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Generation of a sexually dimorphic neuronal population in Drosophila Garner, Sarah Rose C. 2017

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Generation of a Sexually Dimorphic Neuronal Population in Drosophila by  Sarah Rose C. Garner  B.Sc., The University of Victoria, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2017  © Sarah Garner, 2017   ii Abstract Differences in the number, morphology and function of neurons between the sexes underlie sexually dimorphic behaviours and physiology. In Drosophila, neuronal sexual dimorphism is determined by the sex determination cascade, most of which occurs downstream of Transformer (Tra). Tra is only expressed in females where it splices the sex determination effectors, fruitless (fru) and doublesex (dsx), into female-specific isoforms (non-coding fruF and coding dsxF transcripts). In males, Tra is not expressed, which leads to default splicing of fru and dsx into male-specific isoforms (coding fruM and dsxM transcripts). Sex-specific isoforms of Fru and Dsx direct most, if not all, sex differences during development of the nervous system.  The development of the male nervous system has been well-studied, whereas the mechanisms that give rise to female-specific dimorphisms have been less researched. We used a subset of insulin-like peptide 7 (Ilp7)-expressing neurons as a model for studying the development of neuronal sex differences. These neurons express fru but not dsx, and innervate the reproductive organs. Using a genetic approach, we found novel roles for tra and fru in generating a female-specific ventral subset of Ilp7 neurons (FS-Ilp7 neurons). We found FruM-dependent male-specific programmed cell death (PCD) of FS-Ilp7 neurons underlies their female-specific generation. Furthermore, we found that FruM is necessary for serotonergic differentiation and for proper axonal targeting of Ilp7 neurons in males. In females, we show that forcing male-specific splicing of fru is insufficient to trigger PCD, because, unexpectedly, Tra prevents FruM-dependent PCD in two ways to ensure FS-Ilp7 neuronal survival; not only does Tra act canonically in fru splicing, but it also acts non-canonically in parallel or downstream of fru splicing to block fruM-dependent PCD. We conclude that FruM controls both neuronal numbers via PCD and arborization in post-embryonic Ilp7 neurons, and that Tra plays a novel   iii failsafe function in females to establish and then reinforce the decision to generate female-specific neurons.    iv Preface The experiments presented in this thesis were conceived by Dr. Douglas Allan, Dr. Monica Castellanos and myself. Monica Castellanos produced preliminary data suggesting that FS-Ilp7 neurons undergo caspase-dependent PCD in males and that FruM is necessary for this process. These findings influenced our investigation and are included in the thesis. Tianshun Lian constructed the Ilp7-tdTomato transgene presented in this thesis and Monica Castellanos screened flies for P-element insertions. Aside from Dr. Castellanos preliminary work, I performed all the experiments, analysis, and generated the figures presented. Part of the work presented is being submitted as a manuscript, which was written by Douglas Allan and myself. This work was conducted at the Life Sciences Institute at the University of British Columbia, Vancouver. Some experimental findings presented in this thesis were submitted in abstract form, or oral/poster presentation at the following meetings: 2015 CanFly Meeting in Montreal (Quebec), 2016 CELL Seminar at UBC in Vancouver (British Columbia), 2016 Canadian Developmental Biology Meeting in Banff (Alberta), and 2016 UBC Graduate Program in Cell and Developmental Biology Retreat at Loon Lake (British Columbia). I was the lead author of abstracts, posters and oral presentations at these research meetings.    v Table of Contents  Abstract .......................................................................................................................................... ii	Preface ........................................................................................................................................... iv	Table of Contents ...........................................................................................................................v	List of Figures ............................................................................................................................. viii	List of Abbreviations ................................................................................................................... ix	Acknowledgements ...................................................................................................................... xi	Dedication .................................................................................................................................... xii	Chapter 1: Introduction ................................................................................................................1	1.1	 Sexual Dimorphism and Sex Determination Systems .................................................... 1	1.2	 Drosophila Sex Determination Cascade ......................................................................... 2	1.3	 Comparison Between Mammalian and Drosophila Sex Determination Systems .......... 5	1.3.1	 Sex Chromosome Influence on Cell Autonomous Dimorphisms in Mammals and Drosophila .............................................................................................................................. 6	1.3.2	 Conserved Proteins in Mammals and Drosophila .................................................. 7	1.4	 Female-biased Dimorphism in Drosophila Behaviour and Neural Circuitry ................. 8	1.4.1	 Roles for fru in Nervous System Development .................................................... 11	1.5	 Development of a Sexually Dimorphic Adult Nervous System ................................... 14	1.5.1	 Shaping the Nervous System by Programmed Cell Death (PCD) ........................ 14	1.5.2	 Sex-specific Programmed Cell Death and Proliferation Underlie Sex-biased Neuronal Populations ............................................................................................................ 17	1.6	 Ilp7-Neurons as a Model for Studying Sexually Dimorphic Neurogenesis ................. 18	  vi 1.6.1	 Thesis Overview ................................................................................................... 21	Chapter 2: Materials and Methods ............................................................................................22	2.1	 Fly Husbandry ............................................................................................................... 22	2.2	 Molecular Cloning ........................................................................................................ 23	2.3	 Tissue Processing and Immunohistochemistry ............................................................. 23	2.4	 Image and Statistical Analysis ...................................................................................... 23	Chapter 3: Results ........................................................................................................................25	3.1	 Male-specific Programmed Cell Death (PCD) of FS-Ilp7 Neurons ............................. 27	3.1.1	 Investigating the NB origins of FS-Ilp7 neurons .................................................. 29	3.1.2	 Investigating the Genetic Mechanism of Male-specific PCD of FS-Ilp7 Neurons.... ............................................................................................................................. 30	3.2	 FruMC is Necessary for Cell Death of FS-Ilp7 Neurons in Males ................................. 32	3.3	 Rescued FS-Ilp7 do not have a Typical Masculine Neuronal Identity in fruP1>p35 or fruM Null Males ......................................................................................................................... 37	3.4	 Forced Male-specific Splicing of fru in Females is Insufficient to Kill Ilp7 Neurons . 45	3.5	 Tra Blocks FruM-dependent PCD Independently of Splicing to Promote FS-Ilp7 Neuron Survival. ....................................................................................................................... 48	Chapter 4: Discussion ..................................................................................................................51	4.1	 Summary of Major Findings ......................................................................................... 51	4.2	 NB origins of FS-Ilp7 neurons ...................................................................................... 52	4.3	 FruMC Removes Female Neurons from the Male Nervous System by PCD ................. 53	4.4	 The fru4-40 deficiency may abrogate fruM expression in FS-Ilp7 neurons ..................... 55	  vii 4.5	 Role of PCD and fru in Neuronal Number, Identity, and Arborization of Male Ilp7 neurons ...................................................................................................................................... 56	4.5.1	 Neuronal Identity of Feminized or Rescued Post-embryonic Ilp7 Neurons ......... 56	4.5.2	 Role of Ilp7 Neurons in Male Fertility ................................................................. 57	4.5.3	 Role of fru in Proper Arborization of Ilp7 Neurons in Males ............................... 58	4.6	 A Novel Failsafe Mechanism for tra, but not dsx, in Building the Female Nervous System. ...................................................................................................................................... 59	4.7	 Biological Relevance .................................................................................................... 61	4.7.1	 Cellular Mechanisms that Generate Dimorphic Neuronal Number are Conserved… ......................................................................................................................... 62	4.8	 Considerations and Potential Limitations of this Study ................................................ 62	4.9	 Future Directions .......................................................................................................... 64	4.9.1	 Immediate Goals ................................................................................................... 64	4.9.2	 Long Term Goals .................................................................................................. 65	Chapter 5: Major Conclusions ...................................................................................................67	Bibliography .................................................................................................................................68	    viii List of Figures  Figure 1.1. Overview of the Drosophila Sex Determination Cascade in the Nervous System.......................................................................................................................................................... 3	Figure 1.2. A Subset of Insulin-like Peptide 7 (Ilp7) Expressing Neurons are Female-Specific. ........................................................................................................................................ 20	Figure 3.1. The Ilp7-tdTomato Reporter Faithfully Recapitulates Ilp7 Immunoreactivity in Ilp7 Neurons. ............................................................................................................................... 26	Figure 3.2. FS-Ilp7 Neurons are Eliminated by PCD in Males. ............................................. 28	Figure 3.3. dsx is not Necessary in Males or Females for Female-specific Generation of Ilp7 Neurons. ....................................................................................................................................... 33	Figure 3.4. FruMC is Necessary to Eliminate FS-Ilp7 Neurons in Males. ............................... 35	Figure 3.5. “Undead” Ilp7 Neurons Express FruMB and FruMC ............................................. 38	Figure 3.6. Abnormal Differentiation and Arborization of Post-embryonic Ilp7 Neurons in fruP1>p35 and fruM Null Males. ................................................................................................ 41	Figure 3.7. Tra Blocks PCD of FS-Ilp7 Neurons Genetically Downstream of fru Splicing. 46	Figure 4.1. Fru and Tra have Novel and Opposing Roles in Constructing Sexually Dimorphic Neuronal Number of FS-Ilp7 Neurons. ................................................................. 54	    ix List of Abbreviations oC	5-HT	 Degree	Celsius	5-hydroxytryptamine	(Serotonin) A1	 Adult	within	24	hours	after	eclosion	Abd-A	 Abdominal-A	Abd-B	 Abdominal-B	Abg	 Abdominal	Ganglion	ANOVA	 Analysis	of	Variance	APF	 After	Puparium	Formation	AVPV	 Anteroventral	Periventricular	Nucleus	BDSC	 Bloomington	Drosophila	Stock	Centre	bp	 Basepair	BTB	 road-complex,	tramtrack	and	brick-a-brac	cas	3	 Caspase	3	CNS	 Central	Nervous	System	Crz	 Corazonin	CrzR	 Corazonin	Receptor	cVA	 11-cis-Vaccenyl	Acetate	Df	 Deficiency	DMRT1	 Doublesex	and	Mab-3	related,	Transcription	Factor	1	DNA	 Deoxyribonucleic	Acid	dsx	 Doublesex	DsxF	 Female-specific	Doublesex	Transcription	Factor	DsxM	 Male-specific	Doublesex	Transcription	Factor	Ecdysone	 20-hydroxyecdysone	elav	 embryonic	lethal,	abnormal	vision	EMSA	 Electrophoretic	Mobility	Shift	Assay	Fkh	 Fork-head	fru	 fruitless	fruF	 Female-specific	fruitless	transcript;	non-coding	FruM	 Male-specific	Fruitless	Transcription	Factore	FS	 Female-specific	GFP	 Green	Fluorescent	Protein	GluR	 Glutamate	Receptor	h	 hour	Her	 Hermaphrodite	Hid	 Head	Involution	Defective	Hox	 Homeobox	  x Ilp7	 Insulin-like	peptide	7	Ix	 Intersex	Lgr3	 Leucine-rich	repeat	G	protein-coupled	receptor	3	Mind	 Male-specific	mOL	induced	motor	neurons	mOL	 Muscle	of	Lawrence	mRNA	 Messenger	RNA	n-syb	 neuronal	synaptobrevin	nEGFP	 nuclear-localized	enhanced	green	fluorescent	protein	NIG-FLY	 Fly	Stocks	of	National	Institute	of	Genetics,	Japan	NMJ	 Neuromuscular	Junction	PCD	 Programmed	Cell	Death	PFA	 Paraformaldehyde	ppk	 Pickpocket	RNA	 Ribonucleic	Acid	RNAi	 RNA	interfence	RNA-seq	 RNA	sequencing	Robo	 Roundabout	Rpr	 Reaper	SAG	 Sex-Peptide	Abdominal	Ganglion	Neurons	SEM	 Standard	Error	of	the	Mean	Skl	 Sickle	SP	 Sex-Peptide	SPR	 Sex	Peptide	Receptor	SPSN	 Sex-peptide	Sensory	Neuron	SRY	 Sex	Determining	Y	Sxl	 Sex-lethal	TDC	 Tyrosine	Decarboxylate		TDF	 Testis-Determining	Factor	TF	 Transcription	Factor	TH	 Tyrosine	Hydroxylase	tra	 transformer	Tra2	 Transformer	2	TriP	 Transgenic	RNAi	Project	UAS	 upstream	activating	sequence	VDRC	 Vienna	Drosophila	Resource	Centre	VGlut	 Vesicular	transporter	of	glutamate	VNC	 Ventral	Nerve	Cord	   xi Acknowledgements I would like to thank Dr. Douglas Allan for his supervision and guidance throughout my MSc. He has given me many opportunities to develop invaluable skills that I will cherish throughout my career and life. Most importantly, I will be taking away a new sense of confidence and self-assuredness from the support I received from Dr. Allan and colleagues. I am also grateful for funding from NSERC and UBC, without whose support this work would not have been possible.   