The genetic function of Drosophila heterochromatin has been debated since its earliest description by Heitz (1933). To examine the genetic composition of the proximal region of chromosome 2 of Drosophila melanogaster, the generation of proximal deficiencies by the detachment of compound second autosomes appeared to be a promising method. Compound second autosomes were detached by gamma radiation. A fraction of the detachment products were recessive lethals owing to proximal deficiencies. Analysis of these detachment products by inter se complementation, pseudo-dominance tests with proximal mutations and alleleism tests with known deficiencies, provided evidence for at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. These data in conjunction with cytological observations further demonstrate that rolled and light are located within the proximal heterochromatin of the second chromosome. To further this analysis, lethal alleles of the largest 2L and 2R proximal deficiencies were generated, employing, as a mutagen, ethyl methane sulphonate (EMS). Analysis of the 118 EMS induced recessive lethals and visible mutations recovered provided evidence for seven loci in the 2L heterochromatin and six loci in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these loci, one in 2L and two in 2R fall near the heterochromatic-euchromatic junction of 2L and 2R respectively. None of the 113 EMS lethals behaved as a deficiency, thereby confirming that, in Drosophila, the EMS mutagenesis method of Lewis and Bacher (1968) results in true "point" mutations. All of the heterochromatic loci uncovered in this study appear to be non-repetitive cistrons. Thus functional genetic loci are found in heterochromatin, albeit at very low density relative to euchromatin. / Science, Faculty of / Zoology, Department of / Graduate

Testes and paragonial glands of Drosophila melanogaster wild type males were labelled in vitro using ³⁵S-methionine, and the proteins synthesized were analysed by 2-dimensional gel electrophoresis (O'Farrell, 1975). Testes and paragonial glands were also labelled in vivo by feeding male or larvae on ³⁵S-labelled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. ³H-proline was also used to label testes, and no significant differences from the pattern obtained with ³⁵S-methionme were found. Different laboratory stocks were analyzed to examine the degree of genetic heterozygosity of testicular proteins. The variation between patterns was very low, facilitating subsequent studies in which flies of defined genetic constitution, but with different genetic backgrounds, were compared. Testes and paragonial glands from X/0 and X/Y/Y males were labelled in vitro with ³⁵S-methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Non-equilibrium pH gradient 2 dimensional gels (O'Farrell et al., 1977) were also run on testis proteins from X/0, X/Y and X/Y/Y males. These gels will resolve basic as well as acidic proteins and once again no differences attributable to the Y chromosome were seen. Pure sperm was manually dissected from in vivo labelled males and the proteins analyzed. Ninety-two proteins were detected, and all were synthesized in comparable amounts by X/0, X/Y and X/Y/Y males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational factors, or regulatory proteins only present in very small amounts in the adult testes. ³⁵S labelled males were also mated to unlabel led females, and the proteins of the transferred sperm were analyzed by 2DPAGE. The contributions of the testes and paragonial gland to the ejaculate were determined. Testes at various stages of development were also cultured in vitro in ³⁵S-methiomne containing media. A profile of the proteins synthesized during development revealed that the spectrum of proteins synthesized at different stages between third instar larvae and the imago were remarkably similar, despite the morphological changes taking place in the organ. Sperm proteins were localized on the patterns, and the quantitative changes occurring during this period were examined. The basic proteins of the testis were studied in an attempt to biochemically identify a Drosophila protamine. Sperm was isolated by dissection, and the acid soluble proteins were separated on a 15% modified Laemmli SDS gel. No unusually small basic protein was seen upon staining, but a protein was present which comigrated with trout histone H4. This suggests that D. melanogaster males may retain somatic histones in the nucleus during the condensation of the sperm head. Testes were labelled in vitro with ³H-arginine and the basic proteins were analyzed on a 15% modified Laemmli SDS gel. The gel was autoradiographed and a prominent doublet was seen at the front of the gel, suggesting that a small highly basic protein is synthesized in the testis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In order to study and compare spontaneous and radiation induced recombination frequencies of the euchromatic and heterochromatic regions of chromosome 2 Drosophila melanogaster females were exposed to 0, 2, 3, 4, or 5 krads of gamma radiation. Exchange was measured in the b-Bl, Bl-It, lt-rl, rl-pk, and pk-cn intervals. The lt-rl interval defines a wholly heterochromatic segment. Analysis of the recombination data demonstrates that spontaneous recombination occurs in the heterochromatic interval at a frequency of approximately 0.1% and the frequency of induced recombination is primarily increased in the heterochromatic interval. Moreover, the frequency of recombination in the heterochromatic interval is correlated with the dose of radiation. There are slight or no increases in the recombination frequencies of the euchromatic segments and the regions containing the heterochromatic-euchromatic boundaries show responses intermediate to the heterochromatic and euchromatic regions. Testing of the recombinant chromosomes indicates that 22% of them are associated with recessive lethals. The association is greater in eggs laid the first four days after radiation treatment than in thoselaid five-eleven days after the radiation treatment. It is postulated that induced recombination can occur via symmetrical as well as asymmetrical interchange. The interchromosomal effect of chromosome 3_, heterozygous for In(3LR)DcxF, on recombination in chromosome 1_ has been studied. The results show that its effect is not significant in the heterochromatic region. Thus, the alterations in the recombination frequencies owing to radiation treatment appear to be independent of those owing to the interchromosomal effect. Recombination was also measured in the presence of the heterochromatic deficiency - Df(2R)MS2¹º. The results indicate that the frequency of recombination is decreased in the chromosome arm containing the deficiency and in the heterochromatic interval of the left arm. The euchromatic regions of the opposite arm show a slight increase in recombination. A higher number of multiple crossover progeny are recovered than would be expected according to map distances in the presence of the heterochromatic deficiency, Df(2R)MS2¹º, the heterozygous inversion, In(3LR)DcxF, and for double crossovers involving the heterochromatic region, but only at high doses of radiation. / Science, Faculty of / Zoology, Department of / Graduate

