Title: Expression and Functional Analyses of the Entire Cadherin Gene Family in C. elegans
Description: Neurobiologists have sought an overarching logic of circuit assembly for decades. Canonically, piecemeal approaches have led to the discovery of many genetic pathways underlying discrete steps in nervous system development. These findings have cumulatively helped us understand how neurons extend axons, form neighborhoods, and choose synaptic partners to ultimately build sophisticated circuits. Today, advances in connectomics and transcriptomics have placed us in an exciting position to begin to tackle this systemically. This entails not only studying entire circuits and nervous systems, but also entire gene families which coordinate circuit assembly in space and time. The nematode C. elegans provides us with an opportunity to study circuit assembly on both genome-wide and nervous system-wide levels. In the past, C. elegans connectomics has relied heavily on the first wiring diagrams which were established in the 1980s. There is a growing need to scale this approach and study nervous systems across development, in different genetic backgrounds, and in various environmental paradigms.

In this work, we first establish transgenic and in silico tools to facilitate interrogation of a previously understudied region of the C. elegans nervous system, the largest neuropil called the “nerve ring”. Our tools – WormPsyQi and AxoPAL - help study synapses and neuronal adjacencies in a precise and high-throughput manner, therefore overcoming constraints on sample size and phenotypic space. Next, we focus on the cadherin superfamily of cell adhesion molecules (CAMs) and its implications on nervous system structure and function. Across evolution, two families of CAMs have expanded significantly with increasing nervous system complexity: cadherins and immunoglobulins (IgSFs). While many studies have described the expression and function of IgSFs, many cadherins are relatively under-studied in most neuronal contexts. Here, we present an expression atlas of all cadherins encoded by the C. elegans genome. Expression patterns are described with neuron-type spatial resolution and across larval development to define the richness and diversity of the cadherin repertoire in an entire nervous system, which has never been previously done for any model organism.

Our analysis reveals interesting temporal changes and a striking dichotomy between broad- and sparse-expressing cadherins. Some of the most well-conserved cadherin subfamilies - classical cadherin, calsyntenin, fat, and flamingo - are expressed in all neuron types in C. elegans. Furthermore, when analyzed in the context of the well-established C. elegans connectome, the expression atlas unfolds a putative molecular code underlying connectivity and selective adjacency. Altogether, by studying the expression of the entire cadherin family in neuronal and non-neuronal cell types, across several stages of development, this thesis highlights previously unknown salient themes of cadherin expression patterns which likely have functional implications. In addition to characterizing expression, we generated a collection of null mutants for all C. elegans cadherins, and proceeded to characterize them. To our surprise, most single mutants are viable and show minimal obvious phenotypes; we think this will favor studying neuronal functions of these genes since early lethality in other systems has often been a limitation.

We also found that the C. elegans Fat cadherin homolog, cdh-4, has several structural and behavioral phenotypes. Studying neuronal structure defects in single and compound mutants of cadherins implicated by the expression and speculative molecular code will further help delineate the roles of this gene family in various aspects of circuit assembly; these include cell positioning, axodendritic patterning, synaptic partner choice, and downstream behavior. By addressing the question of circuit assembly from multiple directions and with new tools, this thesis provides a generic workflow; we hope that it will bring C. elegans neurobiologists a few steps closer to untangling complex circuit assembly in the context of entire gene families which orchestrate it.