Conversations and meetings with my committee members were integral to the progression of my research project and completion of my M.Sc. degree. I thank Dr. Mike Gordon and Dr. Elizabeth Rideout for their mentorship, and the space they provided for questions and conversations.  Members of the Allan Lab, past and present, along with several members of the Life Sciences Institute were also instrumental to the progression of my research and conferral of my degree. I thank Dr. Monica Castellanos for the foundation she provided to my project, and her mentorship. I am grateful for the support network provided by so many people. To avoid rambling, I would like to especially thank Amanda Pisio, Dr. Luba Veverytsa, Dr. Robin Vuilleumier and Andrea Globa for putting up with me over the last two years.  Finally, thanks to Sam Dodd, and my mother, Colleen Garner, for their love and support.    xii Dedication  I dedicate this thesis to my mother, Colleen Garner. This work would not have been possible without your support.   1 Chapter 1: Introduction  1.1 Sexual Dimorphism and Sex Determination Systems Sexual dimorphism refers to the differences between the female and male state within a species. These differences have implications in how we understand biology including human health and treatment, but are sometimes not fully appreciated. For example, many cancers are treated equally in males and females, but it is becoming evident that cancers behave differently between the sexes. Although occupational and behavioral differences play a role in dimorphic disease states, molecular and cellular dimorphisms are being acknowledged as culprits for differences in cancer incidence and survival between the sexes [1]. Furthermore, certain mental disorders (eg.  autism, Alzheimer’s, schizophrenia, anxiety disorders, depression, and eating disorders) exhibit sex differences in incidence, onset, and symptoms [2-4]. However, the mechanisms that lead to sex differences in mental illnesses remain very poorly understood.  Sex determination pathways establish which sex develops, and can be programmed either by environment or genetic inheritance. Environmental sex determination is initiated by a non-genetic cue, whereas genetic sex determination is initiated by the inheritance of sex chromosomes. Environmental sex determination is seen in a variety of species such as plants, nematodes, worms, amphipods, fish and amniote vertebrates, where temperature, photoperiod, or nutrient availability determines sex shortly after conception [5]. Genotypic sex determination is prevalent in invertebrates and vertebrates, where inheritance of sex chromosomes determines the sex of the species. In this case, sex chromosomes contain all the necessary cues for the correct determination pathway, and meiotic segregation ensures each gamete contributes one sex chromosome to fertilization, ensuring an approximately 1:1 ratio of males to females. Genotypic   2 sex determination pathways can take place in one of two ways; either dosage of sex chromosomes trigger a sex-specific pathway (eg. Drosophila and C. elegans), or sex chromosome-linked gene expression can be sufficient to trigger a sex-specific pathway (eg. mammals).  In this chapter, I will explain how the sex determination cascade works, and how key factors generate sexual dimorphisms in the nervous system. I will highlight cellular and genetic mechanisms that generate sexually dimorphic neural circuitry, with an emphasis on the generation of neuronal populations in only one sex. Where relevant, I will also describe how this is similar or divergent in vertebrates. This will provide context for my studies, and emphasize the utility of using Drosophila for studying dimorphic developmental neurobiology.  1.2 Drosophila Sex Determination Cascade The Drosophila sex determination cascade is comprised of a sequence of alternative splicing that regulates the sex-specific expression of effector proteins that execute the cell determination plan (Figure 1.1). The cascade is initiated by the ratio of X chromosomes to autosomes, such that a ratio of 1:1 leads to differentiation into a female fly and 0.5:1 leads to differentiation into a male fly. Expression of Sex-lethal (Sxl), a splicing repressor, is the “binary switch” for sex determination; Sxl expression is “on” in females and “off” in males. Sxl acts as a splicing repressor and begins the female-specific cascade of splicing, but what regulates how Sxl is only “on” in females? This is unresolved, but Sxl expression is thought to be controlled by transcriptional activator and repressor between genes located on the X chromosome (ie. ‘numerators’) and autosomal chromosomes (ie. ‘denominators’).    3  Figure 1.1. Overview of the Drosophila Sex Determination Cascade in the Nervous System. Left. In females, a ratio of 1:1 X chromosomes to autosomes initiates a female-specific pathway. Sxl is only expressed in females, and causes female-specific splicing of tra. Tra is then female-specifically expressed, and together with its co-factor Tra-2, causes female-specific splicing of fru and dsx; fruF, a non-coding transcript, and DsxF are expressed in females. DsxF is thought to regulate all female nervous system dimorphisms and behaviour. Right. In males, a ratio of 0.5:1 X chromosomes to autosomes initiates a male-specific pathway. Neither Sxl or Tra protein are expressed in males, and subsequently, default splicing of fru and dsx occurs; FruM isoforms and DsxM are expressed in males. FruM isoforms and DsxM are thought to regulate most, if not all, male nervous system dimorphisms and behaviour.  The next steps in the sex determination cascade are controlled by transformer (tra), and then doublesex (dsx) and fruitless (fru). These are all transcribed in both sexes, but sex-specific splicing ensures their proteins are different in each sex. Sxl physically blocks a splice site on tra   4 pre-mRNA to exclude a premature stop codon in exon 2. This ensures that Tra protein is female-specifically expressed. Tra is a splicing activator, containing an Arg/Ser-rich domain, that has only two known direct targets: dsx and fru [6-9]. Tra, along with its co-factor Transformer 2 (Tra2), recognizes specific Tra/Tra2 binding sequences on dsx and fru transcripts. Tra activates female-specific splicing of dsx, leading to DsxF protein expression, whereas activation of an alternative 5’ splice site on fru introduces a premature stop codon (no Fru protein). In contrast, males do not express the splicing factors Sxl or Tra, and therefore default splicing produces coding dsxM and fruM transcripts. In summary, females express the splicing factors Sxl and Tra leading to DsxF protein expression, whereas males do not express these splicing factors and therefore express DsxM and FruM. Dsx is responsible for differentiating most of the somatic tissue sex-specifically. Dsx is expressed in a variety of tissues at embryonic stage 16, but is mostly present in neurons [10, 11]. DsxM and DsxF are transcription factors that differ between the sexes in their 3’ carboxy-termini, and display unique transcriptional regulation [11-16].   The sex-specific Dsx isoforms perform double duty, by repressing differentiation of the opposite sex’s developmental program while also activating its own sex’s sex-specific differentiation. Genetic manipulations of Dsx make this statement clearer. If DsxF were expressed in dsx-expressing cells of a XY male, a phenotypically female fly emerges, and vice versa if DsxM were expressed in a XX female. Furthermore, in dsx mutant males and females an intersex fly develops, one with both male and female characteristics. How Dsx both activates and represses differentiation is well established in the genital disc. There are two distinct genital primordia in Drosophila, which is different than the bi-potential primordia in mammals. In females, one primordium differentiates into most of the female reproductive organs, and the other differentiates into most of male reproductive organs.   5 In females, proliferation and differentiation is activated by DsxF, and the other primordium’s proliferation is repressed by DsxF. In males, DsxM activates proliferation and differentiation of the other primordium, whereas the female’s major primordium is repressed by DsxM. The repressed primordium in each sex develop into subsidiary structures of each sex’s reproductive organs (parovaria and uterine wall in females and eighth tergite in males). In the absence of dsx expression, each primordium enlarges and partially differentiates into male and female reproductive organs, demonstrating that both DsxF and DsxM are needed to activate and repress differentiation of the other sex’s primordium [17-19]. Dsx has a prominent role in nervous system construction too, but this well be expanded upon in section 1.5. Since Fru has a predominant role in the development of nervous system sexual dimorphism, it will be discussed in Section 1.4.1.  1.3 Comparison Between Mammalian and Drosophila Sex Determination Systems Mammalian sex determination is most similar to Drosophila in their differentiation of the gonads, a hormone-independent process. Both Drosophila and mammals use genetic sex determination, but after the gonads differentiate in mammals, sex-specific hormones instruct the sexual differentiation of most somatic cells. Mammals have a bi-potential gonadal primordium, a tissue that can either be differentiated into an ovary or testis. In males, the sex determining region Y (SRY) gene, located on the Y chromosome, encodes the testis-determining factor (TDF), which initiates the development of male testis. In the absence of TDF, the primordium differentiates into an ovary, suggesting that a default program is responsible for ovary differentiation. However, X-linked genes may be necessary to actively repress male differentiation of the gonads in mammals. For example, a small duplication on the X   6 chromosome is responsible for developing ovaries despite being genetically male (XY and functional SRY gene). This is known as “dosage-sensitive sex reversal.” In this case, male-to-female sex reversal was due to a duplication of the gene DAX-1 (the “antitestis” gene); DAX-1 is thought to antagonize SRY action [20]. Although testis differentiation is dependent on SRY, there is evidence that suggests the mammalian sex determination system may be more complex than simple due to SRY presence or absence.   1.3.1 Sex Chromosome Influence on Cell Autonomous Dimorphisms in Mammals and Drosophila In Drosophila, each individual cell’s sexual identity is determined cell-autonomously by the sex determination cascade. Consider an error in segregation in the X chromosomes in a progenitor cell. The resulting lineage of cells will be entirely male in an otherwise female fly. This is called gynandromorphia, where a fly contains clones of cells that are specified by the opposite sex’s determination cascade [21]. This same error in segregation would not have such a profound effect in mammals, because of the dominant effect of hormones in sexual differentiation.  In mammals, sex differences can also be caused by the genetic and epigenetic differences between XX and XY cells in the brain. There are regions of the X and Y chromosomes that do not recombine with each other, leading to Y-linked genes and X-linked genes. Interestingly, there is an unusual concentration of genes required for brain function on primate X chromosomes. Since XX cells inherit a paternal X and a maternal X, they are subject to differential maternal and paternal imprinting, and random X chromosome inactivation in most cells. This leads to a mosaic of cells in females where differential imprinting on each X   7 chromosome between females is seen and differential X chromosome inactivation within a female. In contrast, XY cells only inherit a maternal X chromosome and therefore are not mosaics. For example, XX and XY mesencephalic cells harvested from the brains of mouse embryos behave differently in vitro in the absence of hormonal influence, where XY cultures develop more dopamine neurons than XX cultures. Whether these differences are due to presence or absence of Y-linked genes, dosage of X-linked genes, mosaicism or parental imprinting remains to be seen (reviewed in [22]).   1.3.2 Conserved Proteins in Mammals and Drosophila Although mammals and Drosophila differ in their sex determination pathways, there are some conserved effectors in both species. Both systems utilize a different ‘master regulator’ of sex determination: SRY initiates the male program in mammals, and Sxl initiates the female program in Drosophila. However, two genes that are integral components of sex determination in Drosophila are conserved in mammals; the Drosophila genes dsx and tra2 have orthologs in mammals. As discussed in Section 1.2, the dsx-dependent pathway can alone direct most somatic cell sexual fate decisions. The mammalian ortholog of dsx is Doublesex and mab-3 related, transcription factor 1 (DMRT1). DMRT1 is an autosomal gene that is up-regulated in the developing genital ridge of XY embryos and its expression persists in Sertoli cells (a ‘nurse cell’ of the testicles that assists spermatogenesis). It has been proposed that this gene initiates an independent regulatory network for maintenance of the testis throughout adulthood. Loss of DMRT1 in mouse Sertoli cells leads to impaired testis development and feminization of germ cells. In addition, humans with mutations in or loss of DMRT1 have varying disorders of sexual development (reviewed in [23]). Orthologs of the Drosophila tra2 gene are also present in   8 humans. Tra2 acts as a splicing co-factor in Drosophila that is necessary for sex-specific splicing of the sex determining effectors, fru and dsx [24-26]. There are two Tra2 paralogs in humans, which both are sequence-specific activators of pre-mRNA splicing, but a role in sex determination has not been shown [27]. Although there are important similarities between Drosophila and mammals, cell autonomous sex determination and absence of sex hormone influence in Drosophila can be advantageous for studying the cellular and genetic mechanisms that determine sex.  1.4 Female-biased Dimorphism in Drosophila Behaviour and Neural Circuitry Males and females of most species exhibit distinct behavioral repertoires, many of which are a consequence of dimorphisms within the nervous system [28, 29]. However, showing that sex-specific construction of the nervous system is causal to behaviour is challenging. For example, many mammals do not have absolutely discrete and reproducible sex-specific behaviours. In contrast, Drosophila males exhibit highly stereotyped behaviours governing courtship and aggression. For this reason, and due to the genetic amenability of Drosophila, Drosophila males have become one of the leading models for understanding how dimorphic circuits generate sex-specific behaviours. These two factors allow unambiguous conclusions to be made about how dimorphisms within the nervous system gives rise to dimorphic behaviour. These studies have further identified the gene regulatory networks, developmental processes and circuits that underlie many male-specific behaviours. In stark contrast, Drosophila female-specific neural circuitry and behaviour have been largely unexplored, until recently. The reasons for this lie in the fact that female behaviours were less well defined, and researchers in the field had long believed that the female Drosophila   9 nervous system was a ‘default’ or ‘ground’ state, upon which the male nervous system is built. However, it is important to resolve how female circuits and behaviour are generated, because they represent the second half of the sexual dimorphism story in Drosophila. As my thesis focuses on how female-specific neuronal populations are generated, I will bias my introduction largely to progress made in understanding female-biased neural circuitry and behaviour.  