Browsing by Author "Volkan, Pelin C"

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

In both insects and mammals, odor detection depends heavily on diverse classes of olfactory neurons that organize their axons to converge in a class-specific manner within the central brain’s olfactory bulb, or antennal lobe in flies. The olfactory sensory circuit is characterized by its unique and essential feature—a functionally organized topographic map. This map relies on the convergence of axons from dispersed olfactory sensory neurons of the same type into specific regions known as class-specific glomeruli. Exploring how the identity of neurons shapes this circuit organization is a central pursuit in neurobiology, given its significant implications for neurodegenerative diseases and neuronal dysfunction.In the olfactory system, various cell surface proteins, such as Robo/Slit and Toll receptors, govern numerous aspects of circuit organization, including axon guidance and synaptic matching. In our study, we have identified an atypical cadherin protein called Fat2 (also known as Kugelei) as a regulator of axon organization specific to neuronal classes. Fat2 is expressed in olfactory receptor neurons (ORNs) and local interneurons (LNs) within olfactory circuits, with minimal expression in projection neurons (PNs). Notably, Fat2 expression levels vary depending on neuronal class and peak during pupal development. In cases of fat2 gene mutations, we observed varying degrees of phenotypic presentations in ORN axon terminals belonging to different classes, with a notable trend toward more severe effects in classes with higher Fat2 expression. In the most extreme cases, fat2 mutations resulted in ORN degeneration. Our findings suggest that the intracellular domain of Fat2 is crucial for its role in organizing ORN axons. Specifically, during early stages of olfactory circuit development, Fat2 plays a pivotal role in coordinating axons precisely, facilitating the formation of class-specific glomerular structures. Importantly, our research indicates that the expression of fat2 by PNs and LNs does not significantly contribute to ORN organization. Finally, we have identified potential interactors of the Fat2 intracellular domain, namely APC family proteins (Adenomatous polyposis coli) and dop (Drop out), which likely coordinate cytoskeletal remodeling essential for axon retraction during protoglomerular development. In summary, our study establishes a foundational understanding of Fat2's role in organizing the olfactory circuit and underscores the critical importance of axon behavior in the maturation of glomeruli.

Sensory neuron diversity is a common theme in the animal kingdom. It provides the cellular infrastructure that supports the accurate perception of the external world. Among all sensory systems, the olfactory system demonstrates an extreme in the extraordinarily diversified neuronal classes it holds. The system-wide cellular diversity is in sharp contrast with the individual specialization of olfactory receptor neurons (ORNs) per se. How the nervous system, particularly the olfactory system, uses limited genetic information to generate a huge variety of neurons with distinct properties remains elusive.

The adult Drosophila olfactory system is an excellent model to address this question due to its conserved organizational principles and reduced complexity. The fly olfactory appendages contain 50 ORN classes, each of which expresses a single receptor gene from a family of ~80 genes. Stereotyped clusters of 1-4 ORN classes define about 20 sensilla subtypes, belonging to 3 major morphological types. All cellular components within a sensillum are born by a single sensory organ precursor (SOP) via asymmetric divisions. The molecular mechanisms that determine SOP differentiation potentials to develop into distinct sensilla subtypes and the associated ORN classes are unknown.

From a genetic screen, we identified two mutant alleles in the rotund (rn) gene locus, which has a critical function in diversifying ORN classes. Rn is required in a subset of SOPs to confer novel sensilla subtype differentiation potentials from otherwise default ones within each sensilla type lineage. In rn mutants, ORNs in rn-positive sensilla subtypes are converted to lineage-specific default rn-negative fates, resulting in only half of the normal ORN diversity. This work is described in Chapter 2.

Based on an unbiased time-course transcriptome analysis, we discovered two critical downstream targets of Rn, Bric-à-brac (Bab) and Bar. In light of the knowledge about leg development, we found these genes, along with Apterous (Ap) and Dachshund (Dac), are part of the conserved proximal-distal (PD) gene network that play a crucial role in patterning the antennal precursor field prior to proneural gene-mediated SOP selection. Interactions between these PD genes under the influence of morphogen gradients separate the developing antennal disc into 7 concentric domains. Each ring is represented by a unique combination of the aforementioned transcription factors, coding the differentiation potentials for a limited number of sensilla subtypes. Genetic perturbations of the network lead to predictable changes in the ratios of different sensilla subtypes and corresponding ORN classes. In addition, using CRISPR/Cas9 technology, we were able to add tags to specific rn isoforms in the endogenous locus, and show positive regulation of Bab and negative regulation of Bar by the direct binding of Rn to the promoters in vivo. This work is presented in Chapter 3.

