Information processing in the nervous system relies on precise patterns of synaptic connections between neurons. The level of synaptic specificity within the nervous system is remarkable given its tremendous complexity. How neurons can distinguish their correct synaptic partners from many other neurons during circuit assembly remains a central question in neuroscience. Although important progress has been made on molecular mechanisms regulating neural circuit assembly, the cellular recognition mechanisms mediating synaptic specificity are still poorly understood. The Drosophila visual system is well suited to uncovering the molecular mechanisms underlying synaptic specificity, because of the availability of diverse genetic tools, cell type specific markers and electron microscopic reconstruction data. In the medulla neuropil of the Drosophila visual system, different neurons form synaptic connections in different layers. Within a layer, neurons form synapses with a unique set of multiple neuronal types, which represents only a subset of neurons with processes in that layer. In my thesis research, I sought to identify candidate cell recognition molecules underlying this specificity. I did RNA sequencing on closely related neurons with different layer-specific synaptic specificities, lamina neurons L1-L5 and photoreceptor R7 and R8, at the onset of synapse formation. I showed that each of the seven cell types expresses a unique set of hundreds of genes encoding cell surface and secreted proteins. Using these data and additional localization studies on proteins tagged through modification of the endogenous locus, I demonstrated that 21 paralogs of the Dpr family, a subclass of Immunoglobulin (Ig) domain containing cell surface proteins, are expressed in unique combinations in each of the seven cell types during synapse formation. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in subsets of synaptic partners for each of the seven cell types. Thus, I demonstrated that interacting Dprs and DIPs are expressed in synaptic partners during synapse formation in the Drosophila visual system. Furthermore, I generated null mutants of Dprs and DIPs via CRISPR-based methods, and showed that mutants of two DIPs and their cognate Dprs had similar phenotypes: neurons normally expressing these DIPs had reduced number of cells in corresponding DIP and cognate Dpr mutants. These data suggested that cognate Dprs and DIPs play important roles in the development of synaptic partners expressing them.

The stereotyped synaptic connections that define neural circuit function are established during development. Neuronal activity independent of environmental stimulus contributes to neural circuit assembly in vertebrates, but its precise role remains an open question, particularly at the level of defined cell types. By contrast, invertebrate neurodevelopment was thought to proceed in the absence of such developmental activity. Here I show that developmental activity accompanies synaptogenesis in the Drosophila brain. Using Drosophila genetics, epifluorescence microscopy, two-photon microscopy, immunohistochemistry, and visual behavior, I characterize this developmental activity, determine its role in synapse formation, and identify regulatory mechanisms at the cellular and molecular level. This patterned-stimulus independent neural activity occurs in a brain-wide fashion, and is characterized by stereotyped oscillations and cellular dynamics that reflect synaptic connectivity. Activity patterns instruct synapse development in a cell-type-specific manner. A transient and genetically specified population of neurons propagates this activity throughout the brain. Glia participate with complementary cycles of activity in a cell-type-specific fashion, and astrocytes are necessary for activity to occur. These data indicate that developmental activity is an evolutionarily conserved and fundamental component of neural circuit assembly that is coordinated by multiple molecular and cellular mechanisms.

During circuit assembly in the developing brain, neurons undergo dynamic expression of many neurotransmitter receptor (NR) subunits that are used to establish a series of postsynaptic domains. However, our understanding of the molecular logic that determines synapse composition between classes of inputs remains limited. The Drosophila central nervous system demonstrates particularly striking specificity of synapse types. Here, I report the use of new tools developed to study the localization of endogenously expressed NR subunits in sparsely distributed cells, thus offering a window into the process of synaptogenesis and the establishment of variously defined postsynaptic sites in the brain. I use a combination of genetic methods and expansion microscopy (ExM) to investigate NR localization in the fly brain at the scale of single synapses. In developmental studies, I also present evidence of temporally distinct dynamics of synapse formation that differ between NR type as well as cell type. Lastly, I explore methods to uncover mechanisms that regulate NR localization in these cells. This work describes newly advanced methods that can be leveraged to probe the molecular architecture of synapses in vivo and suggests a complex picture of how synapse formation is coordinated through differential, synapse-specific processes during development.