker (GMR86C10-LexA, LexAop-mtdT) with two

ORN labels, including the ORN class that synapse with VM5v PNs by the expression

of mouse CD8-GFP from the Or98a-mCD8GFP transgene, and the ORN class

projecting to a neighboring glomerulus, VA2, by the expression of rat CD2 from the

Or92a-rCD2 transgene (Figure 3-4 A). In addition to these transgenic elements, the

C155-GAL4 was used to drive the expression of RNAi lines (Dietzl et al., 2007;

Perkins et al., 2015) of predicted transmembrane or secreted proteins (Kurusu et al.,

2008). Around 700 RNAi lines covering over 200 genes whose protein products

contain domain types of leucine-rich repeat (LRR), immunoglobulin, cadherin,

epidermal growth factor repeat, and fibronectin were tested for their necessity in

accurate olfactory neuron wiring. Knocking down one of the LRR genes, Fish-lips

(Fili) showed targeting defects in VM5v and/or VM5d dendrites (Figure 3-4B’, C’,

arrows).

Fili encodes a transmembrane protein that contains multiple LRR domains and

shares sequence similarities with two other LRR proteins, Capricious (Caps) and

Tartan (Trn) (Adachi-Yamada et al., 2005), both of which have been proven to specify

PN targets in the antennal lobe (Hong et al., 2009). However, the function of Fili in

the nervous system has never been documented.

58
Figure 3-4 Identify Fili as a candidate molecule for olfactory wiring.

(A–C) Confocal sections of the adult antennal lobe, with GMR86C10-LexA, LexAop-

mtdT labeling PN dendrites targeting VM5v and VM5d glomeruli (magenta), Or98a-

mCD8GFP labeling ORN axons targeting VM5v glomerulus (green), and Or92a-

rCD2 labeling ORN axons that target VA2 glomerulus (blue). White shows neuropil

staining by the Ncad antibody. Images in the same column are anterior and posterior

sections of the same antennal lobe from the same animal. (A) Control. (B-C) C155-

GAL4 drives two different Fili RNAi constructs (Bloomington28568, VDRC44532

respectively). Ectopic PN targets are indicated by yellow arrows. N=10 for each

genotype. The penetrance of the RNAi phenotypes are 4/10 and 5/10 respectively.

59
 To validate the phenotype induced by Fili knockdown, I used the CRISPR

technique to generate a loss-of-function allele for Fili, in which the first exon and part

of the second exon are removed, including the first two possible starts for protein

translation (Figure 3-5B). According to SMART protein prediction (Schultz et al.,

1998), this excision will cause the elimination of the start codon, the signal peptide,

and the first 6 LRR domains in Fili protein produced from both predicted transcripts

(Fili-RC and Fili-RD in Figure 3-5A), even if the C-terminus of the protein could still

be produced (Figure 3-5C), thus most likely creating a null allele. The animals that are

homozygous for this Fili allele showed dorsal ectopic projection from VM5v and/or

VM5d PNs (Figure 3-6, arrow). This phenotype is similar to that caused by the pan-

neuronal knockdown of Fili (Figure 3-4, arrows). Therefore, the RNAi-based genetic

screen focusing on the ventral-medial region of the antennal lobe identified a cell

surface molecule with a potential novel function in directing wiring specificity of

olfactory neurons.

60
Figure 3-5 Generation of a Fili loss-of-function allele.

(A) Genomic region of Fili (adapted from flybase.org). Two transcripts (Fili-RC and

Fili-RD) possessing different transcription start sites share the identical protein coding

sequence. Dashed rectangle around their start codon is enlarged in (B). Two other genes,

PpN58A and CG43742, are nested within the Fili gene. (B) The first two ATG triplets

in Fili and the DNA sequence in between them are deleted by CRISPR technique,

leaving the adjacent CG43742 gene intact. (C) Fili protein domain prediction by

SMART. The first blue bar denotes the signal peptide, while the second blue bar stands

for a transmembrane domain. Green bars are leucine-rich repeat domains. Pink bars are

low-complexity regions. Protein sequence within the red brackets is deleted due to the

CRISPR mediated excision shown in (B).

61
Figure 3-6 VM5v and/or VM5d PN dendrites target ectopically in Fili-/-.

GMR86C10-GAL4>UAS-mCD8GFP labels VM5v and VM5d PNs in the antennal

lobe of Fili+/- (A) and Fili-/- (B) animals. Images in the same column are anterior and

posterior sections of the same antennal lobe from the same animal. Magenta shows

neuropil staining by the Ncad antibody. N=10 for each genotype. PN ectopic targeting

(arrow) was observed in 9/10 animals.