Although less overt than male courtship behaviour, female receptivity to courtship and changes in physiological state post-courtship are perhaps the best understood female behaviours. Courtship begins with a male chasing a female, and once a female’s attention is held, a male generates wing song by extending and vibrating his wings one at a time. Courtship continues with a male vibrating his abdomen, tapping the female’s abdomen, and finally, male mounting of the female to initiate copulation. In addition to these steps, males detect pheromones to ensure conspecific and opposite sex attraction [30, 31]. Female pre-copulation receptivity to male courtship is also stereotyped; the female will stop fleeing, slow her motion, and allow the male to mount. After copulation, the female will retain the male’s seminal fluid, and peptides within the seminal fluid triggers stereotyped post-mating behaviours (see below). Post-mating behaviour lasts for approximately one week, and is characterized by a female’s rejection of secondary courtship and a female’s egg-laying behaviour. A female who has already mated will persistently flee from, flick her wings at, and repeatedly attempt to kick a male during his mating attempts. In addition, a female will extrude her ovipositor to avoid copulation, and prematurely eject seminal fluids if a male succeeds in copulation [31, 32]. Post-mating, a female will engage an ovipositor motor program for egg-laying, the egg-laying rate will increase, and a female will have a change in egg-laying site selection [33]. Since female courtship acceptance and rejection behaviours are more subtle than males, they are still an active area of characterization. Recently, two new pre-  10 mating behaviour were quantitatively characterized: the frequency of female pausing was correlated to male wing song; and vaginal plate opening was correlated to copulation attempts [34]. Although less overt than male courtship behaviours, female-specific behaviours can be used to study the underlying female-specific nervous system construction. Female courtship behaviours result from an interplay between internal physiological state and female neural circuitry. Seminal fluid transfer to the female, and specifically sex-peptide (SP) transfer, is responsible for the switch from pre- to post-mating behaviour in females (reviewed in [35]). SP storage in females is detected by sex-peptide receptors (SPRs), which are located on SP sensory neurons (SPSNs). The SP neural circuit controls the female receptivity switch. Located on the uterus and oviduct, the afferent SPSNs project to the abdominal ganglion (Abg), where they are presynaptic to SP abdominal ganglion (SAG) neurons that ascend to the brain [36]. SPSNs are silenced by SP, which in turn suppresses the activity of SAG neurons. SPSN and SAG neuron silencing leads to post-mating behaviour and physiological changes in a female [36, 37]. Efferent female-biased octopaminergic neurons in the Abg, innervating the female reproductive tract, also regulate the pre- to post-mating response, potentially by interacting with SPSNs [38]. Changes in female physiology post-mating include increased egg production, and tissue remodeling in the oviduct [39], and these changes are interpreted by female neural circuits. For example, increased egg-laying is detected by mechanosensory neurons on the oviduct, which in turn increase acetic acid attraction for egg-laying site preference [33, 40]. The female-biased SP neural circuit is one of the most understood connections between circuitry, behaviour and physiological changes in females.  Identifying female-specific and female-biased neuronal populations is critical for understanding female neural circuitry. Many sexually dimorphic neuronal circuits contain   11 neurons that express dsx and/or fru, important determinants of sexual dimorphism in the brain (see section 1.4.1 and 1.5.1). For example, some SPSNs express dsx or fru, and their second-order interneurons (SAG neurons) express dsx but not fru [36, 38]. Two other female-specific dsx-expressing neuronal populations were recently identified, which differentially respond to male pheromones and wing song. Zhou and colleagues (2014) found two small clusters of neurons, the pCd and pC1 neurons, that are necessary and sufficient for inducing receptivity [41]. Both clusters respond to male-specific cVA pheromones, but only the pC1 cluster responds to wing song. The previously mentioned pausing behaviour in females, which the authors suspect is females ‘listening’ to male wing song, is activated by another female-specific neuronal population. Abdominal-B (Abd-B)-expressing neurons reside in the Abg and are required for female-specific pausing, but not vaginal plate opening [34]. Surprisingly, the Abd-B-expressing neurons do not express dsx or fru. The characterization of female-specific mating behaviours has allowed these discoveries of female-biased neuronal populations, which is an essential first step to analysis of construction of a female nervous system. 1.4.1 Roles for fru in Nervous System Development fru is almost exclusively expressed in the nervous system, and has been shown to be necessary and sufficient for many male-specific behaviours. The fru locus is complex in that it has at least four active promoters, each producing diverse transcripts that are generated by alternative and sex-specific splicing [42-45]. Three of the four promoters express transcripts that are expressed in both sexes. This set of fru common transcripts, or fruCOM, are necessary for early embryonic development, but are not expressed in the adult and do not appear to be necessary for sexual dimorphism [46]. In contrast, a transcript generated from the fru P1 promoter is transcribed in both sexes (in the same cells in both sexes) during the third instar larval stage, but   12 it is sex-specifically spliced into fruF and fruM isoforms in females and males, respectively [42, 47].  Stockinger and colleagues generated a reporter, fruP1-GAL4, which reports on fru P1 promoter activity in both sexes [48]. Importantly, this genetic tool drives GAL4 into all cells that express fru P1 transcripts, making it a powerful reagent for imaging and regulating gene expression in fruP1-expressing neuronal of both sexes. A number of survey studies have used fru P1 GAL4 lines to drive UAS-GFP to visualize and catalogue all fruP1-expressing neurons. These studies have shown that fruP1 is expressed in ~2% of the nervous system and demonstrates a clear male bias in the number of neurons.  Additional alternative splicing of the fruM transcript generates different transcription factor isoforms whose differential functions have recently become elucidated. Alternative splicing at the 3’ end of fruM transcripts generates four different transcripts with different C-terminal C2H2 zinc-finger domains (fruMA, fruMB, fruMC and fruMD). All isoforms except fruMD are transcribed in the nervous system. Most fruP1-expressing neurons express fruMA, fruMB, and fruMC, but some express just one or two [49]. By engineering the fru locus to manipulate endogenous fru splicing, Demir and Dickson (2005), demonstrated that FruM is necessary in males for male-specific courtship behaviours, and nearly sufficient for all these behaviours in females. The question then arises, if FruM is responsible for male-specific behaviours, how is this achieved? Importantly, the different FruM isoforms appear to have differential contributions to mating success, courtship song, and neural circuitry. All FruM isoforms were necessary to maintain normal neuronal numbers in a context dependent manner (with no reported mechanism), whereas FruMC appeared to be more responsible for male-specific arborization of   13 neurons [49]. To study genomic occupancy of the different isoforms, to investigate their cellular function, the Goodwin group used the DamID method to map the binding of each FruM isoform to DNA or chromatin throughout the genome by fusing each isoform to a DNA adenine methyltransferase [50]. Other groups have also tried to determine the consensus binding sequence of each isoform, but results so far have proven inconclusive [49-52]. Overall, the FruM isoforms seem to have overlapping expression patterns, but studies of genomic occupancy and loss-of-function analysis strongly suggest they have different context-dependent functions. A few studies have demonstrated that FruM and its isoforms regulate transcription. FruMC binds a 42 bp regulatory sequence upstream of robo1 to control neurite outgrowth of mAL neurons in males by repressing robo1 transcription. FruMB also binds to a short intron of leucine-rich repeat G protein-coupled receptor 3 (Lgr3) to inhibit its expression. Lgr3 is the receptor to an insulin-like protein, Dilp8, that is expressed in the fat body and regulates organ growth in Drosophila [53]. From polytene analysis and biochemical assays, FruM has also been shown to recruit two antagonistic chromatin remodeling factors (the histone deacetylase complex 1 and Su(var)205) at the same polytene loci. The authors demonstrated that these complexes fine-tune the sex-specific arborization of mAL neurons, where HDAC1 promotes masculinization (neurite outgrowth and male-biased neuron number) and HP1 promotes feminization [54]. It is incredible that FruM can control three distinct dimorphisms within the mAL neuronal population through different means: FruM (unknown which isoform) prevents PCD of mAL neurons in males (discussed in Section 1.5.2), accounting for 30 neurons in males versus five in females; FruMC activates neurite outgrowth of mAL neurons in males by repressing robo1 transcription; FruM (unknown isoform) promotes chromatin remodeling through HDAC1 in mAL neurons to   14 masculinize bifurcation of terminals.  1.5 Development of a Sexually Dimorphic Adult Nervous System To establish sex-specific neural circuitry within the brain, the nervous system develops differences in neuronal number, morphology, function, and connectivity between the sexes. Two main stages of neurogenesis take place: first, during embryonic development; and second, during post-embryonic development during the late larval to early pupariation periods. 90% of the adult neurons are produced during post-embryonic neurogenesis, whereas embryonic neurogenesis primarily makes up the larval brain. As such, intense remodeling must take place throughout metamorphosis to generate the adult nervous system from the larval nervous system. During this time, flies must form a nervous system that is capable of an entire suite of sensory input, processing and motor output for behaviours that are not seen in larvae, including courtship, leg and wing locomotion etc (reviewed in [55]). In the following sub-sections, I will explain how the nervous system is remodeled by programmed cell death (PCD) and mechanisms that give rise to sex-specific neuronal populations.  1.5.1 Shaping the Nervous System by Programmed Cell Death (PCD) PCD is a programmed fate that assists in the development of a mature nervous system in both larva and adult flies (reviewed in [55]). Remodeling from the larval to adult nervous system by PCD is initiated by steroid hormone 20-hydroxyecdysone (ecdysone), which is highly expressed at the end of the 3rd larval stage. Many cases of either a surge of ecdysone or lack of ecdysone signaling results in PCD of specific subsets of neurons (reviewed in [56]). Ecdysone signaling is used to coordinate several phases of Drosophila metamorphosis, and this signal is   15 interpreted in a cell subtype-specific manner [55]. Absence of a target-derived signal can also lead to PCD. This is seen in neurons that lose their tissue-derived signals during the metamorphosis from larva to adult. Pre-existing neurons from the larval stage may undergo PCD, remodel their axonal and dendritic arbors, or transdifferentiate their gene expression profiles. NBs or newly generated subtypes of neuronal lineages from post-embryonic neurogenesis will also have a pre-determined fate; NB or subtypes of their progeny may either undergo PCD or incorporate into the adult nervous system [55].  These internal or external cues activate a PCD signaling pathway that de-represses caspase activation. The inhibitors of apoptosis proteins (IAPs) directly maintain caspases in an inactive state. Three IAPs have been identified in Drosophila (DIAP1, DIAP2, and Deterin), which bind to caspases and inhibit their activity. The IAPs are themselves repressed by so-called pro-apoptotic proteins Reaper (Rpr), Head involution defective (Hid), Grim and Sickle (Skl). Rpr and Grim have also been shown to suppress DIAP1 translation. Thus, expression of these pro-apoptotic genes represses IAPs, which de-represses caspase activity to execute the cell death program.   The four pro-apoptotic genes are clustered together in the cell death gene locus, containing Rpr, hid, grim and skl, commonly referred to as the RHG locus. Deletion of most of the RHG locus (Df(3L)H99), not including skl, demonstrates the necessity for these genes in PCD, because it blocks all developmental apoptosis. Further, overexpression of each gene is sufficient to induce caspase-dependent apoptosis in both insects and mammals. Rpr, hid or grim have been reported to initiate PCD alone in neurons, but more often they act in a combinatorial, synergistic manner. RHG proteins share similar downstream pathways, but they are not functionally equivalent and do not share overlapping activation pathways. For example, evidence   16 suggests hid expression and activity are negatively regulated by the Ras/MAPK pathway, and rpr expression is regulated by the p53/DNA damage pathway and the EcR-mediated signaling pathway. RHG genes are differentially regulated to respond to different developmental cues (reviewed in [57]).  PCD also removes NBs throughout development. Embryonic neurogenesis ends shortly before larval hatching, where NBs stop dividing and enter a quiescent stage or undergo PCD. NB PCD is initiated by a cis-regulatory element (enh-1, within the NB response region; NBRR) located within the RHG locus. This region restricts expression of RHG genes to abdominal NBs, and is activated by an Abdominal-A pulse at the end of embryogenesis. Embryonic NB’s have also been shown to undergo PCD that is dependent on segment-specific Hox gene expression or a unique TF code. These mechanisms result in only 3 out of 30 embryonic NB’s in A3-A7 abdominal hemisegments persisting in each hemisegment after larval hatching (NB3-5, NB5-2 and NB5-3). Therefore, 27 NB’s in each hemisegment undergo PCD or exit the cell cycle prior to or at the start of post-embryonic neurogenesis. To limit proliferation, Hox genes and unique TF codes can lead to post-embryonic NB death or Prospero-dependent mechanisms can lead to cell-cycle exit (reviewed in [57]). The three persisting NBs in each A3-A7 hemisegment exit quiescence and begin proliferating to create most of the adult abdominal VNC.   