Family of neural wiring receptors in bilaterians defined by phylogenetic, biochemical, and structural evidence

The evolution of complex nervous systems was accompanied by the expansion of numerous protein families, including cell-adhesion molecules, surface receptors, and their ligands. These proteins mediate axonal guidance, synapse targeting, and other neuronal wiring-related functions. Recently, 32 interacting cell surface proteins belonging to two newly defined families of the Ig superfamily (IgSF) in fruit flies were discovered to label different subsets of neurons in the brain and ventral nerve cord. They have been shown to be involved in synaptic targeting and morphogenesis, retrograde signaling, and neuronal survival. Here, we show that these proteins, Dprs and DIPs, are members of a widely distributed family of two- and three-Ig domain molecules with neuronal wiring functions, which we refer to as Wirins. Beginning from a single ancestral Wirin gene in the last common ancestor of Bilateria, numerous gene duplications produced the heterophilic Dprs and DIPs in protostomes, along with two other subfamilies that diversified independently across protostome phyla. In deuterostomes, the ancestral Wirin evolved into the IgLON subfamily of neuronal receptors. We show that IgLONs interact with each other and that their complexes can be broken by mutations designed using homology models based on Dpr and DIP structures. The nematode orthologs ZIG-8 and RIG-5 also form heterophilic and homophilic complexes, and crystal structures reveal numerous apparently ancestral features shared with Dpr-DIP complexes. The evolutionary, biochemical, and structural relationships we demonstrate here provide insights into neural development and the rise of the metazoan nervous system.

The role of lineage, hemilineage and temporal identity in establishing neuronal connectivity in the Drosophila larval CNS

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neuronal morphology and connectivity. Here we map the developmental origin of 78 bilateral pairs of interneurons from seven identified neural progenitors (neuroblasts) within a complete TEM reconstruction of the Drosophila newly-hatched larval CNS. This allows us to correlate developmental mechanism with neuronal projections, synapse targeting, and connectivity. We find that clonally-related neurons from project widely in the neuropil, without preferential circuit formation. In contrast, the two NotchON/NotchOFF hemilineages from each neuroblast project to either dorsal motor neuropil (NotchON) or ventral sensory neuropil (NotchOFF). Thus, each neuroblast contributes both motor and sensory processing neurons. Lineage-specific constitutive Notch transforms sensory to motor hemilineages, showing hemilineage identity determines neuronal targeting. Within a hemilineage, temporal cohorts target processes and synapses to different sub-domains of the neuropil, effectively “tiling” the hemilineage neuropil, and hemilineage/temporal cohorts are enriched for shared connectivity. Thus, neuroblast lineage, hemilineage, and temporal identity progressively restrict neuropil targeting, synapse localization, and connectivity. We propose that mechanisms generating neural diversity are also determinants of neural circuit formation.

Molecular basis of synaptic specificity by immunoglobulin superfamily receptors in Drosophila

In stereotyped neuronal networks, synaptic connectivity is dictated by cell surface proteins, which assign unique identities to neurons, and physically mediate axon guidance and synapse targeting. We recently identified two groups of immunoglobulin superfamily proteins in Drosophila, Dprs and DIPs, as strong candidates for synapse targeting functions. Here, we uncover the molecular basis of specificity in Dpr–DIP mediated cellular adhesions and neuronal connectivity. First, we present five crystal structures of Dpr–DIP and DIP–DIP complexes, highlighting the evolutionary and structural origins of diversification in Dpr and DIP proteins and their interactions. We further show that structures can be used to rationally engineer receptors with novel specificities or modified affinities, which can be used to study specific circuits that require Dpr–DIP interactions to help establish connectivity. We investigate one pair, engineered Dpr10 and DIP-α, for function in the neuromuscular circuit in flies, and reveal roles for homophilic and heterophilic binding in wiring.