62
3.3.3 Fili Expression Pattern in the Developing Antennal Lobe

To understand the mechanism by which Fili functions in neuronal wiring, it is essential

to discover the cellular source and distribution pattern of this protein. To study which

cells produce Fili, I utilized a Drosophila strain in which a MiMIC element (Venken et

al., 2011) is located in a coding intron shared by both transcripts of Fili. An artificial

exon containing a splicing acceptor, coding sequence for 2A peptide followed by GAL4,

and a transcription terminator (Diao et al., 2015) was inserted to this MiMIC locus to

generate Fili-GAL4. With the reporter of UAS-mCD8GFP, the Fili-expressing cells and

their processes were labeled (Figure 3-7). I focused on the developmental stage of 48 h

APF, because 1) protoglomeruli are just formed around this time (Jefferis et al., 2004),

and therefore discerning the identity of neurons based on their projection pattern starts

to be feasible, and 2) neuronal wiring is likely to have finished by then (Jefferis et al.,

2004), and hence the expression pattern at later stages would be less relevant to neuronal

targeting. At 48 h APF, the commissure structure is GFP+ (Figure 3-7B, arrow),

indicating that ORNs are potentially Fili-GAL4+, as ORN axons are known to travel

through commissure to target bilaterally. On the other hand, the cell bodies around the

antennal lobe (Figure 3-7A, arrow) suggest that PNs and/or local interneurons (LNs) are

also Fili-GAL4+.

To distinguish Fili produced in different cells types, I used the intersection strategy

with which an ORN or PN specific FLP recombinase can restrict the flip-out reporter

expression in the cell type of interest. UAS-FRT-stop-FRT-mCD8GFP reporter

combined with eyFLP that is active in ORNs (Chotard et al., 2005; Newsome et al.,

2000) reveals the glomeruli that are targeted by Fili-GAL4+ ORNs (Figure 3-8), while

63
GH146-FLP that is specific for PNs (Hong et al., 2009) can reveal PNs that are Fili-

GAL4+ (Figure 3-9). ORNs and PNs that target the ventromedial glomeruli appear

mostly Fili-GAL4+, which indicates that the loss-of-function phenotype aforementioned

is due to the loss of Fili from the region where Fili is expressed at a high level.

In addition, I generated a number of Fili-GAL4+ MARCM clones and found that

some of the cells displayed a projection pattern distinct from ORNs or PNs (Figure 3-10).

Their cell bodies are located around the antennal lobe and their processes cover multiple

glomeruli — a pattern that is reminiscent of a previously reported category of LNs

(Chou et al., 2010). Lacking pan-LN genetic tools, it is challenging to decide the total

number and complete projection patterns of LNs that express Fili. Nevertheless, the

preliminary discovery of Fili-GAL4+ LNs by random sampling provides a curious

possibility that these neurons may also contribute to antennal lobe wiring — a

possibility that has not been demonstrated before.

64
Figure 3-7 Fili-GAL4 labels a subset of glomeruli in the developing antennal lobe.

An anterior (A) and a posterior (B) section of 48 h APF antennal lobe are labeled by

Fili-GAL4>UAS-mCD8GFP. Magenta shows neuropil staining by the Ncad antibody.

65
Figure 3-8 Fili-GAL4 labels a subset of ORNs during antennal lobe development.

Confocal images of an anterior (A) and a posterior (B) section of the antennal lobe at 48

h APF. Ey-FLP intersecting with Fili-GAL4 together with UAS-FRT-stop-FRT-

mCD8GFP shows glomeruli targeted by Fili-expressing ORNs. Magenta shows

neuropil staining by the Ncad antibody.

66
Figure 3-9 Fili-GAL4 labels a subset of PNs during antennal lobe development.

Confocal images of an anterior (A) and a posterior (B) section of the antennal lobe at 48

h APF. GH146-FLP intersecting with Fili-GAL4 together with UAS-FRT-stop-FRT-

mCD8GFP shows Fili expression pattern in PNs. Magenta shows neuropil staining by

the Ncad antibody.