Often-used to terminate proliferation of neural progenitor cells or to eliminate neurons, PCD is an evolutionarily conserved mechanism observed in flies, worms and mammals [29, 58]. The core cell death machinery is evolutionarily conserved, where orthologous effectors at each level of the cascade is seen in each species. However, mammals are more dependent on cytochrome-c dependent PCD, whereas flies and worms more heavily rely on RHG gene expression [58]. In Drosophila PCD repression is maintained by IAPs, whereas mammals more   17 highly rely on Bcl2 proteins to repress PCD activation. Although IAPs are not the main players in mammalian PCD, eight IAPs have been identified. IAPs in mammals have prominent roles in regulating PCD in the nervous system and also in axon and dendrite pruning (reviewed in [57]).   1.5.2 Sex-specific Programmed Cell Death and Proliferation Underlie Sex-biased Neuronal Populations Supernumerary neurons in males can be accounted for mostly by female-specific PCD and male-specific proliferation. Sanders and Arbeitman (2008) demonstrated the first example of female-specific PCD, and found DsxF expression was both necessary for PCD in females (TN1 cluster in the VNC). Since then, Dsx has been implicated in regulating PCD of several other neuronal populations. P1 neurons, a population that is part of the male courtship initiation circuit, undergo PCD only in females. In this case, PCD was shown to be driven by DsxF, whereas DsxM had no role in their male-specific generation [59]. Birkholz and colleagues (2013) also demonstrated a similar phenomenon, where caudal NBs undergo DsxF-dependent PCD in females, and that DsxM blocks PCD in the male counterparts [10]. Furthermore, expression of the p35 caspase blocker in dsx-expressing cells (in the brain and VNC) rescued many male-biased neurons in females [60]. Another mechanism for female-specific PCD was demonstrated by Kimura and colleagues (2005), who found supernumerary mAL neurons survive in males, and that FruM was necessary for survival. The female counterparts of mAL neurons are eliminated by rpr-dependent PCD, but FruM acts in males to block this PCD [61]. Lastly, the muscle of Lawrence (mOL) is a muscle necessary for male abdomen bending, and is formed by inductive anterograde signals from the male-specific mOL induced (Mind) motor neurons. These Mind neurons express fruP1 and undergo PCD in females, and their male counterparts are likely   18 protected from PCD in males because of FruM [62]. Overall, Dsx seems to have a greater role than Fru in female-specific neuronal PCD. In mammals, sex-specific neuronal proliferation is observed in both sexes [63], but in Drosophila, this has only been reported to occur in males. Taylor and Truman (1992) were the first to demonstrate male-biased proliferation in the Abg in Drosophila [64]. They determined that dimorphic neuronal number was due to DsxM expression in males, leading to male-biased BrdU incorporation. However, Birkholz and colleagues (2013) proposed that this sexual dimorphism in the caudal Dsx-expressing neurons was due to female-specific PCD, not male-biased proliferation [10]. Sanders and Arbeitman (2008) also demonstrated that DsxM-dependent proliferation of NBs in males underlies the male-biased generation of pC1 and pC2, and that DsxF antagonizes this in the female counterparts [65]. In summary, Dsx has been implicated in both female-specific PCD and male-specific proliferation, but Fru has only been linked to blocking PCD. Because of this, Dsx has been touted as the more important sex determination factor in the sexually dimorphic development neuronal number in the Drosophila nervous system.  1.6 Ilp7-Neurons as a Model for Studying Sexually Dimorphic Neurogenesis Insulin-like peptide 7 (Ilp7) expressing neurons are spatially organized in two neuronal clusters and arise from two stages of neurogenesis. Four bilateral pairs of Ilp7 neurons arise from embryonic neurogenesis. Castellanos et al (2013) discovered that Ilp7 neurons also consisted of post-embryonically derived neurons. These neurons were found to innervate the reproductive organs in Drosophila and demonstrate significant sexual dimorphisms in neuronal number, function and target tissues.    19  The dMP2 NB generates one Ilp7 neuron in each A6-A9 hemisegment during embryonic neurogenesis, to generate a total of eight embryonic Ilp7 neurons that persist throughout larval and adult stages [66]. Embryonic Ilp7 neurons are ventrally located in the Abg, where they project to the hindgut in larval and adult stages. They have no observable difference in expression patterns or functions between the sexes, and therefore are not sexually dimorphic. Ilp7 neurons are also generated post-embryonically, and exhibit sexual dimorphisms in neuronal number and function. Post-embryonic Ilp7 neurons are organized into discrete ventral and dorsal clusters in the Abg of females. Dorsal Ilp7 neurons and female-specific Ilp7 (FS-Ilp7) neurons (ventrally located) provide motor input to the oviduct (Fig. 1.2). The oviduct is composed of striated muscle, which receives motor neuron input by post-embryonic Ilp7 motor neurons for rhythmic contractions to push the egg to the uterus. Therefore, post-embryonic Ilp7 neurons are critical for female fertility. Post-embryonic Ilp7 neurons are only present in a dorsal cluster of the Abg in males. These dorsal Ilp7 neurons provide serotonergic input to the male seminal vesicles of the reproductive tract. They are not thought to have a role in male fertility. In my thesis, I use the post-embryonic Ilp7 neurons to study the cellular and genetic mechanisms that give rise to the female-specific neuronal subset, and the role of fru in differentiation and projection of male dorsal Ilp7 neurons.   20  Figure 1.2. A Subset of Insulin-like Peptide 7 (Ilp7) Expressing Neurons are Female-Specific. (A) Cartoon of the adult Drosophila central nervous system (CNS), showing the abdominal ganglion (Abg) highlighted (red box), and the female reproductive tract. The female-specific Ilp7 motoneurons (FS-Ilp7 neurons) reside in the Abg and provide motor input to the oviduct (green) (B-C) FS-Ilp7 neurons are present in females. FS-Ilp7 neurons can be selectively identified in females by the coincidence of Ilp7 immunoreactivity (α-Ilp7; red) and fruP1-GAL4, UAS-GFP (fruP1>GFP; green) reporter activity (arrowheads in B). Embryonic Ilp7 neurons are marked by co-expression of α-Ilp7 and Fork head immunoreactivity (α-Fkh; blue). (C) Cartoon summary   21 and marker profile of embryonic (blue) and post-embryonic Ilp7 neurons (green, FS-Ilp7 neurons; yellow, dorsal Ilp7-neurons) in males and females. V=ventral, D=dorsal, A=anterior, P=posterior 1.6.1 Thesis Overview In this thesis, we performed cellular and genetic analyses to determine the specific roles for tra and fru in sexual dimorphisms of post-embryonic Ilp7 neurons. We find that generation of FS-Ilp7 neurons is underscored by male-specific FruMC-dependent PCD. We find that additional dorsal Ilp7 neurons are rescued in males when PCD is blocked or in fruM mutants. Furthermore, we discovered that rescued Ilp7 neurons in males (FS-Ilp7 neurons and dorsal Ilp7 neurons) are not serotonergic, despite expressing FruM isoforms, and that fru feminized males have aberrant neuronal targeting of Ilp7 projections. However, our genetic analyses demonstrated that the mere presence/absence of FruM does not alone account for differences in FS-Ilp7 numbers between males and females. Unexpectedly, we found Tra is able to block PCD triggered by FruM protein to ensure female-specific survival of FS-Ilp7 neurons. Overall, our results provide insight into how differences in genetic wiring between males and females are utilized to achieve the emergence of a female-specific subset of neurons. Furthermore, we provide evidence that Tra can play a failsafe role, downstream of its canonical fru splicing role, to reinforce the commitment to female-specific nervous system construction.    22 Chapter 2: Materials and Methods 2.1 Fly Husbandry Flies were maintained on standard cornmeal food at 70% humidity at 18°C, or 25°C. Ilp7-GAL4 [67]. Strains from the Bloomington Drosophila Stock Center were: P{10xUAS-IVS-Syn21-GFP-p10} (referred to as UAS-GFP) [68]; UASnlsmycEGFP (referred to as UASnGFP); P{UAS-CD4-tdGFP}8M2 [69]; P{UAS-p35.H}BH1 [70]; P{GawB}elavGal-C155 (referred to as elav> and elavGAL4) [71]; UAS-Dicer2 [72]; P{TRiP.HMC03419}attP40 (referred to as rpr RNAi), P{TRiP.JF03093}attP2 (referred to as skl RNAi) [73]; dsx1 (amorphic allele) [9]; Dp(1;Y)BS;cn tra2Bbw1 (amorphic tra2 allele); Df(2L)trix (tra2 deletion)[8]; and w1118 (referred to as ‘+’). Flies obtained from VDRC: P{GD11287}v21830 and P{GD11287}v22597 (referred to as grim RNAis) [74]. Two hid RNAi lines were obtained from NIG-Fly: UAS-5123R-2 and UAS-5123R-3.  Several alleles were obtained as generous gifts. nsyb-GAL4 [75]. Df(3R)dsx15 (dsx deletion) [76]. The tra alleles used were: UAS-traF [77]; UAS-tradsRNAi [78], traKO (amorphic allele) [79]. Putative or reported severe hypomorphs or nulls of fruP1 transcripts include: fruP1-GAL4 [48], P{PZ}fru3, Df(3R)fruSat15 [43], Df(3R)fru4-40 [42]. The engineered fru alleles that constitutively splice into female- or male-specific isoforms include fruF, fruΔtra and fruM [80]. The following are fruM isoform-specific nonsense mutants: fruΔA and fruΔB [81]; and fruΔC [82]. The following are Myc-tagged FruM isoform-specific alleles: fruAmyc, fruBmyc, and fruCmyc [49].  Balancer chromosomes for stock maintenance and construction of genotypes used were: Cyo. Cyo-Actin>GFP. TM3,Sb. TM3,Ser,Actin>GFP. TM6. TM6B. TM2. SM6-TM6B.   23 2.2 Molecular Cloning To generate the Ilp7-tdTomato reporter, we PCR amplified -2964 to +424 (Ilp7 start codon) relative to the transcriptional start site of the Ilp7 gene. We overlapped the Ilp7 translational start site with tdTomato ORF [83]. This construct was inserted into the psD7-001 vector. Fly transformation by P-element insertion was performed by Best Gene Inc. P element insertions on the second chromosome were recovered and established as stable fly strains.  2.3 Tissue Processing and Immunohistochemistry Verification of correct genotypes in adults was determined by loss of balancer chromosomes and/or by evidence of re-sexualization in appropriate genotypes (eg. chaining behaviour and/or changes in abdominal pigmentation and genitalia). Male and female adult VNCs were dissected within 24 h of eclosion, unless stated otherwise. Standard protocols for immunohistochemistry were used [84]. Primary antibodies used were rabbit anti-Ilp7 (1:1000; E. Hafen, ETH, Zurich, Switzerland); guinea pig anti-Fork head (1:1000; H. Jäckle, Max Planck Institute, Göttingen, Germany); rabbit anti-5-HT (1:1000; Sigma, S5545); rat anti-Myc (1:1000; Abcam JAC6). Secondary antibodies used were: anti-rabbit, anti-guinea pig and anti-rat IgG (H+L) conjugated to DyLight 488, Cy3 or Cy5 (1:400, Jackson ImmunoResearch).   2.4 Image and Statistical Analysis All images were acquired with an Olympus FV1000 confocal microscope. FS-Ilp7 neurons were manually counted in Fluoview Software (FV10-ASW). All representative images in figures were processed using either ImageJ or Adobe Photoshop CS6 (identically for all images being compared), and figures were made in Adobe Illustrator and Photoshop CS6. All images shown   24 are representative z-projections of a given tissue. For images collected from fru>GFP genotypes, we lowered the brightness of the green channel for the readers to easily observe the other channels. Since Ilp7 expression is higher in females than males, we increased the brightness of this channel to better observe these neurons in males. Where small embryonic Ilp7 neurons are indicated (Fig 2.1), the brightness of Ilp7 immunoreactivity was saturated to reveal these weakly expressing Ilp7 neurons in both sexes. Where appropriate, images were false-coloured for clarity, and colours were chosen for colour-blind readers. All statistical analysis and graphing were performed using Prism 7 software (GraphPad Software, San Diego, CA). A minimum n=8 flies was used for each genotype studied, unless indicated otherwise. All data underwent D’Agostino and Pearson normality testing, and data within graphs were compared by one-way ANOVA followed by Tukey post hoc analysis. For comparisons between two genotypes, unpaired parametric T-tests were performed (Student T-Test). Statistical differences are indicated if p<0.05.   25 Chapter 3: Results FS-Ilp7 neurons are generated during post-embryonic neurogenesis, adjacent to a cluster of embryonic-born Ilp7-expressing neurons [67]. To discriminate these neuronal subsets, we took advantage of co-labeling of Ilp7 with anti-Fork head immunoreactivity in embryonic Ilp7-neurons, or with fruP1-GAL4 >10XUAS-GFP in post-embryonic Ilp7-neurons (Fig. 3.1 A-B). In both sexes, fruP1-GAL4 is expressed in cells that express the sex-specific fru transcripts generated from the P1 promoter [48]. In addition, I verified the efficacy of an Ilp7 reporter, in which a genomic region spanning enhancer and promoter elements up to the translation start site is fused to a nuclear localized tdTomato reporter (Fig. 3.1 C-D). We examined Ilp7-tdTomato reporter activity in relation to Ilp7 immunoreactivity, and fruP1>GFP. Ilp7-tdTomato was expressed in fruP1>GFP-positive FS-Ilp7 neurons in females (Fig. 3.1 C) and fruP1>GFP-negative embryonic Ilp7 neurons in both sexes (Fig. 3.1 C-D). Ilp7-tdTomato also labeled all dorsal Ilp7 neurons in both sexes (data not shown). The use of the Ilp7-tdTomato also revealed the presence of faintly immunoreactive Ilp7 neurons in males situated posterior to the embryonic Ilp7 neurons; these were co-labeled with α-Ilp7 (Fig. 3.1 B), Ilp7-tdTomato (Fig. 3.1 D) and fruP1>GFP. From these data, I concluded that the Ilp7-tdTomato reporter labels all Ilp7 neurons, and allows clearer identification of Ilp7 neurons than cytoplasmic Ilp7 immunoreactivity, due to the reporter’s nuclear localization.    26  Figure 3.1. The Ilp7-tdTomato Reporter Faithfully Recapitulates Ilp7 Immunoreactivity in Ilp7 Neurons.  (A-D) FS-Ilp7 neurons can be distinguished from embryonic Ilp7 neurons because of their co-expression of Ilp7 and fruP1. (A-B) FS-Ilp7 neurons (arrows) were observed in A1 adult female (A), but not male (B) VNCs, by their co-expression of Ilp7 (α-Ilp7, red) with fruP1>GFP   27 (green), and absence of Fkh immunoreactivity (α-Fkh, blue); FS-Ilp7 neurons are Ilp7+/Fkh-/fruP1+. Embryonic Ilp7 neurons were observed in both sexes, and were distinguishable by their expression of Ilp7 (α-Ilp7; red), Fkh (α-Fkh; blue), but not fruP1>GFP (green); embryonic Ilp7 neurons are Ilp7+/Fkh+/fruP1-. (B) Large and small embryonic Ilp7 neurons were identifiable in males (Ilp7+/Fkh+/fruP1-ve). FS-Ilp7 neurons were absent in males. Some faintly immunoreactive Ilp7 neurons (α-Ilp7, red) co-expressed fruP1>GFP (green), but not Fkh (α-Fkh, blue). These were posterior to the large embryonic Ilp7 neurons (asterisk). (C-D) Ilp7-tdTomato reporter expression was consistent with Ilp7 immunoreactivity in females (A) and males (B). (C) In females, the Ilp7 reporter is expressed in FS-Ilp7 neurons (arrows), and embryonic Ilp7 neurons. (D) As observed in B, males had embryonic Ilp7 neurons, did not have FS-Ilp7 neurons (arrows), and had some posterior Ilp7 neurons that co-express fruP1>nGFP (asterisk).  3.1 Male-specific Programmed Cell Death (PCD) of FS-Ilp7 Neurons We tested whether male-specific PCD is the primary mechanism responsible for female-specific generation of FS-Ilp7 neurons. Using fruP1-GAL4 (fruP1>) to drive expression of the anti-apoptotic caspase inhibitor, baculovirus p35 (UAS-p35) [70], we could block PCD in fru-expressing cells, if indeed it occurs normally. In controls, females have three to four FS-Ilp7 neurons per fly, whereas males have none. In fruP1>p35 males, we observed a significant increase in the number of FS-Ilp7 neurons, from zero to 2.7±0.4 per fly (Fig. 3.2 A,C). In females, the number of FS-Ilp7 neurons was unchanged from controls (fruP1>+) (Fig. 3.2 B,D). Next, we tested whether PCD occurs in post-mitotic FS-Ilp7 neurons. To test this, we used Ilp7-GAL4 and two well-characterized post-mitotic drivers, elavC155-GAL4 and nsyb-GAL4 to express   28 UAS-p35. No FS-Ilp7 neurons were observed in these males and no significant difference was observed in female FS-Ilp7 neuron numbers. This suggests that the commitment to PCD leading to the loss of FS-Ilp7 neurons in males occurs prior to, or around the time of, the birth of post-mitotic FS-Ilp7 neurons. We conclude that male-specific PCD fully accounts for the female-specific generation of FS-Ilp7 neurons.  Figure 3.2. FS-Ilp7 Neurons are Eliminated by PCD in Males.  (A-D) We blocked programmed cell death (PCD) in cells using the fruP1-GAL4 (fruP1>) Ilp7-GAL4, or postmitotic pan-neuronal GAL4 drivers, elavGAL4 and nsyb-GAL4, to drive expression of the baculovirus p35 caspase blocker (UAS-p35). We quantified FS-Ilp7 neuron numbers in   29 both sexes. (A-B) FS-Ilp7 neurons were not observed in wild type males (fruP1>+), and were always observed in wild type females (fruP1>+) (arrowheads, Ilp7+/Fkh-/fruP1+). In the fruP1>p35 genotype, FS-Ilp7 neurons were generated in males (A, arrowheads), but were not produced at increased numbers in females (B, arrowheads). (C-D) Quantification of FS-Ilp7 neuron numbers per fly (each point in scatter plot) in each sex and each genotype (shown along x-axis). FS-Ilp7 neurons were only generated in males in the fruP1>p35 genotype. No difference in the number of FS-Ilp7 neurons was observed in females in any genotype. All data shown as mean±SEM. ****=p<0.0001 compared to fruP1>+ control.  3.1.1 Investigating the NB origins of FS-Ilp7 neurons Only three NBs are described to generate post-embryonic lineages in each A6-A9 hemisegment in the VNC. As FS-Ilp7 neurons are post-embryonically generated in segments A6/A7 [67], we tried to determine which of these three NB give rise to FS-Ilp7 neurons. We used a technique that immortalizes reporter expression in dpn expressing NBs, and assayed reporters that were reported to label distinct NB lineages [85, 86]. GMR45D04-GAL4 in combination with the patterned recombination tools, labels NB5-2, 5-3, 5-4, 5-7 and 6-2. This combination labelled at least one post-embryonic Ilp7 neuron (dorsal and FS-Ilp7 neurons in females) in 12.5% of VNCs (n=16); in one VNC two FS-Ilp7 neurons were labelled, and in another VNC one dorsal Ilp7 neuron was labelled (data not shown). Since the patterned recombination technique relies on two temporally controlled stop cassette flip outs, we reasoned that it may not be completely effective at immortalizing reporter expression in all lineages. In addition, we found that GMR54B10-GAL4, which labels NB5-3 and 5-6 lineages, labelled one dorsal Ilp7 neuron out of the VNCs analyzed (n=9). The outcomes of these two GMRs,   30 suggested to us that FS-Ilp7 neurons may come from NB 5-3. From this we attempted to look for some markers that are expressed, and were unsuccessful; we did not see co-expression of reporters for vg, gsb, and klu and Ilp7 peptide around 48 h APF in FS-Ilp7 neurons (data not shown). Therefore, we were unsuccessful in these attempts to convincingly validate NB 5-3 as giving rise to FS-Ilp7 neurons.  3.1.2 Investigating the Genetic Mechanism of Male-specific PCD of FS-Ilp7 Neurons Next, we asked whether any of the pro-apoptotic genes are necessary to initiate PCD of FS-Ilp7 neurons in males. Knockdown of rpr, hid, grim, or skl in post-mitotic neurons (using elavc155) or fru-expressing neurons was insufficient to rescue FS-Ilp7 neurons in males (Table 3.1). We next tried hemizygous mutations for the RHG genes. We combined Df(3L)H99, a large deletion that removes all RHG genes, except for skl, with either amorphic or hypomorphic RHG alleles, or RHG deletions. Most combinations did not rescue any FS-Ilp7 neurons (Table 3.2). However, Rpr mutant males occasionally had FS-Ilp7 neurons; Df(3L)H99/XR38 males had 0.9±0.3 FS-Ilp7 neurons, which was slightly more than Df(3L)H99/w1118 males who never had FS-Ilp7 neurons (Tukey’s 1-way ANOVA, p<0.1) (Table 3.2). Therefore, knockdown or hemizygous mutations of individual RHG genes is not sufficient to rescue FS-Ilp7 neurons from PCD in males, suggesting that multiple RHG genes act redundantly.        31 RNAi target Identifier Average of FS-Ilp7 neurons SEM A. UAS-Dicer2, elavc155-GAL4 DRSC	51846 rpr 0.1	(n=9) 0.04 NIG	5123R-2 hid 0	(n=10) 0 NIG	5123R-3 hid 0	(n=9) 0 VDRC	21830 grim 0	(n=7) 0 VDRC	22597 grim 0	(n=11) 0 DRSC	28678	 skl	 0	(n=7)	 0	B. UAS-Dicer2; Ilp7-tdTomato; fruP1-GAL4>nGFP 	 w1118	 0	(n=10)	 0	DRSC	51846 rpr	 0	(n=9)	 0	NIG	5123R-2	 hid	 0	(n=7)	 0	NIG	5123R-3	 hid	 0	(n=6)	 0	VDRC	21830	 grim	 0	(n=9)	 0	VDRC	22597	 grim	 0	(n=9)	 0	DRSC	28678	 skl	 0	(n=10)	 0	 Table 3.1. RNAi against Individual RHG and skl Genes is not Sufficient to Rescue FS-Ilp7 Neurons in Males.  Two GAL4 drivers were used for neuronal knockdown of the pro-apoptotic genes rpr, hid, grim, and skl. (A) elavC155 recombined with UAS-Dicer2 was used for post-mitotic knock-down of PCD genes. (B) UAS-Dicer2; Ilp7-tdTomato; fruP1>nGFP was used for fruP1-specific knockdown of PCD genes, and identification of FS-Ilp7 neurons (Ilp7+/fruP1+). See Materials and Methods for source of dsRNAi targeted against RHG and skl genes.  Genotype Genes completely deleted/disrupted Average of FS-Ilp7 neurons SEM Df(3L)H99/w1118 -- 0	(n=15) 0 Df(3L)H99/Df(3L)XR38 rpr 0.9	(n=8) 0.3 Df(3L)H99/Df(3L)grimA6C grim 0	(n=13) 0 Df(3L)H99/hidA206 hid 0	(n=4) 0    32 Table 3.2. Hemizygous Mutations of RHG Genes is not Sufficient to Rescue Significant FS-Ilp7 Neurons in Males. We investigated whether males that were hemizygous for RHG genes had any rescued FS-Ilp7 neurons. In order to determine if the pro-apoptotic genes are necessary for PCD of FS-Ilp7 neurons, we combined the Df(3L)H99 allele, a large deletion including rpr, hid, but not skl, with alleles for each cell death gene. We counted the number of FS-Ilp7 neurons in pharate or A1 adult male VNCs. The XR38 deletion removes rpr and skl, and when combined with the H99 deletion, rescues on average one FS-Ilp7 neuron in males. In contrast, the grimA6C deletion removes only grim, and does not save any FS-Ilp7 neurons, and the hidA206 hypomorphic allele also did not rescue FS-Ilp7 neurons when combined with Df(3L)H99 in males.   3.2 FruMC is Necessary for Cell Death of FS-Ilp7 Neurons in Males  We wanted to resolve the genetic mechanisms by which FS-Ilp7 neurons are programmed to die in males and survive in females. Our previous work showed that Tra was necessary and sufficient for FS-Ilp7 neuron survival, but our results regarding fru did not resolve its role. Typically, differences in the fru- or dsx-dependent dimorphisms are fully explained by the sex-specific isoform expressed. However, we had found that fruM was necessary for FS-Ilp7 neuron elimination in males, but the lack of fruM in females was not found to be sufficient for the presence of these neurons in females [67]. This indicated that some other mechanism operates to modify FruM activity, either in males or females. We revisited a possible role for dsx, despite our previous expression analysis indicating that dsx is not expressed in FS-Ilp7 neurons or their lineage [67]. We tested dsx nulls and   33 observed no change in the number of FS-Ilp7 neurons in either males or females (Fig. 3.3). Therefore, we eliminated a role for dsx, and returned to a detailed analysis of fru.   Figure 3.3. dsx is not Necessary in Males or Females for Female-specific Generation of Ilp7 Neurons.  Using hemizygous mutants for dsx (dsx1/Df(3R)dsx15) and immunoreactivity against Ilp7 and Fkh, we tested if dsx has a role in FS-Ilp7 neuron generation. (A) In males, no FS-Ilp7 neurons were observed in wildtype (w1118) or dsx hemizygous mutants. (B) In females, there was no significant difference in the number of FS-Ilp7 neurons between wildtype (w1118) and dsx hemizygous mutants. FS-Ilp7 neurons were counted by their immunoreactivity for Ilp7, and absence of Fkh immunoreactivity in the ventral region of the Abg. We expanded our previous analysis of fru to a more extensive allelic series in an effort to shed light on the necessary but insufficient role of fru in male-death and female-survival. To this end, we combined fru alleles that reduce/eliminate FruM expression (fru3, fruF, fruP1-GAL4, fruSat15, or fru4-40), with either a wild type chromosome (+) or an engineered fru allele that forces or prevents male-specific splicing in either sex, fruM or fruF, respectively. The nature of these   34 alleles is depicted in Fig. 3.4 A and referenced in Materials and Methods. Our results show that complete loss of FruM expression in all genotypes (no copy of fru+ or fruM) leads to full FS-Ilp7 neuron survival in males; averaging 4.2 to 6.5 FS-Ilp7 neurons per fly (Fig. 3.4 B). In most genotypes that include a single copy of fru+ or fruM, we observed elimination of most FS-Ilp7 neurons (averaging 0.06 to 1.1 FS-Ilp7 neurons per fly) (Fig. 3.4 B). These data demonstrate that FruM is necessary for PCD of FS-Ilp7 neurons in males, and importantly that fru is not haploinsufficient. Unexpectedly, the fru4-40 allele dominantly rescued FS-Ilp7 neurons in males with + or fruM alleles, where males should theoretically be able to express FruM. This indicates that the fru4-40 deficiency has some unexpected activity that remains unresolved (discussed in Section 4.4).      35  Figure 3.4. FruMC is Necessary to Eliminate FS-Ilp7 Neurons in Males. (A) Schematic of the fru locus (not to scale) showing the fru alleles used (see also Materials and Methods), the fru promoters (white boxes), the exons (coloured boxes), as well as splicing (solid   36 arching lines) and alternate splicing (dotted arching lines). P1 transcripts are sex-specifically spliced so that females express the noncoding fruF transcript (premature stop codon denoted) and males express the coding FruM transcript (magenta/blue box, sex-specific exons). Four FruM isoforms are generated (FruMA, FruMB, FruMC, FruMD) by alternate usage of exons A-D (3’ coloured boxes). (B-C) We tested a role for FruM and its isoforms in male FS-Ilp7 neuron PCD. (B) We placed alleles that either forced male-splicing (M, fruM) or blocked male-splicing (F, fruF), or a control chromosome (+, w1118) over a series of alleles that prevent/reduce FruM protein expression (fruF, fruP1>, fru3, fruSat15, or fru4-40). In these genotypes (shown along x axis), we counted FS-Ilp7 neurons per fly and present these as mean±SEM. FS-Ilp7 neurons were rarely observed in males with one or more FruM-expressing alleles (filled circles). However, FS-Ilp7 neurons were observed in genotypes that have no FruM expression (empty circles). (C) We tested which FruM isoforms are required for PCD. We placed nonsense FruM isoform mutants (fruΔA, fruΔB, fruΔC) over either fruF or fruP1> alleles, and counted FS-Ilp7 neuron numbers, represented as scatter plots and showing mean±SEM. FS-Ilp7 neurons only survived in fruΔC heteroallelic combinations showing that only FruMC is required for PCD. Significant differences are shown compared to pertinent controls (+); **=p<0.01, ****=p<0.0001  FruMA, FruMB, and FruMC differ in their use of alternate 3’ exons that each encodes a distinct C2H2 zinc finger domain, which confer differential DNA-binding and function [43-45, 49, 51, 81, 82, 87]. We tested which isoform is necessary for male PCD, by using isoform-specific mutants that contain a premature stop codon within one of the distinct 3’ exons, referred to as fruΔA, fruΔB, fruΔC (Fig. 3.4 A,C) [81, 82]. We combined these with the fruF or fruP1-GAL4 alleles that both prevent or reduce FruM expression. In males, we did not observe FS-Ilp7   37 neurons in fruΔA or fruΔB heteroallelic genotypes, confirming that FruMA and FruMB are not necessary for PCD. In fruΔC heteroallelic genotypes, we observed survival of FS-Ilp7 neurons to the same extent observed in wildtype females (4.1±0.5 and 4.5±0.4 FS-Ilp7 neurons for fruΔC/fruF and fruΔC/fruP1-GAL4, respectively) (Fig. 3.4 C). Therefore, only the FruMC isoform is necessary for PCD and responsible for the loss of FS-Ilp7 neurons in males.  3.3 Rescued FS-Ilp7 do not have a Typical Masculine Neuronal Identity in fruP1>p35 or fruM Null Males  Since FruMC is necessary for male-specific death of FS-Ilp7 neurons, we wanted to know whether FruM is expressed in these neurons to support it being a cell-autonomous process. To answer this question, we chose to prevent PCD of FS-Ilp7 neurons and probe their expression pattern. Here, I will refer to these as “undead” FS-Ilp7 neurons, a term that is commonly used when referring to neurons that normally died but have their final stages of PCD blocked (reviewed in [57]). We used myc-tagged FruM isoforms and anti-myc immunoreactivity to determine if any are expressed in undead FS-Ilp7 neurons using the fruAmyc, fruBmyc and fruCmyc alleles [49]. We found that undead FS-Ilp7 neurons express FruMB and FruMC, but not FruMA (Fig. 3.5 A-C). Similarly, dorsal Ilp7 neurons also express FruMB and FruMC in males (data not shown).   38  Figure 3.5. “Undead” Ilp7 Neurons Express FruMB and FruMC (A-C) We investigated whether FS-Ilp7 neurons express FruM isoforms in males when PCD is blocked, using fruP1>p35; rescued FS-Ilp7 neurons in this manner are referred to as “undead” (arrows; α-Ilp7, blue; fruP1+, green). Using immunoreactivity of Myc-tagged FruM isoforms (FruAmyc, FruBmyc, and FruCmyc; red), we found that undead FS-Ilp7 neurons express FruMB (B), FruMC (C) but not FruMA (A).   