Genetic studies of cerebellar circuit assembly

I have been broadly interested in the molecular, cellular, and developmental mechanisms underlying neural circuit assembly in mammals. My graduate research has focused on the cerebellum, and I have pursued a variety of genetic and other approaches to investigate the mechanisms underlying the assembly of cerebellar circuits. My work has had three primary directions: (1) characterizing the role of birth timing in the wiring and function of cerebellar granule cells; (2) understanding the role of synapse formation in cerebellar Purkinje cell dendrite morphogenesis; and (3) developing new methods for profiling the cell surface proteomes of specific cell types in mice and applying these methods to characterize the cell surface proteomes of developing and mature Purkinje cells. In the first line of research, I probed the role of birth timing in cerebellar granule cell wiring and function. Cerebellar granule cells comprise over half of all neurons in the mammalian brain, yet there are no reports of molecularly-defined subtypes within cerebellar regions. However, previous work revealed that granule cells project their axons in a manner dictated by their birth timing. Thus, I developed strategies to gain genetic access to early- and late-born granule cells and performed viral tracing and two-photon imaging in vivo to reveal the wiring and functional properties of early- and late-born granule cells. Retrograde monosynaptic rabies virus tracing revealed different patterns of mossy fiber input to granule cells in different lobules, as well as to early- and late-born granule cells of the same lobule. Imaging revealed representations of diverse task variables and stimuli by both populations, with differences in the proportions of early- and late-born cells encoding a subset of movement and reward parameters. Taken together, these data suggest neither organized parallel processing nor completely random organization of mossy fiber--> granule cell circuitry, but instead a moderate influence of birth timing on granule cell wiring and encoding. In the second line of research, I addressed the role of synapse formation in dendrite morphogenesis of cerebellar Purkinje cells. The synaptotrophic hypothesis of dendrite growth, a hypothesis more than 30 years old, posits that dendrites preferentially grow to-ward rich synaptogenic fields. However, this hypothesis had not been causally tested in the mammalian central nervous system in vivo. We reasoned that by disrupting GluD2, a synaptogenic protein expressed in Purkinje cells and involved in formation and maintenance of parallel fiber--> Purkinje cell synapses, we could test this hypothesis in the mammalian brain. Strikingly, sparse but not global knockout of GluD2 resulted in a curious phenotype in which Purkinje cell dendritic arbors grow into an inverted triangular shape, under-elaborating in the deep molecular layer and overelaborating in the superficial molecular lay-er. Developmental, overexpression, structure-function and genetic epistasis experiments, along with modeling, revealed the importance of GluD2's synaptogenic activity in dendritic morphogenesis, supporting an important role for molecular interactions crucial for synapse formation and function in dendrite morphogenesis in the mammalian brain. In the third and final line of research described here, I developed methodology featuring a new mouse line and accompanying protocols for unbiased proteomic profiling of extracellular cell surface proteins of defined cell types in mice via proximity labeling. To date, there have been no methods for cell-type specific, unbiased capture of cell-surface proteomes in situ. Our method works in diverse organs and cell types and has allowed us to generate high quality cell surface proteomes of developing and mature cerebellar Purkinje cells. This work is being followed by a proteome-instructed CRISPR-mediated loss-of-function in vivo screen to discover novel molecular regulators of dendrite morphogenesis. This method constitutes a valuable new approach for profiling cell surface proteins in de-fined cell types in mice. In summary, my thesis work utilizes genetic and other approaches to pursue mechanistic interrogation and technological development to gain insight into the molecular, cellular, and developmental mechanisms of cerebellar circuit assembly

Advisor: Luo Liqun
Ding Jun (Jun B.)
Shen Kang
Südhof Thomas C.