67
Figure 3-10 A Fili-GAL4+ MARCM clone reveals a potential LN.

Confocal images of an anterior section (A), a posterior section (B), and the projection

along the Z-axis (C) of an adult antennal lobe show a local interneuron produced through

MARCM, labeled by Fili-GAL4>UAS-mCDE8GFP.

68
 Next, I generated an antibody against Fili and stained developing antennal lobes in

order to investigate Fili protein distribution pattern. At 48 h APF, Fili protein is enriched

in a few glomeruli including the ventromedial ones (Figure 3-11A). This signal is absent

in Fili-/- animals (Figure 3-11B), which verifies the specificity of the antibody. This

antibody was generated against an epitope from the intracellular domain of Fili, whose

DNA sequence remains in the CRISPR mediated mutant allele. Thus, the absence of

Fili signal in the animals homozygous for this allele strongly supports that the Fili allele

generated by CRISPR mediated excision is a null allele.

Subsequently, I used the Fili-/- animals to pre-absorb the anti-Fili serum, and stained

the developing wild-type animals with the pre-absorbed serum to achieve a better signal

quality. A few ventromedial glomeruli, including VA2, VM5v, and VM5d which are

covered in our RNAi screen show a higher level of Fili expression (Figure 3-11C)

compared to the others, such as the DA1 glomerulus (Figure 3-11C, yellow dashed

circle). It is noteworthy that a few PN classes including VM5v and VM5d are not

positive for GH146-GAL4, a line that covers only 80% of antennal lobe PNs. Therefore,

I co-stained Fili protein with GFP that was driven by GH146-GAL4. As a result, the

VM5v and VM5d glomeruli appeared negative for GFP as expected, and they showed

enriched signal for Fili (Figure 3-11E, dashed circle). This confirms the presence of Fili

protein in the region of the antennal lobe where loss-of-function phenotypes were

observed.

Notably, the Fili antibody does not show a protein distribution pattern identical with

that reveal by Fili-GAL4. For instance, the antibody did not detect any Fili above the

69
background level in DA1 glomerulus (Figure 3-11C, yellow dashed circle); however

Fili-GAL4 in conjunction with GH146-FLP uncovered GAL4 activity in DA1 PNs

(Figure 3-9). It is possible that Fili goes through some post-transcriptional regulations

that are not reflected by the production of GAL4. Alternatively, it is likely that GAL4

is more sensitive than the antibody in revealing low amount of Fili, and therefore some

ORN and PN classes can be Fili-GAL4+ without an outstanding Fili antibody signal.

70
Figure 3-11 Fili is enriched in a subset of glomeruli, including VM5v and VM5d.

(A-B) Before pre-absorption, anti-Fili serum stains wild-type (A) and Fili-/- (B) antennal

lobe at 48 h APF (green). Neuropil is stained by HRP (blue). (C-D) Confocal sections

of wild-type antennal lobes at 48 h APF stained by anti-Fili serum that is pre-absorbed

by Fili-/- brains. (E-E’) Fili+/+ animals with most of the PNs labeled by GH146-

GAL4>UAS-mCD8GFP. VM5v and VM5d glomeruli marked by lack of GFP signal

(dashed line) show enriched Fili signal (magenta).

71
3.3.4 Fili Expressed in ORNs Can Control PN Targeting

Since Fili can be produced by multiple groups of olfactory neurons, it is critical to

attribute the loss-of-function phenotype observed in VM5 PNs to more specific groups

of cells in order to dissect the underlying cellular mechanisms. To this end, I used the

MARCM technique to test whether Fili acts cell-autonomously in VM5 PNs. In this

experiment, GMR86C10-GAL4 would only label VM5v and VM5d PNs after they

became Fili-/- in a Fili+/- background. Compared to the wild-type PNs, no

distinguishable dendritic projection pattern was observed in Fili-/- VM5 PNs (Figure

3-12A). This result indicates that Fili is not autonomously required in these PNs to direct

their dendritic targeting.

Although PNs innervate the antennal lobe prior to ORN axon arrival (Jefferis et al.,

2004), substantial PN dendritic refinement can take place after they interact with their

synaptic partners, including ORNs, and undergo dynamic wiring (Ward et al., 2015).