Castellanos et al (2013) determined that all post-embryonic Ilp7 neurons are serotonergic in males and not serotonergic in females, so we next asked whether rescued FS-Ilp7 neurons have a male-like neuronal fate (ie. serotonergic) when either PCD is blocked or when FruM is not   39 expressed. Using 5-HT immunoreactivity, we confirmed that embryonic Ilp7 neurons are not serotonergic in males (w1118), and we found that the small, weakly expressing Ilp7 neurons (identified in Fig 3.1) are serotonergic (Fig. 3.6 A). Undead FS-Ilp7 neurons in males, surprisingly, were not serotonergic (Fig. 3.6 B), like the male post-embryonic dorsal Ilp7 neurons. Similarly, the rescued FS-Ilp7 neurons in ‘feminized’ fru males (ie. fruM null) also were not serotonergic (Fig. 3.6 C). Therefore, rescued FS-Ilp7 neurons, either by blocked PCD or preventing FruM expression in males, do not become serotonergic.   40    41 Figure 3.6. Abnormal Differentiation and Arborization of Post-embryonic Ilp7 Neurons in fruP1>p35 and fruM Null Males. (A-C) We investigated whether rescued FS-Ilp7 neurons become serotonergic in fruP1>p35 and fruM null genetic backgrounds. We used Ilp7-tdTomato;fruP1>nGFP to distinguish FS-Ilp7 neurons (Ilp7, red; fruP1, green; FS-Ilp7 neurons indicated with arrows), and 5-HT immunoreactivity (blue). (A) Wildtype males did not have FS-Ilp7 neurons, but weakly expressing Ilp7 posteriorly located neurons (identified in Fig 3.1) were found to be serotonergic (Ilp7+/fruP1+/5-HT+; asterisks). (B) Undead FS-Ilp7 neurons were not 5-HT immunoreactive (arrows) in males. (C) Rescued FS-Ilp7 neurons in a fruM null male (fruF/fruP1>) were also not serotonergic (arrows). (D) We quantified the average number of dorsal Ilp7 neurons in males and whether they were serotonergic or not in the following genotypes: Ilp7-tdTomato;fruP1>nGFP with w1118 (left), UAS-p35 (middle), and fruF/fruP1>. We found that dorsal Ilp7 neurons were always fruP1+ and 5-HT+ in wildtype males. In UAS-p35 and fruM null males, we observed a significant increase in the number of dorsal Ilp7 neurons (sum of grey and black column), all of which express fruP1. However, we found that rescued dorsal Ilp7 neurons were not serotonergic (black column). (E-F) The UAS-CD4tdGFP reporter revealed unidentified Ilp7 neuronal projections on the Accessory Gland (AG). A1 adult males of genotype w1118;Ilp7-GAL4,UAS-CD4-tdGFP (Ilp7>CD4-tdGFP) were assessed for Ilp7 neuronal projections on the reproductive organs. (E) Ilp7 projections were observed on the Seminal Vesicles (SV), and previously unidentified projections were observed distally on AGs. (F) The Ilp7 projections on the AG were serotonergic as determined by 5-HT immunoreactivity in Ilp7-labelled synaptic boutons (α-5-HT; red). (G-J) FruM is necessary for proper innervation of the male reproductive organs. Ilp7 projections in fruM null male reproductive organs were varied, and representative images are   42 presented (Ilp7-tdGFP/+;fruF/fruF; n=6). (G) In fruM null males, ectopic Ilp7 neuronal projections were observed on the ejaculatory duct (EJ), although the area of Ilp7 projections varied between males; most males had extensive projections proximally and distally (shown in J). Unlike wildtype projections onto the AG, the Ilp7 projections were less concentrated at the distal tip and more concentrated on the proximal end of the AGs in fruF/fruF genotypes (H). Projections onto the SV innervations appeared similar in wildtype and fruM null males (I). (H-J) In fruF/ fruF males, few Ilp7 boutons (Ilp7>CD4-tdGFP, green) on the AG (H) and SV (I) had 5-HT accumulations (red), and EJ (J) innervations never had 5-HT accumulations pre-synaptically (red). This is in contrast to wildtype boutons that have 5-HT accumulations pre-synaptically in AG and SV projections.  We next asked whether dorsal Ilp7 neurons maintain their serotonin expression when p35 is expressed in fruP1 neurons or in genotypes that prevent FruM expression (Fig 3.6D). Unexpectedly, we found that there were significantly more dorsal Ilp7 neuronal soma in fruP1>p35 and fruM null (fruF/fruP1>) males compared to wildtype; fruP1>35 males had 10.8±0.6 dorsal Ilp7 neurons per fly and fruM null males had 9.5±0.9 dorsal Ilp7 neurons per fly. In contrast, wildtype males have 5.1±1.1 dorsal Ilp7 neurons per fly (1-way ANOVA, Tukey’s Post-Hoc Test; p<0.001). We next asked whether dorsal Ilp7 neurons maintain their serotonergic fate when p35 is expressed (fruP1>p35), or in FruM mutant backgrounds (fruF/fruP1>) (Fig. 3.6 D). Dorsal Ilp7 neurons in wildtype males (w1118) were always immunoreactive for 5-HT. In contrast, we found that only a portion of dorsal Ilp7 neurons were serotonergic; there were non-serotonergic Ilp7 neurons in fruP1>p35 (~24%) and fruM null (~51%) males. Therefore, approximately two-fold more dorsal Ilp7 neurons are generated in fruP1>p35 and fruF/fruP1>   43 genotypes in males, and many of these do not become serotonergic. Although untested, we think that the newly generated dorsal Ilp7 neurons in these genotypes do not become serotonergic, whereas fruM is also necessary for differentiation, leading to loss of serotonin expression in the pre-existing dorsal Ilp7 neurons in fruM null males.  We wanted to determine whether FruM was necessary for normal Ilp7 projections into the male reproductive tract. Castellanos et al (2013) discovered and characterized the sexually dimorphic projections of the Ilp7 neurons. Embryonic Ilp7 neurons were shown to innervate the hindgut, and post-embryonic Ilp7 neurons were found to innervate either the oviduct in females or the seminal vesicle (SV) in males at dimorphic NMJs [67]. Castellanos et al (2013) had used a UAS-CD8-GFP membrane marker to examine Ilp7 neuronal projections. Since then, an improved axonal marker, UAS-CD4-tdGFP, had been developed to better label neuronal processes [83]. I wished to re-examine Ilp7-neuron projections using this enhanced membrane marker construct driven from Ilp7-GAL4. I found that CD4-tdGFP did in fact label finer structures on the hindgut and oviduct with innervations that were consistent with Castellanos et al (2013) (data not shown). The CD4-tdGFP marker labelled structures on the SV that were consistent with Castellanos et al (2013). Surprisingly, CD4-tdGFP also labelled previously unreported Ilp7 projections on the accessory gland (AG) in males (Fig. 3.6 E-F), and, like the SV Ilp7 neurons, these projections express serotonin (Fig. 3.6 F). Aberrant neuronal projections to the male reproductive organ have been well characterized in fruM null males [82, 88]. In wildtype males, extensive innervation on all the male reproductive organs are observable with pan neuronal markers, most of which are fruP1-expressing serotonergic neurons [88]. Castellanos et al (2013) demonstrated that postembryonic Ilp7 neurons are serotonergic and comprise all the neuronal input to the SV. When fruM is not   44 expressed, serotonin accumulation pre-synaptically is lost in most projections, and all neuronal projections observed are either highly reduced or abnormal [82].  We wanted to determine whether FruM expression is necessary for Ilp7 projections onto the SV and AG, and whether we could distinguish rescued FS-Ilp7 neuronal projections in these males. Indeed, fruM null males have abnormal Ilp7 projections (Fig. 3.6 C-F). Most notably, Ilp7 projections were observed on the EJ, which is never seen in wildtype males. Further, we never observed serotonin accumulation pre-synaptically on the medial and distal EJ, and observed reduced, but variable, serotonin accumulation on other organs. Similar to Billeter et al’s (2006) characterization, we saw reduced projections onto the AG, fewer serotonergic boutons, and slightly reduced projections onto the SV. Serotonergic projections were varied on the SV, where some males had strong 5-HT immunoreactivity, and others little to no 5-HT immunoreactivity.  In conclusion, using the Ilp7-tdTomato and Ilp7>CD4-tdGFP reporters, we were able to better define post-embryonic Ilp7 neuronal fates and arborizations in fru mutant backgrounds. First, we found that undead FS-Ilp7 neurons express two of the three FruM isoforms. Second, we found that rescued FS-Ilp7 neurons do not become serotonergic when PCD is blocked (by p35 expression) or when FruM expression is prevented, unlike post-embryonic dorsal Ilp7 neurons in males. Third, we discovered that supernumerary dorsal Ilp7 neurons are generated in males when p35 is expressed or in fruM null backgrounds, and that a portion of these also do not become serotonergic. Lastly, we discovered that when FruM is not expressed, Ilp7 neurons ectopically innervate the EJ, and have aberrant projections on the AG and SV, with weak to no serotonin accumulation pre-synaptically. In summary, p35 and fruM null male Ilp7 neurons have atypical neuronal fates and projection patterns onto the male reproductive organs.    45 3.4 Forced Male-specific Splicing of fru in Females is Insufficient to Kill Ilp7 Neurons In many studies where fruM is necessary for the male-specific phenotype of specific neurons, it is also partially or completely sufficient when expressed in females to confer male-specific neuronal phenotypes [49, 60, 67, 80, 89]. In contrast, we had found that forced fruM splicing in females was not sufficient to eliminate FS-Ilp7 neurons in females [67]. However, as that study had tested one copy of fruM over a large fru deficiency, it left unresolved the possibility that fruM is haploinsufficient in females. To test this here, we generated females with two alleles that each produce FruM protein (fruM and fruΔtra) irrespective of Tra activity [80]. We observed 3.0±0.3 FS-Ilp7 neurons in these fruM/fruΔtra females compared to 4.0±0.4 FS-Ilp7 neurons in wildtype controls (Fig. 3.7 A). Thus, fruM is not able to eliminate FS-Ilp7 neurons in females, as it does in males. We tested a larger fru allelic series and despite one observation of a modest but significant fruM-driven reduction in FS-Ilp7 neurons when comparing fruM/fruF (3.2±0.4 neurons per fly) to fruF/fruF (5.0±0.3 neurons per fly) (Fig. 3.7 A). Our data demonstrates that fruM is not sufficient to trigger PCD of FS-Ilp7 neurons in females.    46  Figure 3.7. Tra Blocks PCD of FS-Ilp7 Neurons Genetically in Parallel or Downstream of fru Splicing.   47 (A-C) We tested if fruM is sufficient for PCD of FS-Ilp7 neurons in females, and for genetic interactions between tra and fru that result in FS-Ilp7 neuron survival in females. (A) We quantified FS-Ilp7 neurons per female in a fru allelic series—similar to Fig 3—showing mean±SEM per genotype (Filled circles, FruM-expressing genotypes; empty circles, genotypes that cannot express FruM). There was no dramatic reduction in FS-Ilp7 neurons in any genotype tested. (B) In males, ectopic Tra expression (fruP1>traF) led to FS-Ilp7 neuron survival, whereas controls exhibited no FS-Ilp7 neurons (fruP1>+ or UAS-traF). We tested if this survival was due to Tra-dependent splicing of fru (preventing FruM expression). We introduced the forced male-splicing allele fruM in this background (fruP1>traF + fruM), and did not observe a decrease in FS-Ilp7 neuron numbers in comparison to fruP1>traF. (C) To determine whether Tra epistatically prevents FruMC action in females, we tested if FruMC is sufficient to kill FS-Ilp7 neurons in females if Tra is absent. We knocked down Tra by RNAi (elav>Dcr2;tradsRNAi;fru+/+), and this killed all FS-Ilp7 neurons. We then tested if FruM is required for PCD in the absence of Tra. We prevented male-splicing of fru in the Tra RNAi background (elav>Dcr2;tradsRNAi), and observed a dosage response of fru in killing FS-Ilp7 neurons; One copy of fru available for FruM expression led to partial survival (fru+/F), and no copies of fru available for FruM-expression (fruF/F) led to full survival of FS-Ilp7 neurons. Introduction of the fruΔC allele over fruF (fruΔC/F) demonstrated full FS-Ilp7 neuron rescue. This demonstrated that FruMC alone is sufficient to elicit PCD in females, but only when Tra is absent. Data presented as number of FS-Ilp7 neurons per fly in scatter plots with mean±SEM. Significant differences within each experimental group are shown compared to pertinent controls (+); *=p<0.05, **=p<0.01, ****=p<0.0001.    48 3.5 Tra Blocks FruM-dependent PCD Independently of Splicing to Promote FS-Ilp7 Neuron Survival. We wished to identify the reason for the lack of fruM sufficiency in females, in order to better understand the genetic mechanisms governing female-specific FS-Ilp7 neuron generation. It is intriguing that Tra, the upstream splicing activator of fru and dsx in females, is necessary in females and sufficient in males for FS-Ilp7 neuron survival [67], but that fruM is necessary in males but insufficient in females for loss of FS-Ilp7neurons. Given our demonstration that this discrepancy is not due to the action of dsx (Fig. 3.3), we hypothesized that Tra may play a novel role in parallel or downstream of fru splicing to block FruM-dependent PCD in females. We genetically manipulated these factors in both sexes to tease apart their genetic relationship and relative roles in each sex in order to test our hypothesis.  First, we tested if Tra can prevent FruM-dependent PCD in males, downstream of its role in fru splicing. Contrary to Castellanos et al (2013), we were unable to rescue a significant number of FS-Ilp7 neurons in males by expressing UAS-traF with the post-mitotic driver elavc155; elavc155>Tra in males produced an average of 0.3±0.1 FS-Ilp7 neurons, n=15. However, when we ectopically expressed UAS-traF from the fruP1-GAL4 driver in males, we observed survival of FS-Ilp7 neurons (2.1±0.4 neurons per fly) (Fig. 3.7 B; fruP1>traF/fru+). This survival was expected, under the assumption that this eliminates fruM splicing in males, and thereby prevents FruM expression. However, in order to uncouple fru splicing from Tra’s function here, we tested the effect of ectopic Tra expression in the presence of forced male-specific splicing (fruM). Remarkably, even in the presence of FruM protein, the co-expression of Tra led to the survival of 2.7±0.3 FS-Ilp7 neurons (Fig. 3.7 B; fruP1>traF/fruM). This suggested that Tra is capable of overriding FruM-dependent PCD in a mechanism unrelated to fru splicing.    