Two types of sex-linked recessive mutations in Drosophila melanogaster have been investigated in the present study. The first type includes 5 different mutations which exhibit a stress-sensitive (ses) phenotype. Flies of all five mutant stocks become paralyzed when their containers are lightly tapped; wild type flies are unaffected by the same treatment. The five mutations form three complementation groups (cistrons). Flies mosaic for mutant and non-mutant tissue were studied to determine the foci of their action in embryos by fate mapping. These studies suggest that the mutation ses D² has 6 foci in the presumptive nervous system of the blastoderm. Each focus corresponds to a site in the thoracic ganglion which controls the movement of one leg. The focus for ses E¹ mutation is rather diffuse and occupies a larger area in the thoracic nervous system of the blastoderm fate map. Focus mapping studies with the ses B¹ mutation were inconclusive because of the highly variable expressions of the adult behavioural phenotype in mosaic individuals. Developmental studies, involving temperature shifts from 22°C to 29°C (permissive to restrictive temperatures) revealed that the ses E¹ mutation has 2 temperature-sensitive periods (TSPs) for lethality during its development, one in the late 2nd larval instar stage and the other in the late pupal stage. In addition to developmental TSPs of ses B² at the embryonic, 1st larval instar and pre-pupal stage, temperature-shift studies also revealed a ts maternal- effect lethal for ses B². The second type of mutation studied has a temperature-sensitive (ts) phenotype of adult death induced by shifting up to the restrictive temperature. The add Atsl flies have normal behaviour and longevity at 22°C but die within 24 hours after shift-up to 29°C. In contrast, the ses E¹ flies are less active and require 168 hours at 29°C to induce death. Both mutations studied have a 3rd larval instar-pupal TSP with add Atsl and ses E¹ also having an additional TSP at the embryonic and 1st larval instar-2nd larval instar stage respectively. Fate mapping studies suggest that the adult lethal phenotype of add Atsl is caused by a lesoon in tissues derived from the mesodermal cells of the blastoderm, and for ses E¹ the lesion is in the neural cells. / Science, Faculty of / Zoology, Department of / Graduate

Neuropeptides are central modulators of many functions including male-specific mating behaviours. Understanding how these chemical messengers modulate the neural substrates are still not well understood but remains important for biological research. In Drosophila melanogaster, two well-defined microcircuits (Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD)), are used to understand the underlying mechanisms of how neuropeptide interactions modulate temporal information in mating behaviours. In our study, we investigated the influence of SIFamide receptor-mediated signaling and its association to both LMD and SMD. We performed several RNAi-based screens where we identified and mapped out seven different types of neuropeptidergic neurons which were found to be important to either LMD and/or SMD. Following this analysis, we highlight three independent signaling pathways which are necessary to describe the cellular mechanics of the neuropeptides involved. Firstly, we infer that synaptic contacts between proctolin and SIFamide neurons in the subesophageal ganglion mediate inhibition in SMD whereas proctoclin as a neuropeptide modulates both LMD and SMD in a non-synaptic manner. Secondly, we describe an existing insulin-related microcircuit that is modulated by the inputs of Dimmed (DIMM), a transcription factor, through adipokinetic hormone, allatostatin A, and leucokinin to exhibit SMD. Thirdly and lastly, we discuss our interpretations of how capability neurons in the central brain resolves a potential disinhibition microcircuit in LMD via olfactory based signaling in the antennae lobe. In summary, our results contribute to establishing a model system to study neuropeptidergic microcircuits in complex mating behaviours.