Hence, it is attractive to hypothesize that Fili originated from ORNs may signal to PNs

and control their targeting pattern. To test this, I used eyFLP in combination with a cell

lethal mutant (Newsome et al., 2000; Zhu and Luo, 2004) in trans to the Fili allele to

generate large ORN clones (85%-90% of all ORNs) that were Fili-/-. VM5 PNs in this

experiment were Fili+/-, and again labeled by GMR86C10-GAL4. As a result, these PNs

partially mistargeted to dorsal regions (Figure 3-12B) when the majority of ORNs are

Fili-/- — a phenotype almost identical to that seen in whole animal mutant and pan-

neuronal RNAi knockdown. This data suggests that ORNs can present the cell surface

cue, Fili, to PNs in order to control PN dendrite targeting.

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Figure 3-12 Fili from ORNs controls PN dendrite targeting.

(A-A’’) GMR86C10-GAL4>UAS-mCD8GFP labels Fili-/- PN clones in a Fili+/-

background. (B) GMR86C10-GAL4>UAS-mCD8GFP labels Fili+/- PNs in the antennal

lobe with the majority of ORNs being Fili-/-. (B’) A central section of the antennal lobe

shows ectopic PN target (dashed circle). (B’’) A projection along the Z-axis also shows

that PN dendrites mistarget to a more dorsal region in the antennal lobe (dashed circle).

Blue shows neuropil staining by the Ncad antibody.

73
3.3.5 Overexpressing Fili Alters PN Dendrite and ORN Axon Projection Patterns

The differential expression of Fili in the antennal lobe raises the hypothesis that the

levels of Fili protein in different classes of olfactory neurons encode their identities and

specify their target selection. Indeed, we observed that upon losing Fili, VM5 projection

neurons mistarget from a Fili-high region to a Fili-low space (Figure 3-6, Figure 3-11E),

while DA1 PNs that normally target a Fili-low territory do not exhibit any targeting

defect (Figure 3-11C, Figure 3-13B). To further assess the effects of altered Fili code in

olfactory wiring, I increased Fili expression level in DA1 PNs by inducing the

expression of UAS-Fili (Adachi-Yamada et al., 2005) specifically in this class via the

MARCM technique. As a result of their elevated Fili level, these PNs target their

dendrites beyond DA1 glomerulus and innervate VA1v glomerulus (Figure 3-13C),

where the level of Fili is relatively high (Figure 3-11C, white dashed circle). These data

support the notion that differential Fili levels in distinct glomeruli can instruct PN

classes to make specific target choice.

74
Figure 3-13 Fili overexpression causes DA1 PNs to mistarget.

(A-B) Confocal sections of Fili+/- (A) and Fili-/- (B) adult antennal lobes with DA1 and

VA1d PNs labeled by Mz19-GAL4>UAS-mCD8GFP. The yellow dashed line circles

DA1 glomerulus. The white dashed line circles VA1v glomerulus, which is not

innervated by Mz19+ PNs. (C) Fili-overexpressing DA1 PNs partially mis-target to a

ventral region, which is normally taken by the class of VM1v. Blue shows neuropil

staining by the Ncad antibody.

75
 Moreover, overexpressing Fili in DM6 and DL4 ORNs by Am29-GAL4 also caused

ORN axon mistargeting (Figure 3-14). A straightforward interpretation for these data is

that as a transmembrane protein, Fili acts as a receptor in this scenario to steer ORN

axons away from their initial targets. However, it is also possible that a changed Fili

landscape caused by transgenic overexpression creates ectopic targets for PN dendrites.

Therefore, in response to the change in their post-synaptic partners, ORNs target

aberrantly. Future experiments will be needed to tease apart these possibilities and

identify the molecular and cellular events that govern Fili-mediated neuronal wiring.

Figure 3-14 Fili overexpression causes ORN mistargeting.

(A) Am29-GAL4>UAS-mtdT labeles DM6 (white dashed circle) and DL4 (yellow

dashed circle) glomeruli in adult antennal lobe. (B) Am29-GAL4 driven Fili

overexpression causes the labeled ORNs to mistarget to ectopic region in the ventral

part of the antennal lobe (arrow). Blue shows neuropil staining by the Ncad antibody.