49 To test if this fruM-modifying activity of Tra is an artifact of its overexpression in males, we tested the same epistatic relationship in females. We repeated and confirmed previous results that RNAi-mediated knockdown of Tra in neurons eliminated all FS-Ilp7 neurons, using elavGAL4 driving UAS-tradsRNAi expression (Fig. 3.7 C) [67]. Further, we tested tra null mutant females (traKO/traKO) and found these also have zero FS-Ilp7 neurons (n=7), whereas traKO/TM6B females have 5.3±0.4 FS-Ilp7 neurons (n=8) (p<0.0001, unpaired student T-test). We also tested whether Tra’s obligatory splicing co-factor, Tra2, was part of this pathway. We found that Tra2 was necessary for the generation of FS-Ilp7 neurons, as expected by its known role as a cofactor with Tra in prevention of FruM expression via fruF splicing; in tra2 hemizygous females (tra2[B]/Df(2L)trix), there were 0±0 FS-Ilp7 neurons (n=13) (p<0.0001, unpaired student T-test). We found that Tra2 is not necessary for PCD of FS-Ilp7 neurons in males, suggesting that Tra2 did not have a role in male-specific splicing of fru P1 transcripts; tra2[B]/Df(2L)trix males had 0±0 FS-Ilp7 neurons (n=14). Mutants for tra2 are known to prevent female-specific splicing, leading to fruM transcript in females, whereas tra2 mutant males do not make significantly less fruM [90]. These results demonstrate that Tra and Tra2 are necessary in females for FS_Ilp7 neuronal survival. Next, we tested if FS-Ilp7 neuronal loss in elav>tradsRNAi females is due to default splicing of fru into fruM. To this end, we examined the number of FS-Ilp7 neurons in an allelic series that progressively reduced the dosage of fruM in a tra knockdown background. We found in elav>tradsRNAi females that two copies of wild type fru (fru+/+), which can generate two copies of fruM, eliminated FS-Ilp7 neurons. Next, we found that the introduction of only one copy of wildtype fru (fru+/F) that can generate one copy of fruM, resulted in 1.3±0.2 FS-Ilp7 neurons. Finally, we found that the total loss of FruM (fruF/F) resulted in full survival of FS-Ilp7 neurons   50 (5.3±0.3 FS-Ilp7 neurons) (Fig. 3.7 C). We conclude that FruM expression in females (generated by switching endogenous fruF expression into fruM expression) is fully capable of eliminating FS-Ilp7 neurons, but only in the absence of Tra.  Next, we tested if the FruMC isoform is necessary in females to trigger PCD in Tra-deficient females, as it is in males. In confirmation, we found that in elav >tradsRNAi females, the presence of the fruΔC mutant allele (fruΔC/F) led to survival of 4.0±0.5 FS-Ilp7 neurons (Fig. 3.7 C). This demonstrates that FruMC is required for FS-Ilp7 neuron PCD in females when Tra is absent. Our results demonstrate a novel activity for Tra, by showing that Tra acts in parallel or downstream of fru splicing to block the function of FruM.   51 Chapter 4: Discussion 4.1 Summary of Major Findings Analysis of fru, dsx and tra function in the Drosophila nervous system has transformed our understanding of the construction of sexually dimorphic neuronal circuits [91]. Due to the elaborate stereotyped behaviours of males, studies have mostly focused on construction of the male nervous system. In contrast, how the female nervous system is constructed is less well characterized. Here, we explore the genetic and cellular mechanisms that generate a population of female-specific neurons in Drosophila. In addressing this, we uncover novel functions for fru and tra that are of general significance to understanding the development of dimorphic nervous systems, and also the interpretation of genetic studies using these factors. Prior to this thesis work, the sexual dimorphisms of Ilp7 neurons were characterized by the Allan Lab, along with discovering the female-specific subset of these, the FS-Ilp7 motor neurons. Additionally, tra was found to be essential for FS-Ilp7 motor neuron generation only in females, and fruM was found to be essential for their loss in males, but the absence of fruM in females was not found to account for the presence in females. This thesis followed up on findings by Castellanos and the Allan Lab to determine the following: 1) PCD is responsible for elimination of post-embryonic Ilp7 neurons in males: • Blockade of PCD by p35 or loss of fruM in males results in survival of FS-Ilp7 neurons and supernumerary dorsal Ilp7 neurons (Fig. 3.2, 3.4 and 3.6 A-D) • Only the FruMC isoform is necessary for PCD of FS-Ilp7 neurons (Fig 3.4) 2) Serotonergic differentiation of male Ilp7 neurons is disrupted after loss of fruM: • Loss of fruM in males results in a reduction of serotonergic differentiation of all Ilp7 neurons (Fig. 3.6 A, C, D)   52 • Despite expression of masculinizing factors (FruMB and FruMC), FS-Ilp7 and dorsal Ilp7 neurons do not become serotonergic when rescued from PCD in males (Fig. 3.6 B and D). 3) Ilp7 axon targeting to the male reproductive tract is affected by loss of fruM  • Serotonergic Ilp7 neurons innervate the AGs in addition to the known seminal vesicle innervations in wildtype males (Fig. 3.6 E-F) • In fruM null males, post-embryonic Ilp7 neurons have aberrant arborizations on the reproductive organs in males, including ectopic EJ projections (Fig. 3.6 G-J) 4) Tra plays a double-assurance role in FS-Ilp7 neuronal survival: • Endogenous expression of FruM in females is insufficient to kill FS-Ilp7 neurons in females (Fig. 3.7).  • In the absence of Tra, FruMC is necessary for PCD of FS-Ilp7 neurons in females (Fig. 3.7).   4.2 NB origins of FS-Ilp7 neurons Each NB has a unique TF code, that ensures the coordinated and unique fate of its progeny. This can often serve as a useful marking system to track the lineage of the post-mitotic neurons being studied. The NB TF profile can be maintained from the neuronal progeny to differentiation or it can be markedly different post-mitotically (at the onset of differentiation). Understanding this code allows for a detailed genetic analysis of how neurons are distinctly generated. Although NB are active during two waves of neurogenesis, it is thought that a single NB lineage produces similar neurons in embryonic and post-embryonic neurogenesis, suggesting their TF code is maintained. From a screen, many GMRs were found to accurately label discrete NB lineages from both embryonic and post-embryonic neurogenesis when used with a technique   53 that restricts expression to dpn expressing neuronal cells and immortalizes their expression. We have preliminary data that suggests FS-Ilp7 neurons and dorsal Ilp7 neurons come from NB5-3 because, although infrequently, they were labelled by GMR 54D04 and 54B10. These GMRs both label NB 5-3 lineages, but otherwise mark mutually exclusive NB lineages. Unfortunately, we have yet to identify new markers that precede Ilp7 expression.   4.3 FruMC Removes Female Neurons from the Male Nervous System by PCD Female-specific PCD or male-specific proliferation are mechanisms that have been demonstrated to underlie the generation of more fruP1-expressing neuronal populations in males. Sex-specific isoforms of dsx have been shown to be largely responsible for both mechanisms [59, 89, 92-94]. However, FruM has been implicated in preventing PCD of mAL neurons, and other research suggests it may have a greater role in controlling neuronal numbers [49, 61]. Our results now provide a contrasting view of FruM function, by showing that the FruMC isoform removes female neuronal components from the male nervous system via PCD (Fig. 4.1). These data are interesting in light of a recent examination of fruP1-expressing brain NB lineages, showing that blockade of PCD using UAS-p35 increased neuronal number in both sexes. This indicated that PCD restricts neuronal number in male lineages [95]. We believe that our observation here that FruMC eliminates neurons in males through PCD provides a potential mechanism to account for neuronal loss in those cases, and indeed that this may represent a relatively widespread function for FruM in males. These findings provide a novel framework that accounts for targeted elimination of neurons in males and for generation of female-specific neurons and circuitry.    54  Figure 4.1. Fru and Tra have Novel and Opposing Roles in Constructing Sexually Dimorphic Neuronal Number of FS-Ilp7 Neurons.  In the male nervous system, FruM isoforms A-C are expressed and direct most male-specific differences in behaviour and neuronal morphology, whereas in the female-nervous system, Tra prevents FruM protein production via alternative splicing.  (Left) In males, we found that FruMC is necessary for PCD of FS-Ilp7 neurons. (Right) In females, we found that Tra not only prevents fruM splicing, by generating fruF, but genetically acts in parallel or downstream of FruMC to prevent PCD of FS-Ilp7 neurons.  We propose that this additional role for Tra outside of fru splicing acts as a failsafe to ensure the survival of FS-Ilp7 neurons, which are required for egg-laying.    55 4.4 The fru4-40 deficiency may abrogate fruM expression in FS-Ilp7 neurons In attempting to clarify the ambiguity around the role of fruM function in eliminating FS-Ilp7 neurons by Castellanos et al (2013), we identified an unexpected, and counter-intuitive activity of the fru4-40 deficiency.  Castellanos et al had used fru4-40 as a deficiency to test the role of fruM in FS-Ilp7 neuron elimination.  However, we found in this thesis that the fru4-40 allele dominantly rescued FS-Ilp7 neurons in males when paired with any fru allele (in the presence of a wildtype fru allele or a forced fruM-expressing allele) (Fig. 3.4 B). These findings raised concerns about the findings of Castellanos et al. First, one may have postulated that FS-Ilp7 neurons in males were rescued by fru4-40, and not the lack of fruM. However, these concerns were alleviated by our rigorous fru allelic series analysis showing that fruM is indeed required for PCD of FS-Ilp7 neurons (Fig. 3.4 B). Second, the lack of PCD that was observed in females may simply have been due to the presence of fru4-40. However, these concerns were again alleviated by our rigorous fru allelic series analysis showing that fruM indeed fails to induce PCD of FS-Ilp7 neurons in females (Fig. 3.7 A).  We conclude that the high-resolution nature of our genetic analysis has unveiled a ‘feminizing’ effect of the fru4-40 allele that had eluded previous analyses. These findings regarding fru4-40 data are consistent with the observations of other studies, showing that fru4-40 heterozygotes have a lower magnitude of male-specific mating behaviors and greater feminized behaviours compared to other heterozygous males in some reports [12, 82]. Perhaps fru4-40 reduces the efficacy of fru on the homologous chromosome [80]. It is unclear how fru4-40 mediates this effect. Based on an untested hypothesis that transvection occurs at the fru locus [96-98], we may speculate that a ~70kb deficiency spanning the P1 to P2 promoters may disrupt   56 fruM transcription on the homologous chromosome. This could occur by loss of trans-regulating cis-regulatory regions or from disruptions to somatic chromosome pairing.  4.5 Role of PCD and fru in Neuronal Number, Identity, and Arborization of Male Ilp7 neurons  4.5.1 Neuronal Identity of Feminized or Rescued Post-embryonic Ilp7 Neurons We demonstrated that blocking PCD and fruM expression in males results in additional post-embryonic Ilp7 neurons (FS-Ilp7 neurons and supernumerary dorsal Ilp7 neurons), and in both cases that many of the neurons do not take on a typical masculine identity (Fig. 3.6A-D). We hypothesized that preventing PCD of post-embryonic Ilp7 neurons would rescue FS-Ilp7 neurons, but we expected them to have a masculine identity. Since dorsal Ilp7 neurons express FruMB and FruMC and differentiate into serotonergic neurons, we expected that undead FS-Ilp7 neurons would also become serotonergic, as they also express both Fru isoforms (Fig. 3.6). Unexpectedly, undead FS-Ilp7 neurons in males were not serotonergic, similar to FS-Ilp7 neurons in females. Furthermore, we noticed that when we blocked PCD, there were supernumerary dorsal Ilp7 neurons produced, and that several of them do not express 5-HT either. These results suggested to us that post-embryonic Ilp7 neurons, both dorsal and FS-Ilp7 neurons, undergo PCD in males, and when PCD is blocked, despite expressing FruM isoforms, do not take on a masculine neuronal identity. Similarly, we found fruM null males have an identical outcome in the FS-Ilp7 and dorsal Ilp7 neurons; fru feminized males have rescued non-serotonergic FS-Ilp7 neurons, and supernumerary non-serotonergic dorsal neurons. However, fruM null males have significantly fewer serotonergic neurons in comparison to wildtype males,   57 as expected from previous analysis on dorsal serotonergic neurons [82]. However, there were also fewer serotonergic dorsal Ilp7 neurons in fruP1>p35 males, despite expressing FruMB and FruMC. These results suggested to us that post-embryonic Ilp7 neurons (dorsal and FS-Ilp7 neurons) undergo FruM-dependent PCD, that rescued post-embryonic Ilp7 neurons are not inherently serotonergic, and FruM expression is necessary for serotonin expression in most dorsal Ilp7 neurons.  4.5.2 Role of Ilp7 Neurons in Male Fertility This thesis found that Ilp7 neurons not only project onto the SV, but also onto the AG (Fig. 3.7 E-F). This new-found AG projection was reminiscent of the Corazonin (Crz) neuropeptide circuit in Drosophila. Crz- and Crz receptor (CrzR)-expressing neurons are serotonergic, express FruM, and their soma are located dorsally in the Abg, all of which are features of post-embryonic Ilp7 neurons in males. Furthermore, CrzR-expressing neurons project to several male reproductive organs, including the AGs and SVs. It is tempting to hypothesize that the Ilp7 neurons and CrzR-expressing neurons are not mutually exclusive. Interestingly, the group discovered that CrzR-expressing projection neurons are involved in seminal fluid transfer and copulation duration; when CrzR-neurons are activated with TrpA1, they cause premature seminal fluid and sperm transfer [99]. The projection neurons identified by Tayler and colleagues (2012) are therefore necessary for full fertility. Ilp7 neurons’ role in male fertility was assayed by mating males who lack Ilp7 neurons with females for 24 h [67], but since very little sperm is needed to fertilize eggs it is possible that these males are less fertile than wildtype males. Tayler and colleagues (2012) found that wildtype male flies require less than 20 minutes to court, whereas males with inactivated Crz neurons require more than an hour and a half. We could take   58 a similar approach to determine if Ilp7 neurons regulate courtship duration or seminal fluid transfer in males.  4.5.3 Role of fru in Proper Arborization of Ilp7 Neurons in Males Having a new reporter for Ilp7 projections, we next asked what happens to these neurons at the male reproductive organs in males when they are fru feminized. Initially, we did not expect an interesting phenotype, because fru null male innervation on the reproductive organs have been well characterized [82]. However, these studies relied on pan neuronal or serotonin markers to characterize changes in innervation. Since we were able to label a discrete neuronal population with Ilp7>CD4-tdGFP we were able to uniquely determine changes to their projection pattern in fru mutant males (Fig.3.7 G-J). Ectopic Ilp7 projections were discovered on the EJ in fruM null males, which are never observed in wildtype males. This was an interesting result because previous characterizations of pan neuronal projections on the EJ have been reported as relatively normal in fru mutant backgrounds. Since we were able to label an individual neuronal population we were able to determine that these projections on the EJ are not typical. We also observed aberrant projection patterns on the AG, which is consistent with earlier reports. Unresolved questions arise however, as to whether Ilp7 projections onto the EJ are due to rescued non-serotonergic dorsal or FS-Ilp7 neurons or if lack of fruM expression causes aberrant arborization of existing post-embryonic Ilp7 neurons in males (discussed in Future Directions, Section 4.9).    