Internal states, such as arousal and hunger, elevate the probability of a set of behaviors and persist on longer timescales than the behaviors that they predict. These states are triggered by sensors (e.g. neurotransmitters, biogenic amines) within the animal that detect internal homeostatic conditions and external factors. However, the sustained nature of internal states and the diversity of behaviors associated with a singular state suggest that state is represented not only by hormonal and modulatory signals but also by the coordinated activity of neurons within the central brain. Additionally, recent evidence suggests that internal states are represented throughout cortex in rodents and in many neuropil regions in Drosophila. In this thesis, I suggest how persistent states are represented globally in the brain by observing the activity of neurons, at the single-neuron level, distributed throughout the brain of Drosophila melanogaster and determining on what timescales their neural activity predicts behavior. To do this, we first establish a strategy to rapidly capture brain-wide activity of an awake, freely behaving Drosophila adult. We employ Swept Confocally Aligned Planar Excitation (SCAPE) microscopy, which has been shown to be an effective tool for volumetric imaging in a wide range of living samples, including zebrafish and Drosophila larvae. SCAPE's volumetric imaging speeds exceed those of point-scanning methods ten- to hundred-fold, and offers additional advantages, such as reduced phototoxicity and high signal-to-noise. The optical geometry of SCAPE consists of a single objective located directly above the sample. Therefore, this single stationary objective lens allows for imaging of intact, behaving animals like adult flies. Here, we characterize the spatial resolution of the system with respect to in vivo imaging of neurons in the adult fly brain. We show that we can achieve single-cell resolution, even in closely-spaced or dense neuronal populations. Additionally, we show that high-speed imaging of calcium activity throughout the whole brain can be performed at 20 fly brain volumes per second. These rates allow us to monitor neural dynamics occurring on the time scale of hundreds of milliseconds, which lets us capture the dynamics of popular calcium indicators like GCaMP. Moreover, we have demonstrated the feasibility of this approach to optically record odor responses of individual neurons in the olfactory circuit, while the animal freely behaves on a spherical treadmill. Having established a system for whole-brain imaging in Drosophila, we then use this methodology to explore the representation of two internal states: arousal, in flies freely running on a spherical treadmill, and hunger, in food-deprived flies consuming sugar. We define internal state as neural activity that predicts behavior on long timescales. To determine the timescale with which individual neurons best predict behavior, we define a regression model in which the activity of each cell is proportional to behavior filtered with unique time constant (tau_i). In freely running flies, we see that the neural activity exhibits a strikingly large dominant mode - nearly all cells across the brain are correlated with locomotion. While the median timescale is short, the distribution of timescales across all cells is broad, with some neurons correlated with locomotion on a much longer timescale, representing arousal based on our definition. In food-deprived flies fed sugar, no dominant mode exists; the neural activity tracking feeding is relatively subtle at the global scale. However, by applying the regression model to determine the timescales of individual cells, we do identify some ensembles of neurons possessing either a short timescale (tau_i < 10s), likely representing reward, or a long timescale (tau_i > 60s), putatively representing hunger. To investigate the populations that make up these different timescales, we used both genetic labeling and hierarchical clustering to determine the identity of neurons of interest. For example, in the freely running flies, we notice that cells in a dorsomedial region called the pars intercerebralis exhibit consistently large tau_i with respect to locomotion. Similarly, by genetically labeling neurons producing the hormone DH44, we see that in food-deprived flies consuming sugar, these neurons exhibit large tau_i with respect to feeding. Thus, we have identified dimensions of global dynamics, including a broadly distributed behavioral state as well as subspaces supporting putative neural correlates of the internal states of arousal and hunger. These data presented in this thesis, and the techniques we have established, have the potential to significantly impact our understanding of internal states at a global level in Drosophila melanogaster and can be extended to other organisms.

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