76
3.4 Discussion

This study reports an LRR transmembrane protein, Fili, whose expression level can

instruct olfactory neuron wiring. Two closely related LRR proteins, Capricious and

Tartan, have been proved key molecules in the formation of the discrete olfactory map

(Hong et al., 2009). They both share over 50% homologies with Fili in terms of

extracellular regions, the majority of which are made up of LRR domains (Adachi-

Yamada et al., 2005). However, their intracellular domains bear little similarity with

that in Fili. Furthermore, the overexpression phenotype documented for Caps in Mz19+

PNs is reminiscent of the Fili overexpression effect in the same neurons, which makes

it intriguing to speculate a functional relation between Caps and Fili. Remarkably,

despite studies in motor neurons (Shishido et al., 1998) and photoreceptor neurons

(Shinza-Kameda et al., 2006) which demonstrated that Caps mediate pre- and

postsynaptic matching by homophilic interactions, a series of evidence suggested that

Caps interacts with a heterophilic ligand in the antennal lobe (Hong et al., 2009).

However, the identity of such ligand(s) remains elusive. Future examination of the

potential genetic interactions among Fili, Caps, and trn will be essential to complete our

understanding of the signaling network of cell surface molecules, particularly the

possible LRR codes that instruct the organization of the olfactory circuit.

77
3.5 Experiment Methods

Immunostaining. Tissue dissection and immunostaining were performed according to

previously described methods (Sweeney et al., 2007). Primary antibodies used in this

study include rat anti-DNcad (DN-Ex #8; 1:40; DSHB), chicken anti-GFP (1:1000;

Aves Labs), rabbit anti-DsRed (1:500; Clontech), mouse anti-rCD2 (OX-34; 1:200;

AbD Serotec), mouse nc82 (1:35; Developmental Studies Hybridoma Bank, [DSHB]),

anti-HRP conjugated with Cy5 (1:200; Jackson ImmunoResearch), and rat anti-Fili

(1:200; custom produced by Thermo Scientific Piece against a peptide epitope

containing Fili residues 684-701 DDEPEHLYERFDHYEYPD). Secondary antibodies

were raised in goat or donkey against rabbit, mouse, rat, and chicken antisera (Jackson

Immunoresearch), conjugated to Alexa 405, FITC, 568, or 647. Confocal images were

collected with a Zeiss LSM 780 and processed with Zen software.

RNAi Screening. The RNAi screen fly was generated as follows: C155-GAL4 was

recombined with UAS-dcr2 on the X chromosome and crossed to males carrying UAS-

RNAi constructs. GMR86C10-LexA, LexAop-mtdT, Or98a-mCD8GFP, and Or92a-

rCD2 located on the second chromosome were recombined to label a subset of ORNs

and PNs. The resulting knockdown flies were kept at 25 ℃ for 2 days after egg laying

and then transferred to 29 ℃ to enhance the GAL4/UAS expression system.

Transgene Generation. Gateway vector containing enhancer sequences (Pfeiffer et al.,

2008) were recombined into the pBPnlsLexAp65Uw vector (Pfeiffer et al., 2010)

through LR reaction (Invitrogen). The resulting enhancer-LexA constructs were

injected into either attP2 or attP40 landing sites by integrase-mediated transgenesis.

78
Or92a-rCD2 was made by cloning the rat CD2 coding region (Dunin-Borkowski and

Brown, 1995) downstream of Or92a promoter sequence from Drosophila genomic

DNA using following primers: Or92a5’FOR, CGA CGG AAA GCA ACG AAA GTA

AGG; Or92a3’REV, GAT TCA GCT GTT TGA TCG GCT GAC (Fishilevich and

Vosshall, 2005).

Generation of Fili Mutant. The gRNA sequence targeting a CRISPR site 5’ to the start

codon of Fili was generated by annealing oligos CTT CGT CGG ACC GTC TAA TAG

TTA and AAA CTA ACT ATT AGA CGG TCC GAC. The gRNA sequence targeting

a CRISPR site 3’ to the second ATG triplet in Fili was generated by annealing oligos

CTT CGC TCA CCT ATT TGA ACC TGG G and AAA CCC CAG GTT CAA ATA

GGT GAG C. The resulting products were ligated into BbsI-digested plasmid pU6-

BbsI-chiRNA (Gratz et al., 2013) (Addgene ID #45946) respectively. Two gRNA

constructs were co-injected into Drosophila embryos carrying transgenic vas-cas9 on

the X chromosome in a background of endogenous yellow gene (y) mutant and

FiliMI02854 insertion containing an exogenous y gene (Venken et al., 2011) on the second

chromosome. Then the females were crossed to balancer flies, and F1 offspring was

selected for loss of y, which indicates the loss of genomic sequence between the two

CRISPR sites (including the MiMIC sequence with the exogenous y gene). Successful

events were confirmed by sequencing the genomic locus.

79
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