59 4.6 A Novel Failsafe Mechanism for tra, but not dsx, in Building the Female Nervous System Our genetic manipulation studies show that Tra can override FruMC-dependent neuronal cell death in both males and females (Fig 4.1). This provides an important new perspective for understanding the development of the female nervous system, and for studies in which forced male-specific splicing of fru is used to test the sufficiency of fruM in females. There has been no evidence that tra acts independently of fru or dsx splicing in the nervous system. Further, no modifier of FruM activity has been identified in females. For these reasons, FruM is assumed to carry out its full masculinizing function when expressed in females [80, 94]. However, this contrasts with numerous reports indicating that fruM expression in females is insufficient for full masculinization [59, 80, 92]. For example, males require fruM for the enlargement of numerous brain regions relative to females, but these are only partially enlarged in fruM mutant females. In contrast, these regions are enlarged in tra mutant females to match males [89]. Also, fruM females do not exhibit the full fruM-dependent behavioral repertoire of males [59, 80, 92]. In contrast, tra mutant females have near full behavioral masculinization in all these behaviours [59, 92, 100, 101]. In both cases, tra mutant females more closely resemble a fully masculinized phenotype than do fruM females. It has been proposed that the other arm of the sex determination cascade, regulated by dsx, accounts for this insufficiency observed in fruM mutant females [59, 92]. However, the combined effect of FruM and DsxM expression to account for fruM insufficiency in full masculinization of the female has yet to be directly tested; instead, only tra mutant females have been tested under the widely-held assumption that tra acts only via fru/dsx splicing in the nervous system [59, 80, 92]. In this light, our demonstration of a previously unappreciated function for Tra in blockade of ectopic FruM activity in certain cellular contexts,   60 offers an important and novel perspective for studies testing fruM, and perhaps even dsxM activity, in females.  What function might such a failsafe role for Tra play? Possibly, it may serve as a safeguard against incomplete splicing of fru (or perhaps dsx) sex-specific transcripts; indeed, RNA-sequencing has shown that fruM transcripts are in fact generated in wildtype females at a low level, and it is also possible that this may be exacerbated in stressful environments such as high temperature or hypoxia [45, 96, 102-105]. Interestingly, a related failsafe mechanism in the sex determination pathway has been reported in males, whereby mir-124 targets tra transcripts for degradation in the male nervous system to ensure the elimination of Tra in males [106]. Tra has been shown to act entirely independently of fru/dsx to promote female-specific properties of tissues outside of the CNS [79, 107, 108]. In the fat body, tra is necessary for the non-cell autonomous increase in growth and body size of females relative to males, in a mechanism that is insensitive to dsx and fru [108]. In intestinal stem cells, that do not express dsx or fru, tra acts to enhance cellular proliferation to expand tissue size [79]. These studies are beginning to provide evidence for non-canonical roles for Tra. Interestingly, these reports show that Tra acts independently of dsx and fru outside of the CNS, whereas in this report we show that Tra plays a double assurance role to block FruM splicing and also FruM function in the CNS. Our results support an emerging literature that Tra and also Sxl direct certain sexually dimorphic properties outside of a strictly linear sex determination cascade that uses fru and dsx as sole effectors [79, 107, 108]. Thus, our findings add to an expanding literature regarding a more expansive role for Tra than previously postulated.     61 4.7 Biological Relevance By using Drosophila as a model for studying sexually dimorphic development of the brain, we are able to study how the sex determination cascade instructs individual neuronal cells to be either ‘male-like’ or ‘female-like’ based on sex chromosome inheritance, and investigate cellular mechanisms that give rise to sexual dimorphisms. In my thesis, we show that the sex determination cascade, is responsible for sex-specific differences in the generation of post-embryonic Ilp7 neurons. We also demonstrate that PCD is a mechanism that prevents female-specific neurons from forming in the male nervous system. Studying cell autonomous sex differences in individual neuronal cells of the mammal brain is challenging because it is difficult to remove the confounding effect of hormones and environment. However, it has been proposed that the inheritance of sex chromosomes, the first dimorphic signal, may play a greater role than widely appreciated to date, not just in the development of reproductive organs but also in sex differences in the brain (see Section 1.3).  Similar to Drosophila, reports of increased neuronal numbers in males have been over represented in mammalian studies, where several neuronal populations are either male-specific or male-biased in terms of function. However, female-biased neuronal populations that are associated with maternal care and physiology have been reported (reviewed in [109, 110]). Sex-biased dimorphisms in males have long been attributed to male-specific hormones, but recently, an active role for female-specific hormones has been acknowledged [63]. In Section 4.7.1, I highlight similarities of some cellular mechanisms leading to sex differences in neuronal number. In order to develop effective treatments diseases that exhibit sex differences, it is critical to understand sex determination and the genetic and cellular mechanisms that generate sexual dimorphisms.   62  4.7.1 Cellular Mechanisms that Generate Dimorphic Neuronal Number are Conserved Sex-specific PCD and proliferation are conserved cellular mechanisms amongst many species including flies and mammals (reviewed in [109] and [110]). In this thesis, we demonstrated that the male counterparts of FS-Ilp7 neurons undergo male-specific PCD, but a female-specific factor, Tra, is also actively required to ensure their survival. Testosterone has been shown to both add neurons in males, but also remove female-biased neurons in male brain. The anteroventral periventricular nucleus (AVPV) of the mouse and rat hypothalamus are prime examples of female-biased dimorphism; this neuronal population is larger in females than in males and exhibits sexual dimorphisms that are linked to sex-specific behaviours. Some examples of female-specific behaviours associated with this region include gonadotropin release for ovulation and tyrosine-hydroxylase expression for maternal care. Testosterone-dependent PCD of neurons in AVPV in males underlies their female-specific generation, which are necessary for the estrous cycle. In addition to this, ovariectomy also reduces the volume of the AVPV, suggesting that ovarian hormones play an active role in generating these female-biased neurons in the AVPV [63]. This example parallels our conclusions about how FS-Ilp7 neurons are generated by male-specific PCD due to a male-specific factor, but that an underappreciated female-specific factor also ensures their survival in females.   4.8 Considerations and Potential Limitations of this Study Finding that male-specific PCD of FS-Ilp7 neurons underlies the female-specific generation of these neurons is an important finding. However, we must keep in mind possible misinterpretations or limitations of our study.    63 One of the considerations that was made during this work was the function of the fruM allele in females. We found that forcing male-specific splicing of fru in females was insufficient to elicit PCD of FS-Ilp7 neurons. Since Dickson et al (2005) concluded that FruM is expressed in females with the fruM allele, we assumed that FruM is expressed in FS-Ilp7 neurons in these mutant females. However, this was untested, because we did not have access to FruM antibody.  Second, FruM has been shown to repress transcription of several target genes [53, 111], but we did not check if FruM downregulates transcription of Ilp7 peptide in males. This could explain why we cannot observe FS-Ilp7 neurons in males, because Ilp7 peptide is downregulated. However, we are confident that FS-Ilp7 neurons are absent in males as evidenced by our p35 data, where FS-Ilp7 neurons are rescued from PCD in males.  Third, we considered that male-specific PCD may be a non-cell autonomous process. It is possible that fruP1>p35 expression in males leads to the survival of another neuronal population that is necessary for FS-Ilp7 neuronal survival.  Fourth, we considered that p35 may have another role, aside from directly preventing PCD. It has been shown that caspases have apoptotic and non-apoptotic roles in Drosophila. It has been demonstrated that p35 can cause normal neuronal arborization, leading to proper targets, and subsequent survival (reviewed in [53]). Although far-reaching, it is possible that p35 inactivates caspases that are important for arborization of FS-Ilp7 neurons in males such that they do not undergo PCD.  Lastly, we considered why knockdown of RHG and their hemizygous mutants do not rescue FS-Ilp7 neurons in males. To our knowledge, there is no PCD pathway that occurs independently of these pro-apoptotic proteins in the nervous system. These results could be explained by compensation by the RHG genes.    64 We propose that the last three considerations could be tested by introducing homozygous deletions of the RHG gene locus clonally into post-embryonic Ilp7 neurons in an otherwise heterozygous genetic background. This method would test cell autonomy, apoptotic versus non-apoptotic mechanism, and whether the RHG genes are necessary for FS-Ilp7 PCD in males (discussed in Section 4.9).  4.9 Future Directions We have determined that FS-Ilp7 neurons undergo FruM-dependent PCD in males, but we have not characterized: when and where these neurons die in males, whether PCD is cell autonomous or not, how FruM initiates PCD, or determined the nature of Tra’s non-splicing role in the survival of FS-Ilp7 neurons.   4.9.1 Immediate Goals We are actively trying to determine the time of death of FS-Ilp7 neurons in males, and further understand how PCD is initiated. To determine the stage of PCD, we are attempting to detect evidence of caspase activation in nascent FS-Ilp7 neurons in males. We are using an antibody that was raised against a human caspase (anti-cleaved Caspase 3), but recognizes cleaved Dronc and Drice (an activated initiator and executioner caspase, respectively) in Drosophila (reviewed in [112]). The challenging aspect of this is to properly identify FS-Ilp7 neurons prior to Ilp7 peptide expression. However, a possibility is that FS-Ilp7 neuronal death in males may be coincident with Ilp7 expression, which we are currently investigating. To that end, we are also trying to identify the NB origins of FS-Ilp7 neurons to obtain more markers for their   65 earlier detection. Since we have some data to suggest FS-Ilp7 neurons may come from NB 5-3, we will look at markers associated with its NB lineage.  In this thesis, we have shown that PCD eliminates FS-Ilp7 neurons in males, however this has only been supported by blocking caspase activity in fruP1 expressing neurons. We intend on determining whether the RHG genes are necessary for PCD by introducing homozygous mutations of Df(3L)H99 into an otherwise heterozygous background with MARCM. With this MARCM technique, we would also be able to determine where undead neurons project to on the male reproductive organs. By labelling the death event in FS-Ilp7 neurons, and confirming the role of pro-apoptotic genes in this process, we will be more confident in our conclusion that FS-Ilp7 neurons undergo cell-autonomous PCD in males.  4.9.2 Long Term Goals In this thesis we have not considered how FruM activates PCD of FS-Ilp7 neurons in males or how Tra prevents PCD independent of fru splicing. If our analysis of homozygous Df(3L)H99 mutant clonal analysis with MARCM demonstrates that the RHG locus is necessary for PCD, we could consider removing small elements of the RHG locus, and potentially identify a new enhancer piece that is necessary for male-specific PCD or otherwise. Furthermore, we could investigate whether FruMC directly bind to a necessary element to directly initiate PCD. Another unanswered question of this thesis is how Tra acts as a fail-safe to ensure FS-Ilp7 neuronal survival in females. In addition to female-specific splicing of fru, Tra could prevent transcription or translation (physically blocking ribosome binding/movement or increasing mRNA instability) of fru. Otherwise Tra could have another target that ensure survival of FS-Ilp7 neurons. To determine if Tra prevents translation, we could use antibodies for   66 FruM in fruM females with ectopic Tra expression. In this genetic background, we could also do an electrophoretic mobility shift assay (EMSA) to determine if Tra associates with fruM DNA (to prevent transcription) or RNA (to prevent translation). In order to determine whether Tra has another target that prevents PCD, we could do a candidate screen. We have discovered that Ilp7 neurons project to the AGs in addition to the SVs. As discussed in Section 4.5, we hypothesize that dorsal Ilp7 neurons may be the same neurons as described in Tayer et al (2012). Considering this, we would revisit the role of Ilp7 neurons in full male fertility. First, we would confirm that CrzR is co-expressed in Ilp7 neurons. Unfortunately, the original CrzR-GAL4 line used for the above study was lost (personal communication Celine Chiu, Anderson Research Group). Despite this, we could still test the role of Ilp7 neurons in sperm and seminal fluid transfer along with copulation duration by testing their necessity by expressing the inward rectifying channel, UAS-kir2.1, and testing their sufficiency by expressing the neuronal activator, dTRPA1 in Ilp7 neurons. Further, we would quantify any defects in sperm (don julio::GFP) and seminal fluid (SP::GFP) transfer during mating under these conditions. These experiments could confirm whether Ilp7 neurons in males regulate copulation duration.    67 Chapter 5: Major Conclusions We have determined that male-specific PCD underlies the female-specific generation of a subset of post-embryonic Ilp7 neurons in Drosophila. Using FS-Ilp7 neurons as a model, we have shown for the first time that FruMC has a definitive role in male-specific PCD. We have determined that undead post-embryonic Ilp7 neurons in males do not have a masculine neuronal fate, despite expressing FruMB and FruMC, and that preventing FruM expression in males leads to ectopic Ilp7 neuronal projections on the EJ in males. We also have evidence to strongly suggest that in females, FruMC expression is sufficient to eliminate FS-Ilp7 neurons when Tra is absent. Our work suggests that Tra has a fail-safe role in keeping FS-Ilp7 neurons alive in females, but we have not elucidated how Tra prevents PCD independent of splicing. 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