We proposed a three-step mechanism to explain ORN diversification, starting from pre-patterning of the precursor field by PD genes, followed by SOP selection by proneural genes, and ended with Notch-mediated neurogenesis. The final outcomes are greatly determined by the pre-patterning phase, which may be modified during evolution to compensate special olfactory needs by individual species. In our model, each step serves a single purpose, which displays context-dependent functions. By changing contexts, reassembly of the same logical steps may guide neuronal diversification in parallel systems with completely different identities. This step-wise mechanism seems to be a common strategy that is used by many other systems to generate neuronal diversity.

The human brain contains over 80 billion neurons that make approximately 100 trillion specific connections. Each neuron must acquire a specific identity that includes its, gene expression, morphology, connectivity, and location. The basic logic and mechanisms that are used to coordinate this process on the immense scale of the brain remain largely unknown. We use the Drosophila olfactory system as model to understand nervous system development because of the powerful genetic tools and the workable level of neuronal diversity, which contains 50 classes of olfactory receptor neurons (ORNs). Each class of neurons is defined by the exclusive expression of typically a single olfactory receptor and connect to 50 class-specific glomeruli in the antennal lobe of the brain. Here we demonstrate that a cross regulatory network of transcription factors patterns the antennal disc, which contains ORN precursors and will develop into the antenna. These transcription factors create seven rings that are each labeled by a unique combination of genes, which generate distinct sets of ORN fates. Manipulation of this network changes the ORN fates that are produced in the adult, thereby demonstrating its necessity for generating neuronal diversity. We next show that the DIP/Dpr family of proteins, which are members of the Ig superfamily of genes and heterophilically interact, is required for axon sorting among classes of ORNs to create 50 class specific glomeruli. The members of this family are expressed in a combinatorial code in ORNs at times that correlate to glomerular formation. Computational analysis of DIP/Dpr expression patterns groups ORN classes into clusters that mimics their relative glomerular positioning in the antennal lobe. Class-specific or combinatorial knock down of DIP and dpr genes causes localized axon sorting defects where effected class invade neighboring glomeruli based upon the similarity of the DIP/Dpr expression codes. Our results highlight two functionally conserved strategies for generating and wiring a diverse neural circuit: prepatterning of precursors through combinatorial transcription factor expression, and the generation of differential adhesion force between classes of axons through combinatorial interactions of cell surface molecules. Not only are these strategies conserved in mammals, many of the genes that govern these processes are conserved as well, with genes like Bar, ap, and dac all having mammalian orthologues, and DIPs and Dprs sharing homology with Kirrel proteins. These studies have advanced our knowledge of the underlying logic that governs how a diverse nervous system is generated and coordinately wired.

How species adapt to their local environments over the course of evolution remains an important question that underlies much of modern biology. Though much has been learned, many questions remain unanswered on the interplay between evolution and development, particularly as to the constraints and limitations that one places upon the other. This thesis is an attempt to address a few of those questions as they pertain to the olfactory system of the common laboratory fruit fly genus Drosophila.

To increase detection of a complex chemical environment, vertebrates and insects express an extraordinary number of distinct olfactory receptor neuron (ORN) classes, each functionally specialized to detect a set of odorants. This is achieved as the olfactory system develops and each of these ORN classes makes developmental decisions defining the olfactory receptor genes they will express and their class-specific connections in the brain. In addition to this high level of ORN diversity, olfactory systems are also very dynamic evolutionarily, with both the number and functionality of olfactory receptor genes as well as the requirement for certain ORN circuits being under ecological constraints. In the first chapter, I discuss molecular and developmental strategies underlying both ORN diversity and evolutionary plasticity as well as present the insect olfactory system as a model for evo-devo research in light of recent findings.

I continue by examining patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species. Organisms have evolved strikingly parallel phenotypes in response to similar selection pressures suggesting that there may be shared constraints limiting the possible evolutionary trajectories. An example of this is seen in the behavioral adaptation of specialist Drosophila species to specific host plants, which show parallel changes in their adult olfactory neuroanatomy. I investigated the genetic basis of these parallel changes by comparing gene expression during the development of the olfactory system of these two specialist Drosophila species to that of four other generalist species. Strikingly, the parallelism observed in neuroanatomy extends to developmental programs; the patterns of expression of the transcription factors specifying olfactory receptor neuron (ORN) fates show broad convergence in species that are ecological specialists. These changes seem to result in convergent expression in only a small number of olfactory receptors in the specialist species. When compared across all six Drosophila species, a subset of olfactory receptor genes is disproportionately variable relative to other olfactory receptor neuronal lineages. A similar pattern is seen with a subset of transcription factors governing ORN development, which also show convergent expression in specialist species. These patterns suggest that a non-random component of the developmental program giving rise to the Drosophila olfactory system harbors a disproportionate amount of interspecies variation. My results suggest that the developmental parallelisms in specialist species and the non-random patterns of variation across species are consistent with the hypothesis that there are a limited number of flexible compartments during development, and that differences in the flexibility of these components shapes how developmental traits evolve.

