A key principle of neural circuit assembly is neurite self-avoidance, a process in which sister axons or dendrites of the same neuron repel each other but interact freely with those from other neurons, indicating they can discriminate self from non-self. In flies the cell adhesion molecule Dscam1 has been shown to be a key mediator for neurite self-avoidance. Alternative splicing of Dscam1 pre-mRNA generates a large number of distinct protein isoforms that interact in a strictly homophilic manner, which mediate contact-dependent repulsion that leads to self-avoidance. However, the vertebrate Dscam genes do no encode significant diversity. Rather, this diversity and function appear to be provided by the clustered protocadherins (Pcdhs). Studies in our lab and others have shown that 1) Stochastic expression of clustered Pcdhs by alternative promoter choice generates single cell diversity; 2) Combinatorial homophilic interactions between clustered Pcdhs at the cell surface generate single neuron identity; 3) Pcdh- ? proteins are required for dendritic self-avoidance in mouse starburst amacrine cells and Purkinje cells. These observations have led to the hypothesis that clustered Pcdhs function similarly as Dscam1 proteins to mediate self-avoidance in vertebrates. In this application, we propose to carry out a comprehensive genetic analysis to determine whether clustered Pcdhs play a central role in neurite self-avoidance in the vertebrate nervous system, whether Pcdh diversity is required for this process, and whether different Pcdh clusters functionally compensate each other in specific cellular contexts. To accomplish these goals, we will generate single cell knockouts of Pcdh- ?, - ?, and - ? gene clusters individually or altogether, and study the loss of function effects in neurite arborizationof individual neurons.
In Aim 1, we will establish and validate two complementary single cell knockout methods, Mosaic Analysis with Double Markers (MADM) and Single Neuron Analysis in Chimeras (SNAC). Both methods sparsely generate and label mutant cells in the otherwise normal neural network, allowing robust morphological analysis and rigorous assessment of cell autonomous gene function in single neurons.
In Aim 2, we will use single cell knockouts of the Pcdh- ? gene cluster to validate previously identified dendritic self-avoidance and arborization phenotypes, address the diversity requirement for these processes by generating a Pcdh- ? allele lacking all alternate isoforms, and identify spinal interneuron lineages that display neurit patterning defects that may be associated with a striking phenotype in the Pcdh- ? cluster deletion mice - complete loss of central pattern generator (CPG) for locomotion.
In Aim 3, we propose to study the consequences of loss-of-function of all clustered Pcdhs on neural development, and perform a CNS-wide screen for neuronal cell types in which axonal or dendritic self-avoidance is disrupted. The proposed studies should provide significant new insights into mechanisms of neural circuit assembly in the vertebrate nervous system, as well as into etiologies of diverse neurological disorders resulting from altered neural circuitries.
Many devastating neurological and mental disorders, such as autism and schizophrenia, result from defects in neural circuit assembly, but their etiologies are currently very poorly understood. The proposed study directly addresses fundamental principles of neural circuit assembly, and is expected to contribute to a better understanding of disease mechanisms and inspire rational designs of novel therapies.
|Rubinstein, Rotem; Goodman, Kerry Marie; Maniatis, Tom et al. (2017) Structural origins of clustered protocadherin-mediated neuronal barcoding. Semin Cell Dev Biol 69:140-150|
|Chen, Weisheng V; Nwakeze, Chiamaka L; Denny, Christine A et al. (2017) Pcdh?c2 is required for axonal tiling and assembly of serotonergic circuitries in mice. Science 356:406-411|
|Mountoufaris, George; Chen, Weisheng V; Hirabayashi, Yusuke et al. (2017) Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly. Science 356:411-414|