Next, I narrow my focus to examine divergent behavioral responses towards a specific odorant, carbon dioxide (CO2), in the context of the evolution and development of Drosophila species. Carbon dioxide is an important environmental cue for many insects, regulating many behaviors including some that have direct human impacts. To further improve our understanding of how the system may vary among closely related insect species, we examined both the behavioral response to CO2 as well as the transcriptional profile of the CO2 system across the Drosophila genus. We found that CO2 generally evokes repulsive behavior across most of the Drosophilids we examined, but this behavior has been lost or reduced in several species. Comparisons of transcriptional profiles from the developing and adult antennae for a number of these species suggest that these behavioral differences may be due to differences in the expression of the CO2 co-receptor Gr63a. Furthermore, these differences in Gr63a expression are correlated with changes in the expression of a few genes known to be involved in the development of the CO2 circuit, namely dac, an important developmental transcription factor, and mip120, a member of the MMB/dREAM epigenetic regulatory complex. In contrast, most of the other known structural and developmental components of the peripheral Drosophila CO2 olfactory system seem to be fairly well conserved across all examined lineages. These findings suggest that certain components of the CO2 system may be more developmentally and evolutionarily labile, and may provide potential targets for direct manipulation of the system in the future.

The Drosophila olfactory system provides an excellent model for studying how complex neuronal circuits are assembled. In Drosophila melanogaster, each olfactory receptor neuron (ORN) class typically expresses a unique olfactory receptor (OR) gene, and synapses with their target projection neurons (PNs) within each class-specific and uniquely positioned glomerulus in the antennal lobe. Understanding how ORN axon terminals and PN dendrites are organized into these defined structural compartments is fundamentally important. It can bring us insights into common principles on how highly diverse neuronal populations are genetically controlled to form hardwired circuits, which might also be shared over evolutionary history. Over the past decades, many critical molecular players have been uncovered to control distinct steps of ORN and PN wiring. Among these, multi-member gene family encoding cell surface proteins are of interest due to their features of forming heterophilic interaction networks and cell-type-specific expression as cell surface codes, with the potential to mediate wiring specificity. Compared to the well-studied DIP-Dpr families in diverse neuronal contexts, the expression patterns and functions of another multi-protein interactome, Beat-Side, remain elusive in assembling the Drosophila olfactory circuits. Here, I thoroughly analyzed the expression pattern of the beat/side gene family in ORNs and PNs by leveraging the publicly available single-cell RNA-seq datasets and generating gene trap transgenic driver lines to faithfully probe the spatial expression pattern in vivo. My results reveal that each ORN and PN class expresses a specific combination of beat/side genes, which appears to be regulated by lineage-intrinsic mechanisms. Though ORNs or PNs from closer lineage tend to possess more similar beat/side profiles, I did find many examples of divergence among closely related ORNs and closely related PNs. This raises an interesting possibility that Beat/Side combinatorial expression might introduce variability on the cell surface that assists in the distinct glomerular targeting of these ORN/PN classes. To explore whether the class-specific combination of beats/sides defines ORN-PN matching specificity, I perturbed presynaptically expressing beat-IIa and postsynaptically expressing side-IV in two ORN-PN partners. However, disruption of Beat-IIa-Side-IV interaction did not produce any significant mistargeting in these two examined glomeruli, pointing to the redundancy and complexity of this multi-member cell surface interactome. Nonetheless, this comprehensive analysis of expression patterns lays a foundation for in-depth functional investigations into how Beats/Sides contribute to ORN-PN circuit formation. Additionally, this transgenic driver line collection offers a valuable resource for examining the beat/side expression and roles outside the olfactory system. This study is presented in Chapter 2. When I studied the functions of beats/sides by a targeted genetic screen using an RNAi collection, I also found an unexpected recurrent phenotype, which turned out to be RNAi-independent but linked to the transgenic docking site where UAS-RNAi transgenes are inserted. This landing site is attP40, which is widely used for the bacteriophage integrase-directed insertion of various transgenic constructs into this specific genomic locus by the Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. However, I found that the homozygous attP40 chromosome disrupts the normal glomerular organization of Or47b ORN class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, and Or47b-GAL4/attP40 transheterozygotes yield a similar glomerular defect. I also found a short chromosome region near the attP40 site, covering only five genes, which likely contains the causal genetic lesion accounting for the attP40-associated glomerular phenotypes. Though the exact causal genes remain undetermined, my findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects, particularly in neuronal contexts. Moreover, the identification of this short chromosomal region provides a promising shortlist of candidate genes for future functional investigation to establish the causal link, which may also bring new biology into how ORNs are organized into morphologically specified glomeruli. This story is presented in Chapter 3.

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to

generate defined motor outputs, or behavior. This integration requires developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown.

Here we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless. During development, the putative chromatin modulator Alhambra (Alh) represses the expression of both fru and of specific olfactory receptors, helping to coordinate and establish both the sensory and circuit identities of the ORNs involved in sex-specific behaviors. In contrast, the maintenance of fru expression and thus the identities of

these ORNs in adults utilize signaling from olfactory receptors through Cam Kinases and the histone acetyl transferase p300/CBP. Our results highlight feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs.These mechanisms might underlie innate and adaptable aspects of odor-guided sex- specific behaviors.

The sense of taste enables animal survival and reproduction by allowing them to detect and discriminate different chemosensory stimuli so as to select for food options that are suitable for ingestion for both themselves and their progeny. A dominant model in the eld suggests that animals' taste coding generally follows a relatively simple and clean scheme - the "labeled-line" model - such that individual taste neurons are predetermined to detect one specific category of tastants (e.g., sweetness) and drive predetermined category-specific behaviors (e.g., acceptance). However, results from several recent studies started to challenge this model, and thus the question of how taste information is processed to drive behaviors remains unsolved. Here, I used the Drosophila melanogaster sweet taste system as a model to address this question. By utilizing multiple approaches of genetic manipulation and neural activity recording, I discovered three unexpected features of the taste system at the molecular, cellular, and circuit levels. First, sweet neurons can sense two categories of taste - sweetness and sourness. Second, the sensitivity of sweet neurons is actively dampened by specific molecules. Third, sweet neurons are composed of at least two functionally distinct subgroups that allow for behavioral responses to sweet taste to be adjusted according to context. Together, this study identifies previously unknown mechanisms by which the Drosophila taste system decodes the identities and the intensities of stimuli and promotes proper behaviors towards them.

Animal behaviors consists of both innate and plastic components. Hardwired neural circuits are established during development and confer animals the innate potential to solve the survival challenges. However, in an ever-changing environment, animals need to modify their behaviors constantly and accurately to increase their fitness. Sensory experience modulates animal behaviors via changes in gene expression and circuits neurophysiology. Olfaction, one of the most ancient modalities, regulates feeding and social behaviors such as courtship. It is intriguing how olfactory experience modifies animal behaviors. To study this question, we use the olfaction-driven male courtship in the fruit fly Drosophila melanogaster, which consists of stereotypical and quantifiable rituals wired by a well-defined, sexually dimorphic circuits. We found that social experience, conveyed by olfactory detection of fly body pheromone, converges with internal hormone signaling, exerts chromatin-based regulation on the important behavioral regulator, fruitlessM. fruM is a transcription factor that regulates male-specific behaviors in flies, and it is both necessary and sufficient for male courtship behaviors. We found that juvenile hormone signaling via its receptors Methoprene-tolerant (Met) and Germ-cell expressed (Gce), works coincidentally with calcium signaling and the histone acetyl transferase p300 downstream of Or47b pheromone signaling, to exert transcriptional regulation on fruM, and subsequently modifies neurophysiology and behaviors. As result, group-reared males gain courtship advantage in Or47b- and fruM- dependent manner. Next, we investigated if Or47b is involved in other aspects of behavioral plasticity. fruM mutant males do not court other flies when held isolation, but they acquire courtship learning with grouping experience in olfaction-dependent manner. We found that Or47b is involved in male-female courtship learning and male-male chaining learning in fruM mutant males. Our work elucidated the roles of Or47b in regulating courtship behavioral plasticity in males reared in different social environments. Pheromone signaling via Or47b enhances courtship success in group-reared males with or without the innate fruM -dependent programs. Our findings validated olfaction-driven male courtship behavior as a good model in studying sensory experience dependent behavioral plasticity, and lays ground for future studies on pheromone-driven modifications on genes and circuits driving male courtship.